Characterization of receptor redistribution and regulatory volume decrease in rabbit alveolar macrophages

The present study was designed to examine the hypothesis that trasnporter/receptor recycling rates were involved in regulatory volume decrease (RVD). The alveolar macrophage is a useful tool for the study of membrane component recycling because it expresses a number of different rceptors including those for differric transferring, aMacroglobulin-protease complex, and mannose-terminated glycoproteins. The cellular distribution of these receptors depends on both the rate of internalization and the rate of exteriorization. Alterations in the rate of either limb"" of the recycling pathway between the cell surface and the endocytic apparatus can lead to an alteration in the distribution of receptors. Using recycling receptors as a model for membrane redistribution, involvement of membrane recycling in the regulation of ion transport during RVD was investigated. Hypo-osmotic incubation of cells as 37 ° C resulted in a rapid and reversible increase in surface receptor number for these three ligands. This increase was time, temperature, and dilution dependent. Examination of both the rates of internalization and exteriorization of receptors revealed that the increase in surface receptor number was due to a transitory decrease in the rate of receptor internalization without altering the rate of receptor exteriorization. Hypo-osmotic incubation of cells inhibited internalization of both occupied and unoccupied receptors. Both the rate of receptor internalization and surface receptor number returned to near control values after 30 minutes in hypo-osmotic media. To investigate whether the inhibition of receptor internalization represented a general inhibition of cellular endocytic processes, the effects of hypo-osmotic incubation on fluid phase pinocytosis were examined. HRP uptake by cells in hypo-osmotic solutions was inhibited to the same magnitude, and with similar kinetics as receptor mediated endocytosis. However, unlike receptor internalization, fluid phase uptake did not recover to control vlaues after 30 minutes of hypo-osmotic inbuation. this result is explained by the proposed existance of independent pathways for fluid phase pinocytosis and receptor mediated endocytosis. These studies represent the first report of RVD in alveolar macrophages. The process of RVD in these cells isdependent on the loss of K+, Cl-, and osmotically obliged water. The characterization of ion loss by ion substitution experiments as well as pharmacological assays indicate that the ion pathways operating during RVD in theese cells are most similar to the independent cation and anion conductance pathways reported to operate in human peripheral blood lymphocytes and platelets. Kinetic analysis of cation loss demonstrated that cation loss was rapid and preceded both the changes in surface receptor number and the initiation of RVD. The loss of K+ was first ordered and extrpolated back to the time of media dilution indicating that ion transporters were already present on the cell surface before receptor redistribution and were activated immediately upon dilution. Preliminary investigation into the cause of inhibition of receptor internalization indicate that although cell volume/morphology changes correlate with changes in surface receptor number, the changes induced by forces of swelling may not be the direct cause of inhibition. Alterations of intracellular pH as a result of hypo-osmotic incubation may be involved in the inhibition of internalization processes. Further investigations are required to determine this. Hypo-osmotic incubation of cells provides a unique ""tool"" for further study into the mechanism of recycling and membrane movement. It is the first reported procedure that affects the internalization ""limb"" of the recycling pathway without affecting the exteriorization ""limb."" In addition, hypo-osmotic incubation provides an experimental means of separating the processes of fluid phase pinocytosis and receptor mediated endocytosis.""

CHARACTERIZATION OF RECEPTOR REDISTRIBUTION AND REGULATORY VOLUME DECREASE IN RABBIT ALVEOLAR MACROPHAGES by Jeanne Marie Novak A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Experimental Pathology Department of Pathology The University of Utah Deceniber 1987 THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMrvIITTEE APPROVAL of a dissertation submitted by Jeanne Marie Novak This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. Odz 1987 i odz 1787 0012.1987 ; ad 2, 1'187 ; Martin C. Rechsteiner Oc/ 2,, It87 THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of The University of Utah: I have read the dissertation of Jeanne Marie Novak m Its final form and have found that (I) its format, citations. and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables. and charts are in place; and (3) the final manuscript is satisfactory to the Supervis­ory Committee and is ready for submission to the Graduate School. Ap~roved for the Major Department Chairman I Dean A pproved for the Graduate Council B. Gale Dick Dean of The Graduate School Copyright (I) Jeanne Marie Novak 1987 All Rights Rese.rved ABSTRACT The present study was designed to examine the hypothesis that transporter/receptor recycling rates were involved. in regulatory volume decrease (RVD). The alveolar macrophage is a useful tool for the study of membrane corrponent recycling because it expresses a number of different receptors including those for diferric transferrin, aMacroglobulin-protease corrplex, and mannose-tenninated. glycoproteins. The cellular distribution of these receptors depends on both the rate of internalization and the rate of exteriorization. Alterations in the rate of either "limb" of the recycling pathway between the cell surface and the endocytic apparatus can lead to an alteration in the distribution of receptors. Using recycling receptors as a model for membrane redistribution, involvement of membrane recycling in the regulation of ion transport during RVD was investigated. Hypo-osmotic incubation of cells at 370C resulted in a rapid and reversible increase in surface receptor number for these three ligands. This increase was time, temperature, and dilution dependent. Examination of both the rates of internalization and exteriorization of receptors revealed that the increase in surface receptor number was due to a transitory decrease in the rate of receptor internalization without altering the rate of receptor exteriorization. Hypo-osmotic incubation of cells inhibited internalization of both occupied and unoccupied receptors. Both the rate of receptor internalization and surface receptor number returned. to near control values after 30 minutes in hypo-osmotic media. To investigate whether the inhibition of receptor internalization represented. a general inhibition of cellular endocytic processes, the effects of hypo-osmotic incUbation on fluid phase pinocytosis were examined.. HRP uptake by cells in hypo-osmotic solutions was inhibited to the same magnitude, and with similar kinetics as receptor mediated endocytosis. However, unlike receptor internalization, fluid phase uptake did not recover to control values after 30 minutes of hypo