Study of the relationship between sodium ion and hydrogen ion regulation in rat skeletal muscle

The purpose of this study was to elucidate the relationship between the active transport of sodium and the regulation of intracellular pH in rat skeletal muscle. Experiments were conducted both in vivo and in vitro in which Na transport and tissue acid-base balance changed independently and concurrently. Ouabain was employed to inhibit the Na transport system. The ability of cell to maintain a constant internal pH was challenged by producing graded intensities of extracellular metabolic and respiratory acidosis. Acidosis alone had no discernible effect on the sodium transport system in in-vivo experiments. No correlation could be demonstrated between intracellular Na+ and either intracellular or extracellular H+, nor was the electrochemical gradient for sodium affected by acidosis in vivo. The electrochemical gradient for sodium was increased by acidosis in vitro, however, suggesting a stimulation of the Na pump by H. Does of ouabain which caused a marked impairment of sodium transport resulting in an accumulation of Na in skeletal muscle did not cause a parallel accumulation of H in in-vivo experiments except in the most severely acidotic animals. In the in-vitro experiments, ouabain did an intracellular acidosis in tissues subjects to acid challenges. Several factors might explain the failure of ouabain to alter cell pH to any significant degree in the in-vivo experiments, even though the drug may have impaired the proposed Na-H transport system. First, the large increase in plasma K which invariably accompanied ouabain administration in vivo very probably antagonized the action of ouabain on the Na-H pump. In in-vitro experiments where external potassium did not vary, ouabain was found to impair the ability of skeletal muscle to resist an acid challenge and under these conditions did cause an accumulation of H+ intracellularly. Second, acidosis per se may have stimulated the pump sufficiently to antagonize the inhibitory influence of oubain of H transport. Third, there was some evidence that the intracellular HCO3 concentration was higher in ouabain-treated animals, possibly because the electrochemical gradient which ordinarily drives this ion out of the cell was reduced. This would mean that even though less H might be extruded in the ouabain-treated animals, the excel H would be buffered by HCO3. Finally, the possibility was considered that a change in cell pH may not be a vailid criterion for determining the impairment of H transport, and that a change in the electrochemical gradient for H may be a better standard for evaluating changes in H distribution. It was clear that ouabain does reduce the electrochemical gradient for H both in vivo and in vitro. The results of these experiments are thus consistent with the hypotheses that 1) H is actively transported out of skeletal muscle cells, and 2) the mechanism responsible for the active extrusion of H is extremely similar to, or perhaps even identical with, the mechanism underlying the active extrusion of Na in skeletal muscle.

A STUDY OF THE RELATIONSHIP BETWEEN SODIUM ION AND HYDROGEN ION REGULATION IN RAT SKELETAL MUSCLE by Trudy Ann Elsmore A thesis submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology University of Utah June, 1968 This Thesis for the Doctor of Philosophy Degree by Trudy Ann Elsmore has been approved September, 1967 Chairman, Supervisory Committee " . Reader, Supervisory Commit , " '. , . f i/ I; ( ' Reader, Superv isory Committee Head, Major Department ,. .' :�;">. :: I� ' .i , . � . 'f . ,/ Dean, Graduate School ':... i . . ' ,// / _.,f / , . ,-- ; : ;." " f . , , ; 4 ,- .. ' . l' . ACKNOWLEDGMENTS The author is deeply appreciative of the instruction and assistance she has received from all the members of the Department of Pharmacology throughout her graduate training. A special debt of gratitude belongs to Dr. C. D. Withrow for the warm personal interest, encouragement, enlightenment and practical assistance he has proffered not only during the period of the thesis research but throughout our association. His advice and criticism during the preparation of this manuscript have been particularly valuable. The patience and warmth of the entire Withrow family: Carol, Ann, Linda and Kent Dean have been greatly appreciated. The author is grateful to Dr. Louis Goodman for the guidance and understanding which he has extended throughout her graduate training. Sincere appreciation is extended to the members of the authors thesis committee, Dr. D. M. Woodbury and Dr. E. Fingl of the Department of Pharmacology and Dr. T. F . Dougherty and Dr. W. Stevens of the Department of Anatomy, for the encouragement they have provided and the suggestions and criticisms they have made during the preparation of this manuscript. Thanks are due Mrs. Carol Withrow for many reasons, but particularly for her efforts in expediting the course of data through the computers and in adapting existing programs to particular needs. iii To Mrs. Donnabelle Swingle the author is especially grateful for her technical skill and assistance during the course of the experimental work, for her good sense and good humor, and particularly for her efforts above and beyond the call of duty in the author's wake. Appreciation is extended to Mr. Aldo Gabardi for his help in designing and constructing the in-vitro chambers used in these experiments. Thanks are due Mrs. Myrna Kaufman for her conscientious efforts in typing this manuscript and the special efforts she has put forth to do so. Thanks are also due Mrs. Lou Ann Thomas for her help in preparation of this manuscript. The author has been most grateful to Mr. Dave Lewis for his excellent cooperation in providing the animals for these experiments and to Mr. Mac Gadd for his care and concern for the animals and for the author over the course of several years. The author is grateful to her family, Mrs. Gertrude Elsmore, Mr. Nephi Elsmore, and especially to George Eslmore, for their help, encouragement, and understanding during the many months of research and writing and for so much else that words cannot express. iv TABLE OF CONTENTS page ACKNOWLEDGMENTS iii ABSTRACT vii LIST OF TABLES x LIST OF FIGURES xi INTRODUCTION 1 Historical Background 1 13 Statement of the Problem METHODS 19 In -Vivo Experiments Experiment 1 Experiment 2 19 21 21 In-Vitro Experiments Experiments 1 to 4 Experiments 5 and 6 Experiments 7, 8, and 9 21 23 24 24 Analytical Techniques 24 Calculations 26 RESULTS 27 Tissue Water Distribution In-Vivo studies In-Vitro studies 27 27 27 Acid-base and Electrolyte Data In- Vivo experiments General comments Plasma data Tissue electrolyte data 30 30 30 31 35 v Tissue acid-base data Transmembrane distribution of ions In-Vitro Experiments Extracellular fluid data Tissue electrolytes Intracellular data 40 46 49 49 49 50 DISCUSSION 55 SUMMARY 85 REFERENCES 87 VITA 100 RESEARCH PROPOSALS vi ABSTRACT The purpose of this study was to elucidate the relationship between the active transport of sodium and the regulation of intracellular pH in rat skeletal muscle. Experiments were conducted both in vivo and in vitro in which Na transport and tissue acid-base balance were changed independently and concurrentl y . Ouabain was employed to inhibit the Na transport system. The ability of cells to maintain a constant internal pH was challenged by producing graded intensities of extracellular metabolic and respiratory acidosis. Acidosis alone had no discernible effect on the sodium transport system in in -vivo experiments. No correlation could be demonstrated between intracellular Na + and either intracellular or extracellular H+, nor was the electrochemical gradient for sodium affected by acidosis in vivo. The electrochemical gradient for sodium was increased by acidosis in vitro, however, suggesting a stimulation of the Na pump by H. Doses of ouabain which caused a marked impairment of sodium transport resulting in an accumulation of Na in skeletal muscle did not cause a parallel accumulation of H in in-vivo experiments except in the most severely acidotic animals. In the in -vitro experiments, ouabain did cause an intracellular acidosis in tissues subjected to acid challenges. Several factors might explain the failure of ouabain to alter cell pH to any significant degree in the in-vivo experiments, even though the drug vii may have impaired the proposed Na-H transport system. First, the large increase in plasma K which invariably accompanied ouabain administration in vivo very probably antagonized the action of ouabain on the Na - H pump. In in-vitro experiments where external potassium did not vary, ouabain was found to impair the ability of skeletal muscle to resist an acid challenge and under these conditions did cause an accumulation of H+ intracellularly. Second, acidosis per se may have stimulated the pump sufficiently to antagonize the inhibitory influence of ouabain on H transport. Third, there was some evidence that the intracellular HC03 concentration was higher in ouabaintreated animals, possibly because the electrochemical gradient which ordinarily drives this ion out of the cell was reduced. This would mean that even though less H might be extruded in the ouabain-treated animals, the excess H would be buffered by HC03 • Finally, the possibility was considered that a change in cell pH may not be a valid criterion for determining the impairment of H transport, and that a change in the electrochemical gradient for H may be a better standard for evaluating changes in H distribution. It was clear that ouabain does reduce the electrochemical gradient for H both in vivo and in vitro. The results of these experiments are thus consistent with the hypotheses that 1) H is actively transported out of skeletal muscle cells, and 2) the mechanism responsible for the active extrusion of H is extremely similar viii to, or perhaps even identical with, the mechanism underlying the active extrusion of Na in skeletal muscle. ix LIST OF TABLES Table Page 1 Distribution of water in rat skeletal muscle, in-vivo experiments 28 2 Distribution of water in rat skeletal muscle, in-vitro experiments 29 3 Plasma acid-base and electrolyte data, in -vivo experiments 32 4 Water and electrolyte content of skeletal muscle, in-vivo experiments 36 5 Intracellular electrolyte composition of skeletal muscle, in -vivo experiments 38 6 Skeletal muscle acid-base data calculated by the tissue C02 method, in -vivo experiments 41 7 Skeletal muscle acid-base data calculated by the DMO method, in-vivo experiments 43 8 Transmembrane distributions of K, H, Na, and Cl in rat skeletal muscle, in-vivo experiments 47 9 Plasma data for small Long-Evans rats and composition of in-vitro bath media 52 10 Water and electrolyte content of skeletal muscle, in-vitro experiments 53 11 Intracellular pH and electrolyte composition of skeletal muscle, in-vitro experiments 54 12 Electrochemical gradients of Hand Na, in-vivo experiments 80 13 Electrochemical gradients of Hand Na, in-vitro experiments x 82 INTRODUCTION Historical Background 'The importance of stability of hydrogen Ion concentration of this internal environment for vital cell function is attested by the precison with which it is regulated and by the numbers of mechanisms concerned with its stabilization. The chemical buffers of the tissues and body fluids constitute a first line of defense of body reaction neutralizing those acids and alkalis which are produced within, or which gain access to the body' (Pitts, 1954). The concept of an internal environment in which an animal actually lives was first formulated by Claude Bernard (1878), often regarded as the father of experimental medicine. Bernard was cognizant that acidity was an important aspect of this environment and one which was continually regulated by the organism. For example, he described his experiments with rabbits which indicated that the kidney played a part in this overall regulatory process (Bernard, 1878). The significance of internal acid-base balance was recognized clinically, however, long before the concept was formalized by Bernard. In 1831 a letter by W. B. 0' Shaugnessy was published in the Lancet in which the physician presented his observations on the acidosis accompanying Asiatic cholera which was rampant in England at that time. O'Shaugnessy was sufficiently astute to recognize a "carbonate of soda" as the principal alkali of the blood and to observe that the concentration of this salt in the sera of cholera patients was less than that in the sera of normal individuals. 2 His observations were turned to practical advantage by another practitioner, Latta, who reported in 1832 the beneficial effects obtained by injecting cholera patients with solutions of 0.5% sodium chloride and 0.1 to 0.25% of either sodium carbonate or sodium bicarbonate. These cases and many of the clinical observations recorded before the turn of the century that pointed to the importance of acid - base balance in normal mammals and the disruption of this balance in some disease states have been discussed by Van Slyke (1966). In the early 1900's initiative in the field of acid-base physiology shifted from clinicans to physical chemists. In 1909 the hydrogen electrode and the symbol pH were introduced by S¢rensen. At almost the same time L. J. Henderson published his classic monograph 'Equilibrium between bases and acids in the animal organism' in which he formalized the concept of bicarbonate as the body's alkali reserve (Henderson, 1908; Henderson & Black, 1908) and developed the Henderson equation: Hasselbalch (1916) put this equation into the logarithmic form in which it is used today, the Henderson- Hasselbalch equation: pH = pK + log rlJHCO3 -J • [H2 C03] Hasselbalch also played a role in developing a hydrogen electrode that could be used in the presence of C02 (Hasselbalch, 1910) with which 3 Lundsgaard made the first measurement of blood pH in 1912 (Hassebalch & Lundsgaard, 1912). The Henderson-Hasselbalch equation, the hydrogen electrode and the concept of pH were the basic developments that facilitated the systematic experimental exploration of acid - base metabolism and which stimulated interest in this field. Another basic tool of acid -base physiologists, the CO2 dissociation curve, was evolved in the 1920's (Warburg, 1922; Peters, Bulger & Eisenman, 1923; Peters and Van Slyke, 1931). Such curves are graphic representations of the Henderson - Hasselbalch equation in which the standard bicarbonate line (the relationship between bicarbonate content and pC02 of a system) is determined experimentally. They are widely regarded as the best means of describing the buffer capacity of a biological system (Fenn, 1961) and they have been of great value in classifying the numerous acid- base states observed clinically, insofar as these conditions can be assessed from blood samples (M¢ller, 1959). Despite the widespread interest in problems of acid - base regulation evident in the 1920's and '30's, relatively few investigators attempted to examine the role of the tissues in this process. Most workers concentrated, instead, on describing how the composition of extracellular fluids, particularly blood, was affected by acid-base distortions. This occurred, as ReIman (1966) has pointed out, because data on blood samples were readily obtained and relatively easy to interpret, whereas reliable methods for the direct or indirect assessment of tissue composition evolved far 4 more slowly. The contributions made by those few investigators who surmounted these technological limitations, however, are extremely significant ones. The hypothesis that cells participate in the process of acid-base regulation was first formulated almost 60 years ago. According to E lkinton (1962) it was originally suggested by Henderson in 1909. The first experi- mental evidence for the hypothesis was provided by another of the pioneers in the field of acid-base physiology, D. D. Van Slyke. In 1917 Van Slyke and Cullen reported the results of experiments in which they had infused dogs intravenously with a known quantity, 15 mEq, of sulfuric acid and 90 minutes thereafter had measured the whole blood pH and pC0 . Since 2 the observed pH was les s acid than could be explained by the change in the blood bicarbonate concentration alone, they concluded that .•.. the greater I part of the injected acid did not remain in the blood, but was at once transferred elsewhere and presumably neutralized by the bicarbonate and phosphate reserves in other parts of the body. I Skeletal muscle, which com- prises the largest single tissue mass of the body, was regarded by them as the major source of such reserves. They thus suggested that a dynamic acid-base equilibrium exists between plasma and tissues and that plasma bicarbonate represents only an index of the total alkaline reserve of the body_ The hypothesis was strengthened by several later studies which suggested that tissues, particularly skeletal muscle, playa role in buffering 5 respiratory as well as metabolic acid loads. As previously indicated, the C02 dissociation curve is a widely accepted means of describing the capacity of a system for buffering CO2 • Fenn (1928), Stella (1929), and Root (1933) presented the first data on the CO 2 dissociation curves of tissues, namely, frog nerve and skeletal muscle. Similar curves were obtained for mammalian muscle by Irving, Foster, & Fergusson (1932). These curves indicated that muscle definitely could buffer CO , but that 2 the CO2 buffering capacity of muscle was less than that of blood on a gramto-milliliter basis. In 1936 Fenn elaborated on the original hypothesis that tissues participate in acid-base regulation by postulating a mechanism for this function. He had observed that the potassium content of frog muscle in vitro varied as a function of the extracellular (bath) pH, i.e., muscle potassium decreased in an acid medium and increased in a relatively basic medium (Fenn & Cobb, 1934). He thus suggested that the bufrering process effected by the tissue and the change in electrolyte metabolism accompanying a change in extracellular pH must be related processes (Fenn, 1936). l By the late 1940 s technological advances facilitated examination of these questions on a far larger scale. In 1946 Darrow demonstrated that samples of skeletal muscle taken from alkalotic rats have a higher N a and a lower K content than do samples obtained from normal animals; he regarded this to be directly related to tissue buffering. Similar results were obtained by Elkinton, Squires, & Crosley (1951) in human subjects. 6 In 1948 Darrow and associates showed that metabolic acidosis as well as metabolic alkalosis induced a change in the electrolyte pattern of rat skeletal muscle. They concluded that there is a direct correlation between plasma bicarbonate and intracellular Na and an inverse correlation between plasma bicarbonate and tissue K (Darrow, Schwartz, Iannucci, & Coville, 1948). Cotlove,Holliday, Schwartz, & Wallace (1951) working with rats; Keating, Weichselbaum, Alanis, Margraf, & Elman (1953) studying the problem in dogs;and Elkinton, Squires & Singer (1951) in humans confirmed the findings of Darrow. Cooke, Coughlin & Seegar (1952), Giebisch, Berger, & Pitts (1955), and Schribner, Fremont-Smith & Burnell (1955) were among the first to describe the effects of respiratory acidosis on the distribution of tissue electrolytes. The net impact of studies such as these was to establish conclusively that tissue electrolyte patterns are related to the acid-base status of the organism. Pitts (1954) and Swan & Pitts (1955) examined the question of tissue buffering from a quantitative standpoint. They infused dogs with known quantities of hydrochloric acid and calculated the magnitude and site of buffering that occurred at various intervals after the beginning of the acid administration. These data established the existence and significance of cellular acid·-base regulation since 60% of the buffering observed in these experiments could be attributed to cells other than erythrocytes. Schwartz, Jenson, & ReIman (1954) reported similar findings in human subjects. Schwartz, <;orning & Porter (1957) furthermore demonstrated by acid-infusion 7 experiments in dogs that the partition of hydrogen ions between intracellular and extracellular buffers is essentially independent of the degree of acidosis. For example, when a total of 4 mEq/kg of acid was infused the mean quantity of acid buffered intracellularly (excluding erythrocytes) was 2.3 mEq/kg or 58% of the total; when 14 mEq/kg was infused an average of 8 mEq/kg was buffered intracellularly, also 58% of the total. In addition to establishing the importance of muscle tissue in the overall buffering process, all these workers found evidence that a substantial part of the buffering done by muscle cells involved the exchange of hydrogen ions for intracellular cations. Whether this exchange reflects a purely physical process, as suggested by some (Fenn, 1936; Nuttal, 1965), or an active mechanism, as proposed by others (Darrow, 1946; Cooke & Seegar, 1952; Hill, 1955; Robin, Wilson, & Bromberg, 1961; Bittar, 1964; Lemann, Lennon, Goodman & ReIman, 1964),has not been definitely established. To define how cells accomplish the regulation of acidity it is first necessary to understand how hydrogen ions are distributed between the cell and extracellular fluids and to elucidate the mechanisms which govern this relationship. A number of experiments temporally parallel to those already cited have clarified certain aspects of this relationship and have led to the current concept that cell pH is regulated by an active mechanism which extrudes H from the cell. This concept is Widely, though not universally, accepted. One of the major reasons for invoking the concept of active H 8 extrusion is the fact that the values of cell pH measured by most investigators are about 6 .9, far more alkaline than would be expected if H were distributed passively as a function of the membrane potential in accordance with the Donnan theory (Wallace & Hastings, 1942; Gardner, MacLachlan, & Berman 1952; Hill, 1955; Caldwell, 1958; Waddell & Butler, 1959; Robin, 1961; Bittar, 1964; ReIman, 1966). Fenn (Fenn, 1928; Fenn & Mauer, 1935) presented the first data on intracellular pH which could be considered technically valid by current standards. The methods of earlier investigators: vital staining, potentiometric measurements of tissue breis, etc., were of dubious value (Caldwell, 1956; Bittar, 1964). Fenn utilized the Henderson- Hasselbalch equation to calculate the pH of frog muscle from the CO 2 content of that tissue and arrived at a value of 6.9. Wallace and Hastings (1942) refined the 'C0 method' and reported that the pH of mam2 malian (cat)skeletal muscle was also 6.9. A second chemical method for the determination of cell pH was introduced by Waddell and Butler (1959). This method, based on the distribution of the weak acid 5,5 -dimethyloxazolidine- 2, 4-dione (DMO) between tissues and plasma, has been widely utilized for studying cell pH in dogs (Waddell & Butler 1959; Brown & Goott, 1963), rats (Irvine, Saunders, Milne & Crawford, 1961; ReIman, Adler & Roy, 1963; Withrow & Woodbury, 1964; Adler, Roy & ReIman, 1965a & b; Sanslone and Muntwyler, 1966) and humans (Manfredi, 1963). The agreement among these studies is excellent in that the intracellular pH of skeletal muscle was found to be about 6.9 in all cases. 9 Direct measurement of intracellular pH with a pH-sensitive glass microelectrode was first attempted by Caldwell (1954, 1958) studying tissues of the giant crab. Mter considerable refinement of the technique, it became possible to measure the pH of amphibian muscle (Kostyuk & Sorokina, 1961) and of mammalian fibres (Carter, 1961; Lavalee, 1964). Most micro- electrode measurements have been in good agreement with the results obtained by chemical methods. Only two studies have been published in which the internal pH (pHi) of resting skeletal muscle was not found to be 6.9 to 7 .0 but 5 .9 as predieted from the membrane potential by the Donnan theory. Conway and Fearon (1944) claimed that the pHi values calculated for muscle by Wallace and Hastings (1942) and Wallace and Lowry (1942) using the tissue CO 2 method were far too high. Conway contended that a large fraction of the acid-releasable CO in tissue was not HC03 -, as Wallace assumed it to be, 2 because it was not precipitated by barium hydroxide. When Conway corrected total tissue CO values by subtracting this barium-soluble fraction, 2 the calculated pHi of skeletal muscle was.§..O or less, rather than 7 . Other investigators (Withrow, 1959; Thompson & Brown, 1960; Fenn, 1961; Butler, Poole & Waddell, 1967) have failed to confirm the existence of a large consistent fraction of barium-soluble CO2 in muscle. If this fraction exists, moreover, its significance to cell pH calculations is dubious since the results obtained by the CO 2 method agree with those calculated by methods which do not rely on the measurement of tissue CO 2 • 10 Recently, Carter, Rector, Campion & Seldin (1967) published data indicating that the mean intracellular pH of rat skeletal muscle measured directly by microelectrode is 5.99 ± 0.14 (S.D. based on 38 observations). If the results obtained by Carter and his associates are correct, then all previous microelectrode studies and all the chemical measurements of cell pH must be in error by the same amount. Until there is some independent support of the findings of Carter's group it would seem reasonable to accept the preponderance of evidence that cell pH is normally 6.9, and that H is not distributed according to the Donnan theory . In view of the foregoing discussion of the pHi of resting skeletal muscle, it must be concluded that either a) the cell is impermeable to H and HC03 and the Donnan theory does not apply or b) the cell is capable of transporting H (or HC03 ) against its electrochemical gradient, as it does Na • Conway (1957), and a few others who have accepted his arguments (Harris, 1960; Wyke, 1963), have supported the first explanation. Conway based his arguments on his calculations of the theoretical size of the hydrated H molecule but provided no evidence that these calculations were valid. On the contrary, there is abundant evidence that exogenously administered acid loads are buffered largely in the intracellular compartment (Pitts, 1954; Schwartz et al.,1957; Lemann, Lennon, Goodman, Litzow & ReIman, 1965), observations which suggest that H must enter cells regardless of how large the hydrated form might be. Thus, active transport of H seems a more likely explanation than does cellular 11 impermeability to H. The elegant experiments of Adler, Roy and ReIman (1964, 1965a, 1965b) on the rat diaphragm in vitro have done much to elucidate the relationship between cell pH and extracellular aCidity. In these experiments pC02 and bicarbonate were varied independently over wide ranges (10 to 200 mm Hg and 5 to 50 mEq/L, respectively) and cell pH was determined by the DMO method. They found that over a certain range of extracellular pH, intracellular pH was maintained at a constant value of 6.9. Cell pH did fall when the extracellular pH (pH ) fell below 6.95 or when e the pC02 was greater than 70 mm Hg. The authors concluded from their data that extracellular respiratory acidos is induced a larger change in cell pH than did an extracellular metabolic acidosis of the same magnitude and that pHi was not simply a direct function of pHe as the Donnan theory predicts. In a preliminary note in 1964 the ReIman group reported that '. . . normal cell [H+] , as well as its characteristic responses to changes in pC0 or HC03 -, is critically dependent upon cell metabolism. These 2 data suggest that the control of cell pH is a metabolic function, rather than the simple result of the interposition of a semipermeable membrane between two buffer systems.' (Adler, Roy & ReIman, 1964). The dependence of cell pH on the status of electrolyte metabolism in skeletal muscle was first examined by Gardner, MacLachlan and Berman (1952). Employing the CO2 method to measure cell pH, they found that when rats became deficient in K the intracellular fluid of skeletal muscle tissue 12 became more acid even though the extracellular pH of potassium-deficient animals wa.s more alkaline than that of normal rats. These findings were confirmed by Schwartz & Wallace (1952). Irvine and associates (1961) repeated these experiments and found that the extracellular pH of K deficient rats was 7.50 compared to 7.39 in normal animals, but that the intracellular pH, measured by the DMO method, in such animals was reduced from normal value of 6.94 to 6. 71 . Others have studied the relationship between Na and H transport systems. The experiments of Schoffeneils (1955, 1956) on frog skin suggested that in this system Na and H compete for a common transport mechanism. Swan & Kossman (1958) presented evidence that there is an inverse correlation between cell pH and the active extrusion of Na in frog muscle . Keynes (1963) confirmed that the Na efflux of frog muscle was a function of intracellular pH. The experiments of Leaf, Keller & Dempsey (1964) showed that increasing H first stimulated and at higher levels depressed Na transport by toad bladder. Drugs known to affect the Na pump have been reported to have effects on cell pH as well. Withrow (1959) found that a drug known to stimulate the active transport of sodium by skeletal muscle cells, desoxycorticosterone acetate (DCA), induced an intracellular alkalosis in the rat. Bondani & Withrow (1965) reported that a drug known to inhibit the active transport of sodium, ouabain, tended to make rat skeletal muscle cells more acid. 13 Statement of the Problem The brief historical review above pointed out the significance of the cellular compartment in the over-all process of acid-base regulation and the importance of defining how H is regulated at the cellular level. Despite the wealth of data available, however, there is atpresent no single, generally accepted theory of the regulation of the distribution of hydrogen ion between the cell and extracellular fluid. There is far more consensus as to the hypotheses put forward in the past which cannot be accepted in view of present knowledge. For instance, the widespread agreement that the intracellular pH of skeletal muscle is normally 6.9 negates the proposal of Conway (1944) that Hand HC03 are distributed across the cell membrane in a Donnan equilibrium. According to the Donnan theory, the intracellular pH of a cell having a transmembrane potential of about 80 mV would be 1.3 pH units lower than that of the extracellular fluid, i.e ., at an extracellular pH of 7 .4, cell pH would be 6.1 not 6.9. Moreover, according to the Donnan theory, cell pH should vary directly with the extracellular pH. There is abundant evidence that this is not the case. Wallace and Hastings (1942) were the first to demonstrate that the intracellular pH of anesthetized cats remained unaffected when extracellular pH was varied by the administration of hydrochloric acid in doses up to 1 mEq/kg. Tobin (1956, 1958) found that muscle cell pH varied little even where acid loads as large as 7 mEq/kg of hydrochloric acid were given to cats. The studies of the ReIman 14 group (Adler, et al., 1965a & b) of rat diaphragm in vitro, the experiments of Elsmore and Withrow (1966) on rats in vivo, and the results obtained by Bailey and Withrow (1965) in rats and Schwartz, Brackett & Cohen (1965) in dogs provide additional support for a non-linear relationship between intracellular and extracellular pH. The notion that the skeletal muscle membrane is impermeable to hydrogen and bicarbonate ions, originally put forth by Wallace and Hastings (1942), has also been rejected by most modern investigators on the basis of the evidence cited previously. Hill (1955) was among the first to propose the only explanation which has not yet been refuted. He discussed at length his own data and those of others and concluded that neither the Donnan theory nor the H - and HC0 3 impermeable cell concept were valid. He suggested that a better explanation might be that H is actively transported out of the cell. The active extrusion of Na from muscle cells is a well-established phenomenon (Hodgkin, 1951; Keynes & Maisel, 1954) and Hill argued that if mechanisms existed for the transport of one cation, it was not unlikely that they might exist for other ions as well. The observations of the ReIman group that maintenance of the characteristic relationship between cellular and extracellular pH requires the expenditure of metabolic energy tends to strengthen the case for an active extrusion of H by the skeletal muscle cell. Withrow (1959) suggested that the transport of hydrogen ion from the cell might be done by the same system which extrudes sodium. This 15 explanation would fit with the observation that DCA, which stimulates the active transport of Na +, causes the intracellular fluid to become more alkaline. This' common pump hypothesis would explain the observations t of various investigators that the rate of Na + pumping varies with pH. It would also be consistent with the numerous studies which have demonstrated that the intracellular fluid of K+-deficient animals is more acid than normal since in K+ deficiency the operation of the sodium pump would be expected to be less efficient (see Withrow and Woodbury, 1964, for a discussion of this point). Finally, it would explain the observations that smooth muscle tissues of the rat, which are believed to have a far less active Na + pump than skeletal muscles (Daniel, 1958; Bohr, 1964), also have an intracellular pH much closer to that predicted by the Donnan theory (Elsmore & Withrow, 1966). Although there is considerable evidence to suggest an active transport system for H+ and to indicate that H+ and Na+ might be transported by a common carrier mechanism, the hypothesis is a difficult one to test since direct methods employed for most problems of this type are not applicable to this problem. For instance, if the problem involved Na+ and some ion other than H+ it could be approached rather simply by the use of radioactive tracers. Two isotopic forms of Na +, Na 22 and Na 24, are readily available which are biologically equivalent to Na + and the same is true of most inorganic cations of biological concern. The techniques for measuring influx and efflux rates of ions by the use of tracers have 16 been established and widely accepted (Ussing, 1947, 1949a & b). Thus it would be relatively easy to determine the total number of ions being transported by the pump per unit time, the relative proportions of two ions being transported by the same system, and, by knowing the intracellular concentrations of the ions, the relative affinities of the two ions for the pump "carrier site". Since the cation under consideration is H the question cannot be solved so simply. First, it has been demonstrated that deuterium ion is not biologically equivalent to H (Katz, 1960; Czajka et al., 1961, Thompson, 1960). Second, H participates in the general reaction: H+ + A- , ;:.\==~ HA of every weak acid inside and outside of the cell. The general problem which this would present in a flux study has been illustrated by Hogben (1955) who pointed out that HC 14 0 3 would measure the combined fluxes of HC0 , H2C03 , and CO because of the relationship between these 3 2 entities. Byanalogy, deuterium would measure the combined fluxes of H, H20, and the undissociated form of every weak acid in the cell. Clearly then, it is not possible to measure the total number of ions being transported by a Na-H pump nor the relative affinities of the two ions for the carrier. The approach employed in the present experiments is less direct than the tracer method outlined above. Since it has been established that cardiac glycosides are relatively selective in their ability to inhibit the 17 sodium transport system (Schatzman, 1953; Johnson, 1956; Horowicz & Gerber, 1962), it seemed logical to select a cardiac glycoside, ouabain, as a pharmacological tool for the present experiments. If the Na and H pumping mechanisms are identical, then inhibition of the Na pump should also impair the ability of the cell to extrude H . Whether a change in the rate of H' transport could be detected was another problem which entered into the design of these experiments. Because the rate of H efflux cannot be measured directly, variations in this parameter must be inferred from changes in the concentration of H in the cell. Accumulation of H in cells, or a decline in cell pH, could be expected only if the capacity of the extrusion system were exceeded. That the H extrusion system normally has some reserve capacity is indicated by the pH -plateau found by the ReIman group (Adler et al., 1965 a & b) and by Elsmore (1965). This could explain why Bondani and Withrow (unpublished observations) were unable to demonstrate a large reduction in the pHi of rats treated with ouabain but not subjected to acid-base distortions. For this reason, animals in these experiments were subjected to acid loads, both metabolic and respiratory, to impose stress on the hydrogen transport system, if such a system in fact exists. It also seemed desirable to control the respiration of the animals in these experiments to prevent the pos sibility of compensatory changes in respiration which, by altering pC0 , might obscure a change in the cells r ability to transport 2 H . 18 Most of the experiments planned for this study were to be done in vivo since the methods and techniques involved in that approach were well developed and it was possible to obtain ample tissue from each animal for all of the analyses desired. It also seemed desirable to conduct a separate series of experiments of similar design in vitro in which extracellular K concentrations could be controlled. This was not feasible in the in-vivo experiments since it is well established that acidosis (penn & Cobb, 1934) and the administration of ouabain (Cattel & Goodell, 1937; Wood & Moe, 1942) both produce large increases in the extracellular K. The control of extracellular K levels is important because it has been amply demonstrated that the active of the Na pump is stimulated by a high extracellular K (Steinbach, 1940) 1951; Harris, 1949; Hodgkin & Horowicz, 1959; Horowicz & Gerber, 1965). Thus it seemed possible that the uncontrollable rise in plasma K in the in-vivo experiments might obscure the full effect of ouabain on cellular Na and/or H transport and should be minimized. METHODS Two experimental approaches, in vivo and in vitro, were used to elucidate the relationship between cell pH and electrolyte transport in skeletal muscle. Each of these general categories included two types of experiments, namely, those conducted to obtain data on tissue water and inulin space, and those in which acid-base and electrolyte measurements were made. Except where specifically indicated, these sub-types differed only in the isotopically labeled compound, C 14 -inulinl or C 14 _DM02, employed. Male rats of the Long-Evans strain were used for all experiments. In-Vivo Experiments One-hundred-forty minutes prior to sacrifice normal, non-fasted rats weighing 250 to 300 grams received 1 ml of an 0.9% (w/v) NaCl solution containing 4 Ilc /ml of C 14 -labeled material. This was injected intraperitoneally as were all drugs and solutions in these experiments. Animals receiving C 14 -inulin were nephrectomized under ether anesthesia immediately prior to administration of the tracer solution. Seventy minutes prior to sacrifice each animal was injected with 1 ml/l00 grams of a 0.9% sodium chloride solution containing 0, 1.0, or 2.0m~iGl~. 1 Inulin-carboxyl-C 14, 0.1 mc/29.4 mg, obtained from New England Nuclear Corporation, Boston, Mass. 2 5, 5-Dimethyloxazolidine-2, 4-dione-2 C 14 , 7.47 mc/mmole, obtained from New England Nuclear Corporation; purity was demonstrated by the company with melting point and radiochromatography determinations. 20 ouabain3 and was anesthetized with pentobarbital, 50 mg/kg. The anesthetized animal was connected by an intratracheal cannula to a Palmer small animal respirator and ventilated for 1 hour at a rate of 50 strokes/min with a tidal volume of 1.6 mI. These ventilation parameters were selected to approximate normal values for animals of this size according to the nomograph of Kleinman & Radford (1964) and prior experience with this system (Elsmore, 1965). Respiratory acidosis was produced by connecting the pump intake to a Collins spirometer filled with 20% 02 and various concentrations of C02 and N2 . All other animals were respired with room air. Metabolic acidosis was produced by the injection of 4.0 or 6.0 mEq of NH 4Cl/kg at the time the animal was placed on the respirator. All other aniJ;Ilals received a corresponding injection of an 0.9% NaCl solution. At the end of the 60-min equilibration period, the animal was sacrificed by exsanguination from the abdominal aorta into an oiled, heparinized syringe. The pH and pC0 of the whole blood sample were measured 2 immediately; the remainder of the sample were centrifuged to separate the plasma fraction for subsequent electrolyte and radioactivity analysis. Samples of hind limb muscle tissue were quickly excised, trimmed of fat and connective tissue, blotted lightly on filter paper, and portions thereof 3 Ouabain, U. S. P. (Strophanthin G), m. w. = 728.8, was obtained from Cal Biochem, lot No. 65028. Solutions were freshly prepared for each experiment. 21 placed in tared vials appropriate to the analysis to be performed. The sampling operations were completed within three minutes after sacrifice. The sequence of treatments in a given experiment was determined from a table of random numbers. Experiment 1 To determine whether ouabain and/or acidosis altered the distribution of water in the tissue under study, 36 animals were divided into three acid-base treatment groups: control, extreme respiratory acidosis (24% CO ), and extreme metabolic acidosis (6 mEq NH Cl/kg). Half the animals 2 4 in each group received the high dose of ouabain (20 mg/kg) one hour after the administration of C 14 -inulin. The other half was given a control saline injection. Experiment 2 To study the relationship of pH and electrolytes in vivo, 108 animals were divided into six experimental groups. The control group was subjected to no acid-base distortions; respiratory acidosis was produced in three groups by ventilating animals with 6, 12, and 24% CO2 gas mixtures; and metabolic acidosis was produced in two groups of animals by administration of 4.0 and 6.0 mEq of NH4 Cl/kg. Each treatment group was divided into thirds and treated with saline or 10 and 20 mg ouabain/kg. In-Vitro Experiments Intact diaphragms of rats weighing 80 to 120 grams were incubated in a modified Krebs - Ringer bicarbonate medium aerated with 6 to 12% CO2 22 and 88 to 94% 02 as dictated by the CO 2 tensions required in the various experiments. This preparation has been des cribed in detail by ReIman and coworkers (1961). The composition of the medium bathing the tissues in their experiments was modified in the present studies to conform as closely as possible to the extracellular fluid of the rats being used. Extracellular fluid parameters for the purpose, as well as in-vivo tissue values, were obtained as follows. A group of 10 animals was anesthetized with 50 mg pentobarbital/kg and sacrificed 2 hours after the administration of 2 j.lc C 14 -DMO/rat. Blood pH and pC0 were measured and plasma electrolytes 2 and radioactivity were determined by methods given below. Fresh diaphragms from these animals were analyzed for electrolytes and radioactivity by the tissue methods described later. Based on the results of this experiment and the directions of ReIman, Gorham, & Levinsky (1961) the bath medium employed in these experiments was compounded as follows: NaCI, 105 mEq/L; NaHC0 , 25 mEq/L; KCl, 4 mEq/L; CaC1 2 , 1.3 mEq/L; 3 MgS04 , 1.2 mEq/L; NaHP04, 1.2 mEq/L; glucose, 1 giL; streptomycin, 100 units/L; chloramphenicol, 62.5 units/L. In experiments where metabolic acidosis was desired, the concentration of NaHC03 in the bath was reduced to 6 mEq/L, and that of NaCl increased to 125 mEq/L. At 30 min intervals during the first two hours of incubation, one liter of the bath medium was withdrawn from a total volume of 2.5 liters in the chamber and replaced with fresh solution. During the final two hours of incubation, the bathing medium was not changed and contained 20 to 25 j.lc/L of C 14 -labeled material 23 and, where indicated, 80 or 160 mg ouabain/L in addition to the constituents listed above. The temperature of the bath was maintained between 37 0 and 39 0 C throughout the experiment. In all experiments, two baths containing ten diaphragms apiece were run in parallel. The baths were identical except that one contained ouabain during the final two hours of incubation. During the last hour of incubation, 10 m1 samples of bath fluid were taken at half-hour intervals, analyzed for pH and pC02 immediately, and retained for radioactivity and electrolyte measurements. At the end of the incubation period, the diaphragm muscles were quickly dissected free from the rib cage outside the bath, blotted lightly on filter paper, and placed in tared vials. Tissue water determinations required a portion of one diaphragm; two portions of muscle were pooled for electrolyte analysis. Individual whole diaphragms were taken for radioactivity determinations. Experiments 1 to 4 To ascertain whether the distribution of tissue water was altered by any of the treatment procedures extracellular volumes measurements were made in diaphragms bathed with four different media- normal, low HC0 -, high CO , and hypotonic (NaCl 20 mEq/L less than other baths). 2 3 In each instance, one bath contained 160 mg/L ouabain, the other contained no drug. The extracellular volume of the tissues was determined with C 14 _ labeled inulin. 24 Experiments 5 and 6 To examine the effect of ouabain on cell pH in ---...::. diaphragms were incubated in baths with normal pH, pC02 , and potassium content. Two doses of ouabain were studied. Experiments 7, 8, and 9 To evaluate the cells r ability to handle an acid challenge when the integrity of the sodium-transport system was impaired by ouabain, experiments were done in which diaphragms were incubated iL. acid bathing media with potassium levels approximating control values and containing C14 DMO. In experiments 7 and 8, the effects of two doses of ouabain on tissue in low HC0 - medium (metabolic acidosis) were measured. In 3 experiment 9 the acidosis was induced by a high CO concentration (res2 piratoryacidosis), and only one ouabain concentration, 80 mg/L, was used. Analytical Techniques The analytical procedures followed in the in-vivo and in-vitro experiments were identical. A single sample was required for the determination of both the pH and the pC02 of a sample of whole blood or bath fluid. These measurements were made anaerobically at 37 0 C using an Instrumentation Laboratories Model 123 pH-pC0 Meter. 2 Plasma water content was not measured, but was assumed to be 92% in all experiments. Total muscle water was determined by drying fresh tissue samples at 105 0 C for approximately 48 hours. 25 Tissue electrolytes were extracted by incubating wet tissue in 9 volumes of 0.11 N HN0 for 12 to 18 hours at 60 0 C. The fine fibers of 3 tissue residue settled out spontaneously leaving a clear supernatant. Chloride contents of the tissue extract supernatant and of plasma or bath fluid were determined with an Aminco-Cotlove chloride titrator according to the method of Co-tlove and associates (1958). Potassium and sodium concentrations were measured in the same samples with an Instrumentation Laboratories Flame Photometer Model 143 using lithium as an internal standard (Boling, 1964). The relative concentrations of C 14 -labeled material in tissue and extracellular fluid were determined by liquid scintillation cOlUlting in a Nuclear-Chicago Liquid Scintillation System Model 725 operated at 100 C. The probable cOlUlting error was less than 1%. Samples were prepared for counting as follows: fresh wet tissue samples were digested 12 to 18 hours at 60 0 C in 9 volumes of 1 M piperidine; samples of plasma or bath fluid were mixed in 9 volumes of 1 M piperdine and warmed slightly. Aliquots of the piperdine solutions, 200 j.lL, were transferred to low-potassium counting vials containing 19 ml of a solvent-fluor mixture which was 79% toluene, 21% methanol (v/v) with 4.0 g of PPO and 50 mg of POPOP in each liter of the final mixture. Vials were sealed, shaken to effect a homogeneous solution, and counted. The channels ratio method as described by Hendler (1964) was used to correct the observed count rate of each sample for the quenching effect of that sample. 26 The total acid releasable CO2 content of tissue samples was measured by the method of Eckel, Botschner and Wood (1959). Calculations Tissue electrolyte distributions were calculated by the method of Hastings & Eichelberger (1937), and also by a modification of that method in which inulin, rather than chloride, was used to estimate the extracellular volume. The Henderson-Hasselbalch equation was used to derive plasma bicarbonate from the observed pH and pC0 values. The solubility factor 2 for CO2 in plasma was assumed to be 0.0301 (mEq/L)/mmHg. Calculations of intracellular pH and bicarbonate by the tissue CO 2 method were carried out as described by Wallace and Hastings (1942), although inulin, as well as chloride, was utilized to estimate extracellular space. Derivation of cell pH values from the distribution of C14-labeled DMO in plasma and tissue water was done according to the suggestions of Waddell and Butler (1959). Cell pH calculations were also made by use of both chloride and inulin spaces. The inulin space values used in all calculations were mean values observed in separate experiments of identical design. Statistical comparison and expression of the da.ta was done by several different methods. These techniques are described in connection with the data to which they were applied. RESULTS Tissue Water Distribution In-vivo studies Mean values of tissue water (TW), inulin space (ECW), and intracellular water (lCW) calculated for each of the treatment groups and the Grand Mean ± S.E. of all observations are listed in Table 1. Subsequent acid-base and electrolyte calculations were based on the pooled values labeled as Grand Mean in the table. This procedure was justified by statistical analysis, a two-way analysis of variance, of the data which indicated that there were no significant differences between the treatment groups with respect to any of these parameters. In -vitro studies Mean values + S.E. of tissue water (TW), C l4 -inulin space (ECW), and intracellular water (lCW) of rat diaphragm after 4 hours in -vitro exposure to various media are listed in Table 2. An analysis of variance of these data showed that significant (P<O .02) differences existed among the groups in all three parameters. Because of the numerous differences found among treatment groups with respect to TW, E CW, and lCW, pooled means were not used in subsequent calculations. Instead, acid-base and electrolyte calculations for a particular treatment group were based on the mean values of TW, ECW, and lCW applicable to that group as determined in the se experiments and listed in Table 2. 28 Table 1. Content and distribution of water in samples of skeletal muscle from rats pretreated with saline or ouabain, 20 mg/kg,and exposed in vivo to various acid-base distortions. ii-" if Treatment Tissue water I IAcid-base n Ouabain (TW) Inulin space (ECW) ml/kg tissue ml/kg tis sue 43.2 " Cell water (leW) ml/kg tissue -_......_-,- 0 6 754.2 t 20 6 764.9 0 6 752.1 45.1 707.1 20 6 758.5 45.4 712.8 CO2 0 6 752.5 43.8 708.8 .24% 20 6 759.3 42.8 716.3 36 757.0 44.5 712.2 1.4 1.4 2.1 'j 710.8 ': Control I , NH4Cl 46.4 717.3 I i 6 mEq/kg --~--.-- ---- . Grand Mean:f n indicates the number of observations on which means are based. t Values for individual groups are simple means. :f Values are means + S. E . 29 Table 2. Content and distribution of water in samples of rat diaphragm muscle incubated for 4 hours in vitro in the presence of saline or ouabain, 160 mg/L bath medium,and subjected to various acid-base conditions. _._----_..Treatment Tissue water Inulin space Cell water (TW) n (ECW) (ICW) Acid-base Ouabain ml/kg tis sue ml/kg tissue ml/kg tissue 'r.~ ___ "~"~ .. ~_ •• "~'" .. " _ _ ---"-- "<'" . . . . . ._ . _ _ _ • _ _ , 0 8 v-~"_ -;;;.. ...... 759.3+ _~ ____ " _ _ •_ _ _ _ _ ..,~ _ _ ..... ~_.~* F _ _' ... ",0',.,_.:... .,,~ ___ .... _... ~. __ .-- ~ 186.8 571.3 4.7 7.8 5.9 772.4 203.1 569.4 4.0 4.9 8.0 782.2 183.1 599.1 3.5 5.5 6.3 785.3 188.6 596.8 5.8 5.9 6.8 764.3 204.3 534.8 3.4 13.8 8.9 774.1 239.3 522.3 7.8 9.8 Control 160 0 8 8 High CO2 160 8 (pC0 2 = 90 mmHg) Low HC03 - 0 160 8 8 -- ......... (6 mEq/L) 11.0 -..._---------..- .---_. . 0 8 795.9 181.7 612.1 2.6 3.9 5.8 781.8 187.0 594.8 3.8 3.8 Hypotonic (NaCl ~ 20 mEq/L) 160 8 6.8 . _ - - - . - " " ."< - - " . - . _ - . - n indicates the number of observations in a group. + All values are given as means ±S . E . 1 30 Acid-base and Electrolyte Data In-vivo experiments General comments. The two major variables in the experimental design were acid-base status and ouabain dosage. In presenting the experimental results it would seem desirable to assess the effects of each variable independent of the other. Therefore, in presenting the tables which follow, certain conventions have been followed. First, the effect of acidosis on the various parameters has been considered and any differences between the effects of metabolic and respiratory acidosis have been indicated. To do this, animals subjected to the same dose of ouabain (or to saline) and various acid- base distortions were compared to the non-distorted group for that dose level. Second, the effect of ouabain on the same parameters has been considered. This effect was deduced by comparing the ouabaintreated animals to the saline-treated animals within each of the acid- oose groups. Third, any pertinent interaction of acid-base treatment and ouabain treatment has been mentioned. The results have, for the most part, been presented in the form of descriptive narrative. Formal statistical treatment of the data, such as two-way analysis of variance for each parameter, was not done for the following reasons: 1) changes in certain parameters were sufficiently obvious that such analysis would have been superfluous; 2) changes in some parameters were not really relevant to the problem at hand, and thus the effort required to analyze these data did not seem justifiable; and 3) in 31 some instances, the question was not how a parameter varied among the various groups, but how it varied with respect to some other parameter. In the last instance, data were subjected to formal statistical treatment other than analysis of variance and these will be presented as the results become relevant to the discussion. To make the tables more readable, standard errors have been included only for the mean values of the control group pretreated with saline. Values for the other groups are presented as the means, although standard errors were calculated for these groups. Unless otherwise stated, the standard errors of all groups may be assumed to be of the same order of magnitude as those for the control groups. Plasma data. The mean values of plasma acid-base parameters and electrolyte concentrations in animals subjected to various acid-base distortions and two doses of ouabain are summarized in Table 3. This table has been divided into two portions according to the nature of the acidbase distortion imposed. Each of these portions has been subdivided according to the magnitude of the distortion. Three groups are included within each of these subdivisions corresponding to the doses of ouabain employed in these experiments. The above conventions have been followed in Tables 4 through 8 as well. The number of animals per treatment group is not uniform for the following reasons. In a few instances, animals scheduled to receive ouabain were not injected with the drug. Data for these animals were included in the 0 ouabain group. Further, three plasma Table 3. Plasma acid-base and electrolyte data of rats pretreated with saline or with ouabain, 10, or 20 mg/kg body and subjected in vivo to various gradations of respiratory and metabolic acidosis • wt., ---.- - Treatment Acid-base n Ouabain 0 8 Control NH4Cl 4 mEq/kg NH Cl 6 m4E q/kg pH pC0 2 [H+] mmHg nM/L ~ [HC03 -] ( Na+] [ K+] [Cl"] mEq/L 7.46+ ·35.4 34.6 24.4 140.4 4.46 111.6 + 1.1 + 0.9 - -+ 0.8 + 1.9 -+0.32 + 1.7 -+0.01 10 9 7.41+ 36.7 39.6 22.4 131.0 11.13 102.9 20 8 7.38 38.9 42.0 22.1 131.1 10.81 106.2 0 9 7.32 33.1 49.4 16.7 141.9 4.97 114.9 10 7 7.24 35.5 55.8 15.5 132.8 11.28 113.4 20 8 7.23 36.1 59.6 14.0 132.1 11.68 115.8 0 10 7.22 34.7 61.3 13.4 142.1 5.98 121.0 10 7 7.15 35.1 67.1 14.7 133.3 11.26 117.3 20 9 7.09 40.0 82.0 12.4 132.4 12.81 118,,5 ~ Table 3. (continued) I I Treatment n I T pH ACid-base Ouabain I [HC03 -] pC02 [H+] t mmHg nM/L [ Na+] [K+] mEq/L - ...... -,~-- ..... [CC] ------ "..,......" ---- ...-~ .... 0 10 7.24 60.4 57.8 25.4 143.7 4.23 105.3 CO2 10 8 7.18 64.8 66.1 23.0 134.1 10.79 103.8 6% 20 6 7.14 65.8 73.5 21.5 131.6 11.81 ! ! ,_,_ ..... _ . . . _ . _ _ . 0 ·_ _ _ • _ _ _ .. , . . . . _ , ... _ _ .... _ _ " ' ' ' ' . . . '''' • • ' . • .......... _ 104.4 . . _ _ _ It_ ~-.~- CO2 0 8 7.08 87.2 83.6 25.2 148.2 4.52 109.5 10 9 7.04 89.8 91.2 23.7 135.7 11.23 103.2 20 8 7.02 92.4 95.3 22.4 135.1 12.76 106.0 9 6.90 . 136.4 128.2 27.8 143.9 6.08 106.7 12 % I 0 CO 2 i 10 7 6.85 139.6 141.6 24.1 136.2 12.51 102.9 20 I6 6.84 143.8 144.6 24.3 134.3 13.29 103.3 24 % n indicates the number of observations in each group. + Values are means + S 4= Values are means only: S.E. may be assumed to be of same order of magnitude as that for control group. w w 34 samples were lost and all data on these animals were excluded from the tabulation. Finally, the groups subjected to extreme acidosis had a high death rate even though they were being respired artificially; the combination of acidosis and the high dose of ouabain was particularly toxic. Plasma pH and [HC03 -] both decreased as expected in metabolic acidosis. Plasma pC02 was very slightly less in these acidotic animals than in the control group. In NH 4 Cl-treated animals, plasma H , Na , K , and Cl concentrations were higher than control values. Changes in [Na+] were relatively small, however. Plasma pC02 , [HC0 -] , and [H1 increased, and plasma pH de3 creased when the concentration of CO in the inspired air was increased. 2 Changes in plasma electrolyte patterns accompanying respiratory acidosis were more complex than those observed in metabolic acidosis. Plasma rNa1 and [K+] tended to increase in these groups, but the trend was less clear-cut than in metabolic acidosis. Plasma [CI-] did not increase in respiratory acidosis Ouabain had a slight but consistent effect on plasma acid-base parameters. Plasma pC02 was 4 to 8 mmHg higher and plasma [ HC0 -] was 3 2 to 5 mEq/L less in ouabain-treated animals than in untreated animals subjected to the same acid-base regimen. Consequently, plasma pH was lower and plasma [H+] higher in ouabain-treated animals. Ouabain induced a substantial decrease of [ Na 4 J and increase of [K+ in plasma regardless of the acid-base treatment. The drug caused a reduction in plasma [Cl-] 35 except in those groups where NH 4 Cl had been administered. Tissue electrolyte data... The means of muscle solid material, [ Na 1, [K+], [Cl-] , and chloride space are listed in Table 4.. In muscle of animals not treated with ouabain metabolic, but not respiratory, acidosis resulted in a reduction of tissue [Na +], the magnitude of which appeared to parallel the severity of acidosis.. Tissue [ K+] decreased slightly in both types of acidosis. Tissue [Cl-] increased in the groups subjected to the most severe metabolic or respiratory acidosis .. None of the acid-base distortions produced in these experiments altered chloride space to a great extent. Mean tissue Na values for animals treated with ouabain were 8 to 10 mEq/kg higher than those for untreated animals of the same acid-base group. Tissue K values were 7 to 12 mEq/kg lower in the ouabain- treated groups. Tissue Cl values and mean chloride spaces were con- sistently greater in ouabain-treated animals. The data presented in Table 5 are means of values derived from total tissue values and other data.. The intracellular electrolyte concentrations in Table 5 were calculated on the basis of two different assumptions regarding extracellular volume (ECW), namely, that ECW=chloride space, or that ECW=inulin space.. While the absolute values for intracellular electrolytes calculated on the basis of chloride space differed from those calculated on the basis of inulin space, the qualitative implications of the Na and K data obtained by the two methods were the same .. 36 Table 4 .. Skeletal muscle water and electrolyte data of rats pretreated with saline or ouabain, 10 or 20 mg/kg body wt .,and subjected in vivo1D various gradations of respiratory and metabolic acidosis. Treatment Dry wt. n % Acid-base Ouabain 0 [ Na 41 [ K+] 1[cr] mEq/kg wet tissue 8 25.42+ 19.9 119.2 0.49 0.7 0.9 12.1 0.42 Chloride space ml/kg 10.5 0.4 Control NH 4 Cl 10 9 24.90* 31.0 109.5 15.2 14.3 20 8 25.23 29.1 109.4 13.8 12.8 0 9 25.98 18.8 117.3 12.3 10.6 10 7 25.15 27.5 114.7 16.1 13.7 20 8 24.98 28.2 113.3 16.1 13.5 0 10 25.66 17.8 116.8 13.1 10.5 10 7 25.41 27.9 113.1 15.9 14.0 20 9 25.38 26.0 112.8 16.4 13.4 4 mEq/kg NH 4 Cl 6 mEq/kg 37 Table 4. (continued) .. '~."-' -------_..- Treatment .. -----_ ... Dry wt. [Na+] n % Acid-base Ouabain CO2 1[1(+1 J[ecT" mEq/kg wet tissue .. Chloride space ml/kg 0 10 25.35 19.7 118.4 11.2 10.3 10 8 25.72 31.0 109.8 15.1 13.8 20 6 25.26 28.3 110.3 15.6 14.4 0 8 26.14 19.6 116.7 12.1 10.7 10 9 24.64 29.5 109 . 3 15.4 14.5 20 8 25.55 27.4 104.2 15.2 13.9 0 9 25.89 19.4 115.4 13.0 11.5 10 7 25.36 28.5 108.7 15.0 14.3 20 6 24.39 27.5 104.1 14.4 13.5 6% CO2 12% CO 2 24% ""'" ~-1".';lt~~J!Je;;::;:_~-==:-:-""::~::'::.~:;';"_ n indicates the number of observations in each group. + Values are means + S.E. * Values are means. 38 Table 5. Intracellular Na+, K+, and CI- concentrations of rat skeletal muscle from animals pretreated with ouabain and subjected to various gradations of respiratory and metabolic acidosis. Calculations were based on the assumption that a) chloride space or b) inulin space approximates the true extracellular space. Treatment n Acid-base Ouabain 0 8 Chloride Soace Na+ I K+ I CImEq/L cell H 2 O 7.3+ 1.1 Control NH CI 4 186.0 0 4.1 Inulin Space CI.Na+ I K+ mEq/L cell H O 2 I 19.3 0.98 170.3 3.5 10.2 0.67 10 9 18.9:J: 178.9 0 34.8 156.0 14.5 20 8 16.8 174.5 0 30.5 155.5 12.7 0 9 5.3 184.2 0 16.4 170.7 9.6 10 7 13.4 185.5 0 29.6 162.7 14.7 20 8 16.9 179.9 0 30.8 160.9 15.2 0 10 4.8 180.5 0 17.2 170.3 10.6 10 7 15.7 186.1 0 31.2 164.1 16.3 20 9 12.1 180.6 0 28.4 163.7 15.4 4 mEq/kg NH 4CI 6 mEq/kg 39 Table 5. (continued) Treatment n Acid-base Ouabain Chluride Soace Na+ K+ ( CImEqjL cell H2O I Inulin Snace K+ Na -t1 CImEq/L cell H 2O I 0 10 6.8 183.6 0 18.5 168.7 9.0 10 8 20.2 178.1 0 35.9 155.5 14.3 20 6 16.0 180.2 0 31.6 156.5 15.2 0 8 7.7 183.1 0 18.6 168.4 9.4 10 9 15.1 175.0 0 32.6 152.3 16.3 20 8 13.8 180.9 0 31.4 157.4 15.2 0 9 4.1 181.5 0 19.1 163.7 11.5 10 7 15.6 178.3 0 33.0 155.8 14.5 20 6 14.6 162.2 0 29.8 143.2 13.5 6% CO 2 12% CO 2 24% CO2 n indicates the number of observations in each group. + Values are means .::!: S.E. of concentrations in mEq/L cell water. ::f: Values are means of concentrations in mEq/L cell water. 40 The data in Table 5 indicate that intracellular Na concentration ([ Na+] i) decreased slightly in both respiratory and metabolic acidosis and that intracellular K ([K+] i) was essentially unaffected by either type of acidosis. According to the inulin space data, intracellular chloride ([Cl- Ji) decreased slightly in those groups subjected to moderate degrees of acidosis (4 mEq NH4Cl/kg, 6 and 12% CO2 ) and increased slightly in those groups subjected to the most severe acidosis. Ouabain induced a large increase in [Na + ] i and a marked decrease in [K+] i in all instances. According to the inulin space calculations, [Cl-] i was higher in ouabain-treated animals, but the increase in [Cl-] i was far less than the increase in [Na+] i. Tissue acid-base data. Intracellular pH (pHi) and [H+] ([H+]i) were calculated by two methods in these experiments. Data obtained by the C02 method are presented as means in Table 6, those obtained by the DMO method appear as means in Table 7. Intracellular values calculated on the basis of both chloride space and inulin space appear in both tables. In general, the qualitative implications of chloride and inulin space data were the same; the few exceptions to this rule are specifically indicated. The data in Table 6 indicate that the total CO2 content of skeletal muscle increased in respiratory acidosis and decreased in metabolic acidosis. The same changes occurred with respect to intracellular CO 2 content. The mean intracellular pH of control animals was 7 .26 :!: 0 .02. Mean values of pHi for animals subjected to metabolic acidosis were only slightly higher 41 Table 6. Skeletal muscle acid-base data obtained by the tissue CO method 2 for rats pretreated with ouabain and subjected in vivo to various gradations of metabolic and respiratory acidosis. Treatment n Acid-base Ouabain 0 Control NH4 Cl 8 Tissue total CO2 Chloride S :>ace * HT CO2 pH roM/L mM/L 13.48+ 16.5 0.36 0.6 Inulin Space* H+ CO2 pH nM/L rr1M/L 17.58 7.26 55.2 2.9 0.36 0.02 2.3 7.23 59.4 0.02 nM/i 10 9 14.18:f: 17.0 7.24 58.7 18.22 7.25 54.4 20 8 14.20 18.2 7.2561.1 19.07 7.27 57.4 0 9 12.59 16.7 7 .27 53.4 16.89 7.27 51.5 10 7 12.88 16.9 7.25 59.1 17.32 7.26 57.2 20 8 13.13 17.7 7.25 56.5 17.50 7.25 56.1 4 mEq/kg -.- NH 4 Cl 0 10 12.43 17.1 7.29 53.7 16.93 7.29 53.6 10 7 12.80 17.6 7.17 71.8 18.29 7.17 71.9 20 9 14.22 18.6 7.23 61.2 18.64 7.23 61.3 6 mEq/kg 42 Table 6. (continued) Treatment n Acid-base Ouabain CO 2 Chloride Space H+ CO2 pH Tissue total CO2 mM/L mM/L Inulin Space H+ pH CO2 nM/L mM/L nM/L 0 10 16.76 21.1 7.09 82.2 21.9 7.10 78.9 10 8 16.98 21.8 7 .08 83.6 22.5 7.10 80.6 20 6 17.43 22.5 7.08 82.8 23.1 7.10 80.2 0 8 19.43 25.1 7.00 101.1 25.6 7.06 98.6 10 9 20.36 26.4 6.99 103.2 26.8 7.00 101.3 20 8 19.75 26.5 7.02 97.0 26.8 7.02 0 10 23.50 31.3 6.88 130.4 31.5 6.89 129.2 10 7 22.89 31.2 6.87 136.6 31.2 6.87 136.5 6 23.42 29.5 6.89 149.6 30.0 6 .. 89 145.0 6% CO 2 12% 95.7 t CO2 24% 20 I i I n indicates the number of observations in each group. + Values are means + S.E. :f Values are means. * Values pertain to intracellular water. 43 Table 7. Skeletal muscle acid-base data calculated by the DMO method for rats pretreated with ouabain and subjected in vivo to various gradations of metabolic and respiratory acidosis. n DMO ratio tissue plasma 8 Treatment Acid-base Ouabain 0 Control NH4Cl Chloride Space * H+ pH nM/L Inulin Space * H+ pH nM/L 0.301+ 6.85 141.9 6.96 108.3 0.006 0.02 8.6 0.02 5.9 10 9 0.330* 6.76 177.6 6.95 112.8 20 8 0.342 6.81 157.5 6.95 114.1 0 9 0.376 6.89 131.2 6.96 111.8 10 7 0.381 6.76 176.2 6.88 133.3 20 8 0.419 6.76 176.2 6.86 140.0 4 mEq/kg -- NH4Cl 0 10 0.428 6.85 144.3 6.88 126.6 10 7 0.438 6.70 204.2 6.80 162.5 20 9 0.518 6.81 175.8 6.87 142.4 6 mEq/kg 44 Table 7. (continued) Treatment n Acid-base CO2 Ouabain DMO ratio tissue plasma Chloride Space H;pH nMjL Inulin Space H+ pH nMjL 0 10 0.357 6.73 190.3 6.82 154.4 10 8 0.385 6.68 216.3 6.81 156.3 20 6 0.389 6.62 251.2 6.75 178.6 0 8 0.415 6.66 228.0 6.73 190.6 10 9 0.422 6.55 292.4 6.68 218.2 20 8 0.429 6.57 272.0 6.69 206.1 0 9 0.476 6.54 297.3 6.60 255.7 6% CO 2 12% 1 j 1 \ CO 2 10 7 0.464 6.44 386.2 6.54 307.1 20 6 0.525 6~55 280.9 6.61 235.9 24% n indicates the number of observations in each group. + Values are means + S.E • * Values are means. * Values pertain to intracellular water. 45 than this. In respiratory acidosis groups, pH. decreased as a function of 1 the amount of CO2 added to the atmosphere with which the animals were respired. Compared to the control group values, mean [H4 i decreased slightly in metabolic acidosis and increased progressively in respiratory acidosis. In the control group and in groups subjected to metabolic acidosis, ouabain produced an increase in both tissue and intracellular CO2 • No such effect was found in the respiratory acidosis groups. In all acid-base groups the mean [H+]i for ouabain-treated animals was higher than the value for animals not receiving ouabain. In most'instances the difference was very slight but in the 24% CO2 group it was substantial. The data in Table 7 indicate that the mean tissue plasma ratios of DMO were higher in acidotic groups than in the control group_ The mean intracellular pH in the control group was 6.96 ± 0.02 by inulin space calculations and 6.85 ±0 .02 according to chloride space calculations. Chloride space data indicated that metabolic acidosis had no effect on intracellular acidity, but the inulin space data indicated that severe metabolic acidosis did produce an intracellular acidity. Both methods of calculation indicated that cell pH is reduced and [H +] i increased by respiratory acidosis. In all instances the DMO ratios of ouabain-treated animals were greater than the ratios for untreated animals of the same acid-base group. The mean pHi values for ouabain groups were consistently lower than those of corresponding controls while [H1 i values of the ouabain groups were ·46 consistently higher. For the most part these differences were small and of questionable significance. Transmembrane distribution of ions. The ratios of the concentrations of K , H , Na , and Cl in cellular and extracellular water, and the transmembrane potential corresponding to the K ratio (EK') were calculated for individual animals. The means of these values for the various treatment groups are presented in Table 8. The intracellular values employed in these calculations were those based on inulin space. Metabolic acidosis reduced the ratio (inside cell/outside cell) of all cations measured in these experiments, the ratio (outside cell/inside cell) of Cl , and the mean E K • The effect of respiratory acidosis on these parameters was mQre complex. As the CO2 content of the respiratory mixture increased, the K ratio and EK decreased, but only in the 24% C02 group was the change substantial. Mean H ratios decreased as CO2 was increased. The same trend was apparent in the Na and Cl ratios but the magnitude of these changes was small. Ouabain caused a marked reduction of the K and CI ratios and mean EK in all acid-base groups and a substantial increase in the Na ratio. The H ratios tended to be reduced as a function of ouabain administration and these seemed some suggestion of a dose-effect relationship in most groups. However, the effect of ouabain on the H ratio was in no instance as dramatic as the 100% increase in the Na ratios which ouabain produced regularly. 47 Table 8. Ratios of the concentrations of K , H., Na and Cl in intracellular and extracellular fluids and the potassium equilibrium potential of skeletal muscle. Rats were pretreated with ouabain and exposed in vivo to various gradations of respiratory and metabolic acidosis. ., Treatment Acid-base Ouabain n I K H in/out I ..... - ~-~ Na Cl out/in EK mV '"---0 Control NH Cl 4 8 38.18+ 3.11 0.133 12.09 95.34 2.62 0.11 0.007 1.04 1.83 3.00 0.258 7.45 65.07 t 10 9 14.03 20 8 14.21 2.72 0.259 8.70 67.69 0 9 34.11 2.40 0.119 13.01 89.91 10 7 14.07 2.39 0.216 7.90 66.93 20 8 13.42 2.30 0.228 8.27 66.28 0 10 26.71 2.06 0.112 11.97 84.01 10 7 14.23 2.16 0.225 7.43 67.87 20 9 12.24 1.70 0.205 8.16 64.90 4 mEq/kg NH 4Cl 6 mEq/kg 48 Table 8. (continued) Treatment Acid-base Ouabain CO2 nl I ! K I H J Na Cl out/in In/out EK 0 10 38.99 2.79 0.124 12.71 mV -.-93.73 10 8 14.01 2.39 0.259 7.57 67.60 20 6 12.91 2.48 0.234 7.21 65.44 6% j I CO 2 0 8 36.54 2.30 0.121 12.21 92.00 10 9 13.23 2.26 0.233 6.92 66.08 20 8 11.95 2.16 0.226 7.34 63.54 0 9 27.80 1.96 0.131 11.21 84.70 10 7 10.41 2.10 0.227 7.81 64.64 6 11.92 1.72 0.212 7.62 60.89 12% CO2 24% !'; 20 '. n indicates the number of observations in each group. + Values are means + S.E. * Values are means. 49 In -vitro experiments Extracellular fluid daa. The mean values + S.E. of acid-base and electrolyte parameters determined for the plasma of small (80 to 100 g) rats are listed in Table 9. These data were used to formulate the standard Ringer-bicarbonate solution employed in all in-vitro experiments. The actual measured compositions of the bathing media at the end of each experiment are also indicated in this table. These data are largely self-explanatory and most differences among the various media are attributable to variations (deliberate or inadvertent) in preparation of the bathing medium. However, the initial concentration l of K in the bath was 4 mE q/L and K+] of the medium inva-r:iably increased during the final 2 hour incubation period. The largest change in K+ ob'3erved in vitro,was 1.7 mEq/L compared to changes as great as 10 mEq/L in in-vivo experiments. Bath Na + tended to decrease during the final 2 hour period but this was a variable finding. 1' Tissue electrolytes. The mean [Na [K+] , and [Cl-] / kg wet tissue of rat diaphragm from freshly sacrificed animals and from rib cage preparations incubated in various media are listed in Table 10. Comparison of the fresh tissue data in this table to the data in Table 6 for the control group indicates several differences between the skeletal muscles of diaphragm l' and [Cl-] of the former are higher and [K+] is about 20 and thigh. [Na mEq/kg less than the values found for thigh muscle. The extracellular space of diaphragm (see Table 2) is also larger (approximately 200 ml/kg compared '50 to 45 rnI/kg for thigh muscle). The [K+] of the in-vitro control group was 15% less than that of fresh diaphragm, while [Na1 was 10% higher and [Cll 30% higher after incubation. Acidosis in vitro produced a further increase in tissue [Na+] and a further reduction in tissue [K+] but no change in tissue [Cl-] in comparison to the control group. Addition of ouabain to the in-vitro enviromnent led to a large reduction in tissue [ K+] and a marked increase in tissue [Na+] • Tissue [Cl-] was affected quite variable by ouabain in vitro. Intracellular data. Mean values of intracellular pH, H' , Na , K , and Cl calculated for fresh tis sue and for the in-vitro preparation are presented in Table 11. The electrolyte composition of the in-vitro control group was almost identical to that of the fresh tissues, but the intracellular fluid of the former group was considerably more alkaline (7.21 compared to 6.93). In ac id media, [Na +] i and [H+] i increased while [K+] i and [Cl-] i decreased. Cell pH was reduced by both metabolic (low HC0 -) and res";' 3 piratory (high CO ) acidosis in vitro. 2 Ouabain evoked a substantial decrease in [K+Ji and a large increase in [ Na +] i in all acid-base groups in vitro. No clear cut pattern was evident with regard to[Cl-] i. In the control medium in vitro, ouabain had no significant effect on cell pH or [H +] i. In both types of acid media, the cells of tissues incubated in the presence of ouabain were clearly more acid than 51 those not expos ed to ouaba in. 52 Table 9. Comparison of plasma data for small (80 to 100 g) Long-Evans rats with the composition of the modified Ringer- bicarbonate solutions employed in in-vitro experiments. Bath temperatures were 37-39 0 C in all instances. -- Sample Ouabain Acid-base I pH pC02 mmHg W HCO'.1 - Na+ I K+ I mEq L nM/L 1 Cl- 7.45 36.0 35.1 24.3 135.2 4.19 104.1 0.01 0.8 0.7 0.7 0.8 0.15 0.8 0 7.46 33.0 34.7 22.8 128.5 5.15 103.2 80 7.48 33.3 33 .. 1 23.0 129.5 5.40 101.2 160 7.44 33.5 36.1 22.3 129.0 5.70 98.7 0 6.85 33 .. 0 141.3 5.7 128.5 5.30 120.4 80 6.86 32.0 138.1 5.3 123.0 5.10 119.6 160 6.86 34.0 138.1 5.7 125.0 5.20 116.3 0 7.02 91.0 95.5 35.5 130.0 4.85 101.6 80 6.96 91.0 109.7 22.6 118.0 4.85 96.3 Plasma* Control * Low HC03 - I !High I 1 :C 02 + Values indicate concentration of ouabain in the tissue bath, mg/L, during the final 2 hours of incubation. :f Values for plasma are means * ±S.E" of 8 observations. Values for bath media are based on analysis of samples of bath fluid near completion of the tissue incubation period; for the initial composition of bath media, see METHODS in text. 53 Table 10. Water and electrolyte content of rat diaphragm muscle from freshly sacrificed, normal animals and after incubation of tissue in vitro in the presence of ouabain and/or various acid-base regimen. TW ClI mEi}/kg wet tissue Na+ I K+ Sample Acid-base Ouabain ml/kg Fresh tissue+ 773.2 44.4 97.4 31.1 6.2 0.8 1.2 0.5 759.3:t: 48.2* 80.8 40.0 80 772.4 83.3 50.0 44.3 160 772.4 98.3 35.7 27.7 0 764.3 53.0 74.2 40.7 80 774.1 62.3 51.1 36.4 160 774.1 76.7 48.0 51.7 High 0 782.2 53.8 75.2 38.6 CO2 80 785.3 69.7 49.3 35.7 0 Control -- Low HCO 3 + Values for fresh tissue are means + of 8 observations * * Tissue water values listed are those used for subsequent calculations Values for in..vitro electrolytes are means of 4 observations. 54 Table 11. Intracellular acid-base and electrolyte composition of diaphragm muscle from freshly sacrificed normal rats and after incubation of tissue in vitro in the presence of ouabain and/or various acid-base regimen. I ~~__ H+ nM/L Na+ 6.93 117.8 46.6 135.1 31.6 0.02 5.6 1.1 1.6 0.6 0 7.21 61.5 42.6 133.1 37.1 80 7.30 50.1 100.3 79.2 41.7 160 7.19 63.8 126.7 55.5 8.3 0 6.88 130.6 50.2 117.4 30.2 80 6.75 177.9 63.0 79.4 14.9 160 6.78 167.2 89.5 78.8 45.8 High 0 6.91 122.5 44.2 121.2 33.1 CO2 80 6.75 167.8 80.2 80.6 30.2 pHi* Sample: Ouabain K+ mEq/L cell H2O Fresh tissue+ Control:f Low HC03 - + Values for fresh tissue are means + S.E. of 8 observations. *In -vitro values are means of 40 observations. * pHi was calculated by the DMO method. ! ~ I DISCUSSION These data represent the first attempt to examine the relationship between the mechanism(s) of intracellular pH regulation and the Na transport system of cells (the Na - K pump) by imposing varying degrees of stress on the two systems both singly and in combination. Previous investigators have attempted to control one of the other of these systems but not both. This approach seemed well suited to examination of the hypothesis that the Na pump is the mechanism underlying the active extrusion of H from the cell. The shift of potassium from the intracellular compartment which accompanied acidosis in the present experiments was consistent with the observations of numerous other investigators (penn & Cobb, 1934; Pitts, 1954; Schribner et al., 1955; Brown, 1955; Burnell et aI., 1956; Brown & Goott, 1963; Lade & Brown, 1963; ReIman et al., 1961). It has been established that in acute acidosis this increase in plasma K is attributable solely to the loss of K from muscle since under these conditions bone K does not decrease and may even increase slightly (Levitt, Turner, Sweet & Pandiri, 1956; Clancy & Brown, 1963). At one time it was reported that K moves into cells in acidosis (Darrow, 1946; Darrow, Schwarz, Iannucci & Coville, 1948; Cotlove, Holliday, Schwartz & Wallace, 1951; Cooke, Coughlin & Segar, 1952), but Schribner, Fremont-Smith & Burnell (1955) point out that the discrepancy is only apparent since these workers did not distinguish the effects of acid-base imbalance on total body 56 K balance in chronic experiments from the acute effects of acid-base distortions on the relative distribution of K. This movement of K from tis sues to plasma in acidosis has been regarded by some as a reflection of the effect of H on the Na-K pump (Dac.ries & Keynes, J961). The results of the in-vivo experiments indicate that metabolic acidosis produced a larger shift in K from the intracellular to the extracellular compartment than did respiratory acidosis of comparable severity. Administration of 6 mEq/kg of NH Cl reduced plasma pH about 0.25 pH 4 units from the control value of 7 .46 and increased plasma K from 4.5 to 6.0 mEq/L. Plasma K increased approximately 1.5 mEq/L in the 24% CO2 group as well, but the mean plasma pH for this group was 0.62 pH units less than the control value. Animals respired on 6% or 12% CO 2 showed no increase in plasma potassium. This difference between respiratory and metabolic acidosis with respect to K has been reported by other investigators (Fenn & Asano, 1956; Rogers & Wachenfeld, 1958; Liebman & Edelman, 1959; Brown & Goott, 1963; Spurr & Liu, 1966), although Simmons & Avedon (1959) could detect no such difference in experiments on dogs. To explain these observations Brown and Goott (1963) have suggested that the ratios (H+]i/[ H+]o and (K+Jil[K+]o tend to move in the same direction and, although the two are never truly equal, that 'The Donnan equilibrium for potassium and hydrogen across the cell membrane may be masked by other forces operating on these two ions, nevertheless the 57 forces tending to push these ratios toward equilibrium should be in opera- J for skeletal muscle decreases in both tion.' The ratios [H+] i/[ H+ 0 respiratory and metabolic acidosis, but the change in this ratio per unit change in H+ o is far greater in metabolic than in respiratory acidosis. Thus, on the basis of the Brown and Cbctt hypothesis, the ratio of [ K+J i/[ K+Jo (and, in consequence, plasma K) would be expected to change more per unit change of [ H+Jo in metabolic acidosis than in respiratory acidosis. To facilitate examination of how this hypothesis applies to the present data, the ratios of the intracellular to extracellular concentrations of potassium and hydrogen ions have been plotted as a function of extracellular pH in Fig. 1. Data for the ouabain-treated and control groups have been presented separately. It is evident from this figure that the curves for Hand K are very similar in shape. In the upper graph, which depicts results in the control groups, it is clear that two distinct curves exist for both Hand K and that the curves for both ions in metabolic acidosis are steeper than those observed in respiratory acidos is. Thus, these data are consistent in all respects with the hypothesis of Brown and Goott. If the upper and lower portions of Fig. 1 are compared, it can be seen that ouabain profoundly lowers the ratios of potassium between intracellular and extracellular fluids, and alters the way in which acidosis affects the distribution of both hydrogen and potassium ions. The differential effect of metabolic and respiratory acidosis on K distribution is totally abolished by ouabain. The difference between the effects of metabolic and Fig. 1. Transmembrane distributions of potassium (filled symbols) and hydrogen ion (open symbols) as a function of extracellular pH in saline-treated control groups (circles, upper graph), and in ouabain-treated groups (triangles = 10 mg/kg, squares = 20 mg/kg, lower graph). Relationships observed in respiratory acidosis are indicated by solid lines; those for metabolic acidosis are indicated by broken lines. Curves were fitted by visual inspection. 58 [K1 j [H'i ~~ [K1o ~jo o 30 3 20 2 ~o 10 [K1 j 6.9 [H~i 7.1 7.0 7.2 7.3 7.4 pHe ~~ [Kio [H\ 30 20 10 3 2 ,,, /,r ~ • ~ 6.9 7.0 .".,..""'" ".",. ./ • o t:::.-/l. 0 7.1 pHe rf' 7.2 ~- 7.3 7.4 59 respiratory acidosis on H distribution is substantially reduced by ouabain. With regard to potassium, this effect might be explained by the fact that the drug itself produced such a large reduction of the K ratio that the smaller effect of acidosis on this parameter is prevented or obscured. A similar explanation cannot be invoked for hydrogen ion, however, since ouabain alone does not produce a large decrease in the [H+] il [H+] 0 ratios. A consideration of the sequence of events and the cellular factors involved in the two types of acidosis may suggest a possible mechanism for the reduction of the difference between the effects of metabolic and respiratory acidosis on H distribution by ouabain. The reason that the ratio[ H+] i/[ H+] 0 changes more in metabolic than in respiratory acidosis can be explained briefly as follows. Both intra cellular and extracellular pH are functions of the ratio [HC03 -] I [H 2C03] . When H is added to the extracellular fluid as a strong acid, extracellular pH declines immediately. The added H+ reacts with the plasma HC0 to 3 form H2C03 which is rapidly dehydrated, leading to a transient elevation of plasma pC02 . Respiratory mechanisms rapidly restore pC02 to normal values and the net result is that plasma HC03 is reduced, while H2C03 remains constant. Intracellular pH changes little unless metabolic acidosis is unusually severe. Presumably, this is because the cell is capable of actively extruding or buffering the H which would enter the cell at a greater rate as [ H+] 0 increased. Only when the capacity of cells to handle the added H was exceeded would these ions combine with intracellular bicarbonate 60 to alter the ratio [H CO3 -] I [H2CO3] and, hence, cell pH. On the other hand, when extracellular H+ is increased by administering CO2 , the concentration of H2C0 is immediately increased in both the intracellular and extracellular 3 compartments and the ratio [H+] if [H+] 0 changes less than it would if only [ H+]o were increased. If the mechanism(s) whereby cells 'handle' the additional number of H entering them by diffusion in metabolic acidosis were eliminated or inhibited, the intracellular pH would more closely parallel the extracellular pH and the curve relating [H+] if[ H+] 0 to pHe in metabolic acidosis would tend to resemble the curve observed in respiratory acidosis. As noted in the preceding paragraph, ouabain did reduce the difference between the effects of metabolic and respiratory acidosis on H distribution. This could suggest that the mechanism for handling H in metabolic acidosis is an active process susceptible to inhibition by ouabain. The slight increase in plasma Na observed in acidosis in these experiments is scarcely significant, either statistically or physiologically, and the majority of this Na probably comes from bone rather than muscle tissue (Pitts, 1954; Swan & Pitts, 1955; Levitt et aI., 1956). The changes observed in plasma acid - base parameters consequent to the acid-base treatments are, qualitatively, as would be predicted. The quantitative aspects of these changes, however, merit some comment. The CO 2 dissociation curve is, as previously mentioned, widely used to describe the buffering properties of tissues. The whole blood pH and HC03 data for the in-vivo experiments have been utilized to construct such curves for 61 the control and ouabain-treated groups in the present series of experiments. These are presented in Fig. 2. The whole blood buffer curves obtained by this author in a different strain of rats (Elsmore, 1965) and by Brown & Clancy (1965) in dogs have been included in Fig. 2 for comparative purposes. The curves obtained in different experiments and for different tissues are most conveniently compared in terms of Slykes, a unit describing the slope of the CO2 dissociation curve in terms of 6 [HC0 -] (mEq/L)/6pH 3 (W oodbury, 1965). Most observers have reported that the CO2 dissociation curve of whole blood for animals exposed to various concentrations of C02 in vivo has a slope of 8 to 18 Slykes (Cohen, Brackett, & Schwartz, 1964; Brown & Clancy, 1965; Elsmore, 1965). The CO dissociation curves obtained 2 in the present experiments and the control, 10 mg/kg and 20 mg/kg ouabain groups had slopes of 5.6, 3.9 and 4.3 Slykes,respectively. Why these curves are so much more shallow than those reported by other observers is not known. It seems unlikely that the discrepancy can be attributed to errors of measurement since the techniques used to measure pH and pC02 in these experiments have been employed in similar experiments on dogs in this laboratory and the CO dissociation curves obtained in those experiments 2 had a higher slope of about 9 Slykes. Nor could the discrepancy be attributed to an inadequate equilibration period since the timing used in these experiments was identical to that in experiments by this author from which the SpragueDawley curves in Fig. 2 were taken. A logical conclusion would seem to be 62 that the flat blood buffer curves obtained in these experiments are simply a characteristic of this strain of rats. The slight but consistent changes in plasma acid-base parameters induced by ouabain are best described as a mixed respiratory and metabolic acidosis. Several factors, alone or in combination, can be invoked to explain this. The reduced plasma bicarbonate concentration could result from the stimulation of lactic acid production which has been reported to occur in tissues exposed to ouabain (Kein & Sherrod, 1960; Wollenberger, 1962; Kriesberg & Williamson, 1964). This reduction might also be explained by a change in the distribution of HC0 between the intracellular and 3 extracellular fluids, namely, a retention of bicarbonate in cells as a consequence of ouabain administration. The evidence supporting this suggestion will be discussed below. Finally, the reduced plasma bicarbonate levels could be explained by an increased renal excretion of bicarbonate. Orloff & Burg (1960) and Orloff & Berliner (1961) have reported that ouabain does increase the renal excretion of HC03 because it blocks the renal secretion of H. The acid retained consequent to the renal effects of ouabain would tend to lower plasma bicarbonate even further. Ordinarily, as plasma HC0 3 combines with H to form H2C0 , respiratory reflexes are stimulated and the 3 plasma pC02 is lowered to normal, or lower than normal values. In the present experiments, however, the respiration of animals was controlled mechanically. Thus, reflex alterations in respiration were prevented. Under these circumstances, the addition of acid to plasma resulting from Fig. 2 _ CO2 buffer curves of blood from Long- Evans rats pretreated with saline (circles); ouabain, 10 mg/kg (triangles); and ouabain, 20 mg/kg (squares), and respired in vivo for one hour on room air or 6, 12, and 24% C02- For comparative purposes, blood buffer curves obtained in in-vivo experiments on dogs (Brown & Clancy, 1965) (asterisks) and on Sprague-Dawley rats (Elsmore, 1965) (plus marks) have been depicted as well. The slopes of these lines were as follows: Group Slykes Control 5.6 Ouabain, 10 mg/kg 3.9 Ouabain, 20 mg/kg 4.3 Sprague- Dawley rats 9.5 Dogs 16 Curves were fitted by visual inspection. 63 30 [HeOi] mEq/L Plasma H20 25 20 * 6.8 6.9 7.0 7.1 7.2 pH 7.3 7.4 7.5 7. 64 decreased renal excretion or from increased lactic acid production as a consequence of ouabain would be expected to increase pC02 as well as decrease HC03 , as was observed in these experiments. In the present study, chloride space invariably increased in the ouabaintreated animals as Cl moved into cells along with Na. Under these circumstances, chloride space could no longer be considered a valid estimate of the extracellular volume. Thus, the discussion of intracellular electrolyte concentrations and pH data in the following paragraphs will be based on values derived using inulin space as the estimate of extracellular volume. Also, for the most part, discussions of cell pH will be based on the results obtained by the DMO method. The CO2 data are interesting because of what they indicate about the total CO2 content of the tissues and cells, but the values of pHi derived from CO2 data are very probably more alkaline than the true values of pHi because the true pC02 of the intracellular fluid is probably higher than the pC02 observed in arterial or even venous plasma CElsmore, 1965). In the present experiments acidosis tended to cause a decrease in the mean intracellular Na content which, if real, would imply that acidosis stimulated Na transport. To explore further this possibility, the data on [H+] e and [Na +] i were subjected to analysis of covariance. The plot of these data for individual animals and the regression lines fitted to these data by the method of least squares for each of three groups: control; ouabain, 10 mg/kg; and ouabain, 20 mg/kg are presented in Fig. 3. The 65 slopes of the three lines describing the regression of intracellular Na + on extracellular H+ were: -0.008, -0.027, and -0.024. None of these lines is significantly different from 0; thus it was concluded that no correlation existed between intracellular sodium and extracellular hydrogen ion concentration. If acidosis had any effect at all on intracellular sodium, as some have reported, (pitts, 1954; Rogers & Wachenfeld, 1958; Keynes, 1963; Adler et al., 1965a), it was too small to be detected by the methods employed in these experiments. In contrast to the rather insignificant effect of acidosis, ouabain treatment caused a very large increase in intracellular sodium which was accompanied in all instances by a reduction in the intracellular potassium concentration. This is consistent with the known effects of ouabain on the Na-K transport system and indicated that the doses of ouabain employed in these experiments were adequate to impair significantly the Na pump. The very slight difference between the effects of the 10 and 20 mg/kg doses of ouabain on intracellular electrolyte concentrations suggests that the maximal effect of the drug was attained with 10 mg/kg. It seems evident from the data in Table 7 that doses of ouabain which markedly impaired Na transport in skeletal muscle had relatively little effect on intracellular pH. In the in-vivo experimental series the mean pHi values for ouabain -treated animals were on the average lower than those of their untreated counterparts. The difference, however, was quite small in most instances and could reasonably be attributed to the slight extracellular Fig. 3. Relationship between [ H+] e and [ Na+]i plotted as individual values and resultant curves for control animals (circles); animals treated with ouabain, 10 mg/kg (triangles); and ouabain, 20 mg/kg (squares). The mean point of each line is denoted by a large symbol of the appropriate shape, as defined above. 1i on The slopes of the three lines for the regression of [Na [H+] e were as follows: Group b Control -0.008 Ouabain, 10 mg/kg -0.027 Ouabain, 20 mg/kg -0.024 66 o o N II II <J <J <3 II . <fIJ 0 II <k3 II 0 ~ II <J S 00 lO 0 lC) 0 rt") rt') C\J N -l0 N I' f 0- c:::J Z J :::r: L1J= E Q) u ....J "'- 0 :e c: 0 Q) r ~ 0 II -a..0 0 0 0 II IJII I E en 00 00 <J <J 0 0 II <J II 0 0 0 II ~ N ::I: 0 <J <J II 0 0 <J ~ 10 0 II II 0 0 0 0 0 lO 0 1.0 +:::c J J Fig. 4. Intracellular pH (pHi) of rat skeletal muscle plotted as a function of extracellular pH (pH ) for control (circles); ouabain, 10 mg/kg e (triangles); and ouabain, 20 mg/kg (squares) groups subjected in vivo to various gradations of respiratory acidosis (points connected by solid line) and metabolic acidosis (points connected by broken line). Curves were fitted by visual inspection. 88 • t-- • t-- o• · <D . an CD 69 acidosis consistently observed in the ouabain -treated groups. This fact becomes quite evident when the pH. values calculated by the DMO method on 1 the basis of inulin space are plotted as a function of pH , as has been e done in Fig. 4. The relationship between pHi and pHe observed in these experiments in respiratory and metabolic acidosis is in agreement with the observations made previously by this author and others (Adler et al., 1965a & b; Elsmore, 1965), i.e., there is a range of values over which changes in pHe do not affect pHi; values of pHi are linearly related to pHe at pH values more acid than this range; and the acid limit of the e plateau is lower in metabolic than in respiratory acidosis. It is evident from this figure that a single line may be fitted to the ouabain-treated and control points in either respiratory or metabolic acidosis experiments. This finding suggests that the drug did not alter the relationship between pHi and pH • Thus, any apparent effect of ouabain on pHi could possibly e be explained by the effect of ouabain on pH . e Regardless of how ouabain produced the small reduction of intracellular pH it seemed desirable to test whether this change were correlated with the change in intracellular Na+ induced by ouabain. The plot of the [ Na +] i - [ H+]i values for individual animals and the regression lines fitted to these data by the method of least squares are shown in Fig. 5. The slopes calculated for the control; ouabain, 10 mg/kg; and ouabain, 20 mg/kg data are 0.01275, -0.045 and -0.067, respectively_ None of these slopes was significantly different from 0 when the data were analyzed Fig. 5 Relationship between [H1i and [N4 i plotted as individual values and resultant curves for control animals (circles), animals treated with ouabain, 10 mg/kg (triangles), or ouabain, 20 mg/kg (squares). The mean point of each line has been indicated by a large symbol of the appropriate shape. Regression lines were calculated by the method of least squares. The slopes of the lines for the regression Of[Na 4 i on [H+] i were as follows: Group . Control b -0.0002 Ouabain, 10mg/kg -0.0134 Ouabain, 20mg/kg -0.0083 70. <J o o v <J 0 0 0 rt') 0 <J CD 0 ....J 0 C "'::i c: ~ 00 0 0 0 C c <l 0 1.0 0 ~ rP 0 0 0 c c <J 0 ~ <J 0 ~ <J 0000 0 0 ~"'C 2 0 0 0 0 N ._....J 0 rt') N 0 C <fJ 0 0 COO C <J <J O <J N :t: u D<J C 0 N cr:Z: W_ '---' E -Q) u +:z: '---' 71 further. The fact that ouabain had no discernable effect on cell pH in these experiments, however, does not negate the possibility that ouabain did have an effect on H transport. The first probability which should be considered is that ouabain did lead to an accumulation of H by the cell but that this additional H load in the cell was buffered so that cell pH did not change • The CO2 data indicate a consistent increase in the intracellular bicarbonate content of animals treated with ouabain. This bicarbonate accumulation could relate to the fact that ouabain consistently reduces the transmembrane potential of skeletal muscle. Direct measurements of the transmembrane potential of muscle with microelectrodes have shown that cardiac glycosides do cause a reduction of this potential (Kostyuk & Sorkina, 1961; Draper, Friebel & Karzel, 1963). Furthermore, it is known that over the range of [K+]o values employed in these experiments the cell behaves as a potassium electrode, this is, Em is a direct function of the potassium ratio (Hodgkin, 1951). Thus the potassium ratios calculated for the present experiment and which were markedly reduced by ouabain suggest that the drug reduced the transmembrane potential as well. The electrochemical gradient for HC03 is such that this ion would tend to move out of the cell, and probably does so (Woodbury, 1965) continually, at a rate which is limited by the relative impermeability of the cell membrane to bicarbonate. Reduction of the transmembrane potential would thus necessarily reduce the gradient which tends to drive HC03 72 out of the cell and an accumulation of bicarbonate might result. The plausibility of the proposal outlined in the preceding paragraph was examined by calculating tissue buffer curves from the data of the invivo experiments. These are shown in Fig. 6. Two different sets of curves are depicted in this figure to emphasize the major problem encountered in constructing buffer curves of this type for tissues other than blood, namely, what value of intracellular HC0 3 should be used since this parameter can be calculated in several different ways. Two commonly used means of calculating [HC03 -] i have been applied to the present data. To construct the lower set of curves in Fig. 6, [HC03 -] values were calculated from the measured plasma pC02 values and the pHi calculated by the DMO method. With this method it is necessary to assume that the pC02 of intracellular and extracellular fluids are equal (Kibler, O'Neill & Robin, 1964; Roos, 1965; Clancy & Brown, 1966). As has already been pointed out, the validity of this assumption is questionable. To construct the upper curves in Fig. 6, [HC0 3 -]i values were calculated from the pHi measured by the DMO method and the total intracellular CO values 2 derived from tissue CO2 data. The absolute values of [HC0 -] i calculated 3 by the two different methods are clearly quite different. The buffer curves derived by the two methods also differ in slope. When calculations were based on pC02 and pHi' the tissue buffer curve for the control group had a slope of 9 Slykes, that for the ouabain group had a slope of 8 Slykes. When calculations were derived from tissue CO 2 and pHi data, the resultant Fig. 6. CO buffer curves of skeletal muscle of rats pretreated wit....... 2 saline (circles); ouabain, 10 mg/kg (triangles); or ouabain, 20 mg/kg (squares) and respired on atmospheres containing various amounts of CO2 in vivo. Curves for the control group are indicated by solid lines; curves for the ouabain groups are indicated by broken lines. The upper curves use [HC03 -] i values calculated from the total intracellular CO2 and pHi I S calculated by the DMO method. The lower curves use [HC03 -] i values calculated from plasma pC0 2 and pHi I S calculated by the DMO method. Curves were fitted by visual inspection. 73 24 22 20 18 . ~COfJ 16 mEq/L cell H2 0 14 o 12 10 8 6~----~--~--~----~--~----~--~ 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.: 74 curves had slopes of 14.5 and 13.0 Slykes,respectively. Clancy & Brown have reported that the tissue buffer curve of dog skeletal muscle has a slope of 19.5 Slykes. Although the slopes of the ouabain and control curves in Fig. 6 do not differ substantially, the ouabain curve is shifted to the left of the control line. That is, among animals respired with a given concentration of CO , 2 those pretreated with ouabain had a higher intracellular pC0 2 and higher [ HC03 -] i than those pretreated with saline. This is consistent with the proposal that cells of ouabain-treated animals retain more bicarbon&te and are thus capable of buffering more acid than the· cells of normal animals. Since the equilibration of CO 2 between intracellular and extracellular fluid is not instantaneous, as was once supposed (Woodbury, 1965), it seems reasonable that as acid was buffered by bicarbonate to form carbonic acid, the intracellular pC0 would increase as it evidently does in the ouabain 2 groups. The possibility also exists that the increase in plasma K which followed the administration of ouabain in vivo might stimulate the Na-H pump and thus mask the depressant effect of ouabain on the transport of H by this system. It is well established that K does stimulate sodium transport (Steinbach, 1951) and the antagonism of ouabain's action on heart muscle by K is also well known (Lown, Whipple, McLemore & Levine, 1961). The in-vitro experiments were conducted for the specific purpose of testing this hypothesis. In the in-vitro experiments, where 75 K levels remained low throughout the incubation period, ouabain had no effect on intracellular pH when extracellular acid-base parameters were normal. When tissues were exposed to extremely acid environments (pH<7), those incubated in the presence of ouabain did have a lower intracellular pH than those which were not exposed to the drug. This lends some support to the notion that the high plasma K levels encountered in vivo might have had some influence on the results obtained in those experiments. Thus far, in examining the data for some evidence that ouabain affects the transport of H by the Na pump, it has been assumed that a change in cell pH was valid criterion of such an effect. A brief consideration of the characteristics of the Na pump itself and the factors involved in regulating its rate may suggest another way of looking at the H data and of evaluating whether ouabain alters H transport. The rate of Na transport by the Na pump is regulated by several factors including internal [Na+] (Conway, 1960 a & b; Steinbach, 1961), and external [Na+] (Keynes & Swan, 1959; Mullins & Frumento, 1963; Armstrong, 1965). It was first suggested by Conway and his associates (Conway, 1960 a & b; Conway, Kernan & Zadunaisky, 1961) that the total electrochemical gradient for Na, rather than the chemical gradient alone, was a crucial consideration and that the function of the Na pump is to maintain this gradient at a constant value, the so-called' critical energy barrier'. Recent experimental evidence has provided support for this concept. The studies of Horowicz and Gerber (1965) showed that Na 76: efflux was, indeed, voltage dependent. Fozzard & Kipnis (1967) examined the proposal more thoroughly by determining the electrochemical gradient for Na in rat diaphragm under steady state conditions after altering the electrical or chemical gradient of Na. Their data show that the rate of Na t~ansport is stimulated by any procedure which would tend to lower the total gradient of Na, and that the enhanced pumping of sodium continues until a new steady state value of [Na+ Ji is attained and the total electrochemical gradient of Na is restored to control values. One implication of their data which the authors did not specifically test was that the electrochemical potential of Na would necessarily be reduced by a drug which interfered with the active transport of Na . In addition to the effect of Na on Na pumping, the concentration of K in body fluids is known to have a significant effect on the rate of the Na pump (Steinbach, 1952; Armstrong, 1964; Horowicz & Gerber, 1965). Potassium probably affects the rate of sodium pumping by two different mechanisms. One of these is a direct influence of K on the A TP-ase which is an integral element of the Na-K pumping mechanism (Davies & Keynes, 1961; Skou, 1961). The other is probably an indirect effect of [ K+] e on sodium transport because the skeletal muscle membrane functions as a potassium electrode when [K+]e is greater than 4 mEq/L, that is, Em is directly related to the ratio [ ~] i/ [ ~] 0 and, as F ozzard and Kipnis (1967) demonstrated, a reduction of Em stimulates the Na pump. 77 On the basis of the foregoing discussion of the regulation of sodium pumping, it seems reasonable to make some predictions about the factors which would be expected to affect a Na-H pump. Potassium would be expected to have a non-selective influence on the transport of both ions by the pump. There is experimental evidence that this is the case. Several investigators have demonstrated that experimental potassium depletion, which would be expected to impair Na transport in all tissues, does in fact cause an intracellular acidosis as measured by the CO2 method (Cooke, Seegar, Cheek, Coville & Darrow, 1952; Eckel, Botschner & Wood, 1959; Gardner, MacLachlan & Berman, 1952; Hudson & ReIman, 1962), or by the DMO method (Irvine, Saunders, Milne & Crawford, 1961; Sanslone & Muntwyler, 1966). Also, Miller, Tyson and ReIman (1963) found that raising the [K+] of the medium bathing isolated rat diaphragms (a procedure expected to stimulate Na pumping) caused an increase in the internal pH of both normal tissues and those obtained from animals previously depleted of K. The data of the present experiments also suggest that [K+] has an influence on the rate of H pumping. In view of the fact that the Na pump is influenced by the concentration of Na both inside and outside the cell, it seems reasonable to speculate that a Na - H pump would be affected by the concentrations of both these ions. In the present experiments large variations in external Na concentrations did not occur so this could have had little influence on the rate of the pump. Hydrogen ion concentrations, however, did vary markedly in these experiments 78 and it is not inconveivable that this may in and of itself have increased the overall rate of the Na-H pump_ That H can stimulate Na pumping has been observed in toad bladder (Leaf et al., 1964) and in frog muscle (Keynes, 1963). Finally, it would not seem unreasonable to expect that if a Na pump is regulated by the electrochemical gradient of Na, a Na - H pump might be regulated by the combined electrochemical gradients of Na and H _ The design of the experiments in the present study was well suited to exploring this possibility since in these experiments both the electrical and chemical gradients of H were varied while extracellular Na remained virtually constant. The total electrochemical gradients of Na and H rna y be calculated from the existing data and should provide some insight as to whether the cell maintains a constant gradient of H as it does of Na, despite large variations in the extracellular concentration of the ion. It should be possible to determine by this approach whether the electrochemical gradient of HO , like that of Na , changes after the administration of ouabain. The total electrochemical gradients for Na and H were calculated for each of the experimental groups, both in vivo and in vitro according to the formula: Gradient (mV) =[ (RFT In Na 0 Na . 1 ) - 1 (Fozzard & Kipnis, 1967). Since Em was not measured in the present experiments, the potassium equilibrium potential, E K' was substituted for this term in these calculations. Such a substitution seemed valid since, 79 as discussed previously, the skeletal muscle membrane behaves as a K electrode at concentrations of extracellular K employed in these experiments (Hodgkin, 1951). The electrochemical gradients of Na and H calculated for the invivo groups from the data on the ratios of these ions presented in Table 8 ]0' along with the mean values of [K+ [H+] 0' [Na+] i' and Ek for these groups are presented in Table 12. The values in part A of Table 12 pertain to the control (saline pretreated) groups while those in part B are for the groups pretreated with ouabain, 20 mg/kg. Considering part A of Table 12, it is evident that the extracellular [H+] was altered by all of the treatment procedures and that the electrical gradient, as estimated from E , tends to decline in acidosis. There is K little variation of the intracellular Na concentration among these groups. The electrochemical gradient for Na also remains the same except in the two most acidotic groups. In the groups subjected to NH4 CI, 6 mEq/kg, and to 24% CO2 the electrochemical gradients for Na are lower than those of the other groups. This may be indicative of competition between Na and H for the transport mechanism in these groups. The fact that the electrochemical gradients of hydrogen ion in all of the se groups are virtually the same strongly supports the suggestion that the rate of pumping adjusted to the rate required to maintain a constant electrochemical gradient of H' • If the H pump and the Na pump were actually independent, the rate of each being regulated by the electrochemical gradient of the ion transported, 80 Table 12. Electrochemical gradients for Hand Na calculated from mean concentration gradients for these ions and the potassium equilibrium potential determined in in-vivo experiments. Treatment Ouabain AcidBase A. 0 B. [Hi [Kt [Na' nM/L mEq/L Control 34.7 4.47 6% C02 54.8 12% CO2 EK Electrochemical gradient (mV) mV H+ Na 19.3 -95.34 65.71 147.9 4.23 18.5 -93.73 66.93 148.1 83.6 4.51 18.6 -92.00 70.04 147.0 24% CO2 128.2 6.08 19.1 -84.70 67.16 137.6 LoNH4Cl 49.4 4.97 16.4 -89.91 67.12 145.4 HiNH 4CI 61.3 5.97 17.2 -84.01 67.82 141.1 Control 42.0 10.81 30.5 -67.69 41.6 102.9 6% CO 2 73.5 11.81 31.6 -65.44 41.8 103.3 12% CO2 95.3 12.76 31.4 -63.54 43.5 102.3 24% CO 2 144.6 13.29 29.8 -60.89 46.7 101.3 LoNH4Cl 59.6 11.68 30.8 -66.28 44.6 104.8 HiNH4Cl 82.0 12.81 28.4 -64.05 44.2 105.3 ~Omg/kg 81 then the administration of ouabain might affect Na transport without affecting the H transport. The effect of ouabain on sodium transport is evident in part B of Table 12. Not only does Na accumulate intracellularly as the transport system is impaired, but the cell is unable to maintain the high electrochemical gradient of Na observed in the control situation. The reduction of the membrane potential should stimulate pumping in a system regulated by an electrochemical gradient. Thus, if ouabain had no effect on H transport, H pumping should increase as the membrane potential declined and the electrochemical gradient of H would not be expected to change from its control values. In fact, the electrochemical gradient of H was substantially lower in the ouabain treated animals. This is not definitive proof that the Na and H pumps are identical but it does indicate that the function of both is impaired by ouqbain. Electrochemical gradients of Hand Na were also calculated from the data of the in-vitro experiments and the results are presented in Table 13. The potassium equilibrium potentials calculated for the tissues not exposed to ouabain approximate those calculated by F ozzard and Kipnis for this tissue, and agree with the actual measured transmembrane potential values of normal resting diaphragm muscle in vitro (Creese, Scholes, & Taylor, 1958; Creese, Scholes, & Whalen, 1958). Although the extracellular K concentration remained essentially the same in all groups, it is clear that the ratio [K+]/[ K+]o' and presumably Em,was reduced by ouabain. The potassium ratio was not, however, reduced in vitro by 82 Table 13. Electrochemical gradients for Hand Na calculated from mean concentration gradients for these ions and the potassium equilibrium potentials determined in in..vitro experiments. "_~ •• ~ •• 4 ••~. Treatment Ouabain 0 AcidBase [Ht nM/L [K1 [Na+]i mEq/L EK ___ . _ Electrochemical gradient (mV) mV H Na Control 34.7 5.15 42.6 -84.7 69.9 113.5 Low HC03 141.3 5.30 50.2 -80.7 82.7 115.2 High CO 2 95.5 4.85 44.2 -83.9 77.4 112.0 Control 33.1 5.40 100.3 -70.0 59.2 76.6 138.1 5.10 63.0 -71.5 64.9 88.9 High CO2 109.7 4.85 80.2 -73.2 62.2 83.3 Control 36.1 5.70 126.7 -59.3 44.5 59.9 138.1 5.20 89.5 -70.8 65.8 79.6 80 mg/L Low HC03 l60mg/L Low HC03 83 acidosis as it had been in vivo. As was the case in vivo, the electrochemical gradient for Na was not reduced by acidosis in vitro, but it was lowered markedly by ouabain. The fact that ouabain reduced the electrochemical gradient of Na more in the control group than in groups exposed to acid media is of interest since this observation lends support to the suggestion made earlier that acidosis may antagonize the inhibitory influence of ouabain on the Na pump_ It is also interesting to note that the electrochemical gradient of H was higher in the tissues exposed to acid media than in the control tissues. Fozzard & Kipnis (1967) postulated that the positive electrochemical gradient of an ion is maintained by the active transport of that ion and, hence, the magnitude of the gradient of an ion should afford some index of the efficiency of the mechanism transporting it. On this basis, it might be concluded that increasing acid loads enhance the efficiency of the H pump since the data show that they do increase the electrochemical gradient of H. Ouabain not only reduced the electrochemical gradient of H in the in -vitro experiments, but eliminated the apparent difference between the H electrochemical gradients of control and acidotic tissues. Moreover, the cell pH data in Table 11 show that ouabain actually caused an accumulation of H in vitro in tissues exposed to an acid load. All of these observations tend to reinforce the proposal that ouabain, in doses known to inhibit the Na transport system, impair the ability of cells to handle H. This suggests not only that H is actively transported out of skeletal muscle, but that it 84 is transported by a mechanism very similar to, if not identical with, the Na pump. SUMMARY The relationship between the Na pump and the regulation of intracellular hydrogen ion concentration was studied in experiments conducted in vivo and in vitro. ill these experiments, the two systems were stressed independently and concurrently, the sodium pump by ouabain, the hydrogen regulatory mechanism by extracellular acid loads, both respiratory and metabolic, of various intensities. It has been found that acidosis produced in vivo causes no detectable accumulation of sodium in rat skeletal muscle although the increase in plasma potassium concentration observed in severe acidosis is suggestive of some impairment of the Na - K transport system. Ouabain did not induce an accumulation of hydrogen ion intracellularly in either control animals or those subjected to moderate degrees of acidosis. The drug, however, did appear to interfere with H transport in severe acidosis, and did significantly impair the ability of muscle cells to maintain a constant electrochemical gradient of H. Several factors might explain the failure of ouabain to alter cell pH to any significant degree in the in-vivo experiments, even though the drug may have impaired the Na-H transport system. First, the large increase in plasma K which invariably accompanied ouabain administration in vivo very probably antagonized the action of ouabain on the Na -H pump. In invitro experiments, where external potassium did not vary, ouabain was found to impair the ability of skeletal muscle to resist an acid challenge and 86 under these conditions did cause an accumulation of H intracellularl y • Second, acidosis per se may have stimulated the pump sufficiently to antagonize the inhibitory influence of ouabain on H transport. 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(1959), Calculation of intracellular pH from the distribution of 5, 5-dimethyl-2, 4-oxazolidinedione (DMO). Application to skeletal muscle of the dog. J. clin. Invest. 38, 720-729. Wallace, W. W. & Hastings, A. B. (1942). The distribution of bicarbonate ion in mammalian muscle. J. bioI. Chern. 144, 637 -649. Wallace, W. W . & Lowry, O. H. (1942). An in vitro study of carbon dioxide equilibria in mammalian muscle. J. biol. Chern. 144, 651-675. Warburg, E. J. (1922). Carbonic acid compounds and hydrogen ion activities in blood and salt solutions. Biochem. . 16, 153-340. Withrow, C. D. (1959). The direct effects of desoxycorticosterone on skeletal muscle electrolyte metabolism. Ph. D. Thesis: U. of Utah. Withrow, C. D. & Woodbury, D. M. (1964). Direct and indirect effects of desoxycorticosterone (DOC) on skeletal muscle electrolyte and acidbase metabolism. In Hormonal Steroids, Biochemistry, Pharmacology and Therapeutics: Proc. 1st Int. Cong. on Hormonal Steroids, vol. 1, pp. 503-513. New York: Academic Press. Wollenberger, A. (1962). Digitalis: Action on metabolism and the contractile system. In Ciba Found. Symp. Enzymes & Drug Action, pp. 127 -132. Boston: Little, Brown. 99 Wood, E. H. & Moe, G. K. (1942). Electrolyte and water content of the ventricular musculature of the heart lung preparation with special reference to the effects of cardiac glycosides. Am. J. Physiol. 136, 515-522. Woodbury, J. W. (1965). Chapter 46: Regulation of pH. In Physiology and Biophysics, 19th ed., ed. Ruch, T. C. & Patton, H. D., pp. 899934. Philadelphia & London: W. B. Saunders. Wyke, B. (1963). Brain Function and Metabolic Disorders. London: Butterworths . RESEARCH PROPOSALS PHARMACOLOGICAL CHARACTERISTICS OF THE H TRANSPORT SYSTEM OF RAT DIAPHRAGM IN VITRO: EFFECTS OF DRUGS WHICH STIMULATE NA TRANSPORT IN SKELETAL MUSCLE ALONE AND IN COMBINATION WITH OUABAIN. Recent evidence suggests that ouabain, a drug which inhibits the Na pump (Johnson, 1956), also impairs the ability of cells to handle an acid load (Elsmore, 1967), a process believed to require the active extrusion of H. This is consistent with the suggestion that a single mechanism is responsible for the active transport of both Na and H ions out of the cell (Woodbury, 1960). Additional support for this concept might be provided by elucidating the effects of several drugs known to stimulate Na transport in skeletal muscle on H transport in this tissue and by determining whether these agents were able to reverse the impairment of one or both of these transport mechanisms by ouabain. Withrow and Woodbury (1964) reported that deoxycorticosterone (DOC) does, indeed, stimulate both Na and H transport in skeletal muscle by a direct action. Another corticosteroid, aldosterone, is known to stimulate Na transport (Woodbury & Koch, 1957; Porter & Edelman, 1964), but its effects on H transport are unknown. It has been reported that aldosterone does not reverse the loss of K from heart muscle induced by ouabain (Levy & Richards, 1963). It would be interesting to determine, however, whether aldosterone or DCA reverses the inhibition of either Na or H transport, or both in skeletal muscle. It would also seem desirable to examine the effects on skeletal muscle H transport of triamterene, a drug believed to inhibit the Na - K- H exchange mechanism of the kidney (Baba, Tudhope, & Wilson, 1964), and to explore the possibility of antagonism between the effects of triamterene and the corticosteroids on Hand/or Na transport. Baba, W. 1., Tudhope, G. R. & Wilson, G. M. (1964). Site and mechanism of action of the diuretic, triamterene. Clin. Sci. 2, 181-193. Elsmore, T. A. (1967). A study of the relationship between sodium ion and hydrogen ion regulation in rat skeletal muscle. Ph. D. Thesis; University of Utah. Johnson, J. A. (1956). Influence of ouabain, strophanthidin, and dihydro strophanthidin on sodium and potassium transport in frog sartorii . Am. J. Physiol. 187, 328-358. Levy, J. V. & Richards, V. (1963). Aldosterone-ouabain actions on isolated rabbit atria. Arch. int. Pharmacodyn. 146, 363-373. Porter, G. A. & Edelman, 1. S. (1964). The action of aldosterone and related corticosteroids on sodium transport across the toad bladder. J. clin. Invest. 43, 611-620. Withrow, C. D. & Woodbury, D. M. (1964). Direct and indirect effects of deoxycorticosterone (DOC) on skeletal muscle electrolyte and acid-base metabolism. In Hormonal Steroids, Biochemistry, Pharmacology and Therapeutics: Proceedings of the First International Congress on Hormonal Steroids, vol. 1, pp 503-513. Woodbury, D. M. & Koch, A. (1957). Effects of aldosterone and desoxycorticosterone on tissue electrolytes. Proc. soc. exp. BioI., N. Y. 94, 720-723. Woodbury, J. W. (1960). In Medical Physiology and Biophysics, 18th ed., pp. 2 - 30. ed. Ruch, T. C. & Fulton, J. F. Philadelphia: Saunders. RELATIONSHIP BETWEEN THE H PUMP AND THE ELECTROCHEMICAL GRADIENT OF H IN SKELETAL MUSCLE. Fozzard and Kipnis (1967) recently presented evidence that the function of the Na pump in skeletal muscle is to maintain a constant electrochemical gradient of Na between intracellular and extracellular fluids. Their results indicate that when tissues are subjected to any procedure which would tend to lower this gradient, i.e., a reduction of [Na+]0 or an increase in the transmembrane potential, the Na pump is stimulated, and a new, lower steady state value of [Na + ] i is achieved such that the total electrochemical gradient of Na does not change. The data obtained by Elsmore (1967) in studies on the intracellular pH of skeletal muscle are consistent with the hypotheses 1) that H is actively transported out of skeletal muscle and 2) that the H pump of muscle may, like the Na pump, be regulated by the total electrochemical gradient of the ion being pumped. It seems important to examine this question fully and directly since, if this is the case, it means that the appropriate criterion of whether the cells are functioning normally with respect to acid-base regulation is not the internal pH, but the electrochemical gradient of H they maintain. These studies will be conducted in vitro employing the intact rat diaphragm preparation (ReIman, Gorham & Levinsky, 1961). The steady state chemical gradient of H in tissues exposed to acidic and basic media will be measured from the distribution between tissue and bath medium of C14_DMO (Waddell & Butler, 1959). Transmembrane potentials will be measured directly with microelectrodes rather than estimated from potassium equilibrium potentials. Voltage clamp studies would afford an extremely useful approach to this problem since they would permit Em and [K+] 0 to be varied independently. The proposed investigation should answer several questions. First, is the H pump of skeletal muscle regula ted by the electrochemical gradient of H between intracellular and extracellular fluids? Second, what is the critical value which the H pump functions to maintain? Third, is the ability of the cell to maintain this critical value unlimited, and if not, how severe must acidosis (or alkalosis) become to exceed the ability of the H pump to adjust to it? Fourth, to what extent is the influence of [K+] 0 on the H pump direct, and to what extent does [K+]o influence H pumping indirectly because of its effect on the transmembrane potential? Elsmore, T. A. (1967). A study of the relationship between sodium ion and hydrogen ion regulation in rat skeletal muscle. Ph.D. Thesis; University of Utah. Fozzard, H. A. & Kipnis, D. M. (1967). Regulation of intracellular sodium concentration in rat diaphragm muscle. Science 156, 1257-1260. ReIman, A. S., Gorham, G. W. & Levinsky, N. G. (1961). The relation between external potassium concentration and the electrolyte content of isolated rat muscle in the steady state. J. cUn. Invest. 40, 386-393. Waddell, W. J. & Butler, T. C. (1959). Calculation of intracellular pH from the distribution of 5, 5-dimethyl-2, 4-oxazolidinedione (DMO). Application to skeletal muscle of the dog. J. cline Invest. 38, 720-729. INVESTIGA TION OF THE ACTIVE TRANSPORT OF CHLORIDE IN SKELETAL MUSCLE. At one time it was believed that chloride existed solely in the extracellular phase of tissues (Fenn, 1936), the basic assumption underlying intracellular calculations based on CI space. With the development of better analytical procedures, however, it became evident that a substantial fraction of tissue chloride was, in fact, intracellular (Heilbrunn & Hamilton, 1942; Shenk, 1950). The most widely accepted explanation for this intracellular CI has been that proposed by Boyle and Conway (1941), namely, that chloride is distributed between intracellular and extracellular water in accordance with the Donnan theory as a function of the transmembrane potential. While this passive distribution of chloride may be the case for some muscles, it is not the case for all tissues. Active transport of CI has been demonstrated in the gastric mucosa (Hogben, 1955), rumen epithelium (Stevens, 1964), frog cornea (Zadunaisky, 1966), and thyroid gland (Woodbury, personal communication). In recent experiments on rat diaphragm muscle in vitro, this author measured intracellular chloride values far higher than those which would be predicted for a passively distributed cation from the K equilibrium potential in the same tissue. The possibility that chloride is actively transported in some, if not all, skeletal muscle tissues, and the functional implications of this phenomenon if it does exist, require elucidation. Several different skeletal muscles would be examined in the course of this study with particular attention to the rat diaphragm, since this is a preparation almost ideally suited to study ionic fluxes of skeletal muscle in vitro (Creese, 1954). A combination of tracer techniques, for determin- ing flux rates (Us sing, 1947). and chemical determinations of tissue electrolytes will be employed in this investigation. Transmembrane potentials will be measured in vitro with microelectrodes as extracellular Cl concentrations are varied. To elucidate fully the characteristics of the Cl transport system if it does exist will require the use of pharmacological toolF such as metabolic inhibitors and ouabain, drugs known to have effects on other active transport mechanisms. These studies should provide considerable insight as to 1) whether chloride is actively transported in any, some or many skeletal muscle tissues, 2) the role of extracellular chloride concentrations in determining the membrane potential of those tissues in which Cl is actively transported, and 3) the pharmacological characteristics of the Cl transport system. Boyle, P. J. & Conway, E. J. (1941). Potassium accumulation in muscle and associated changes. J. Physiol., London, 100, 1-63. Creese, R. (1954). Measurement of cation fluxes in rat diaphragm. Proc. Roy. Soc. B., 142, 497-513. Fenn, W. O. (1936). Electrolytes in muscle. Physiol. Rev.~, 450-487. Heilbrunn, L. V. & Hamilton, P. G. (1942). The presence of chloride in muscle fibres. Physiol. 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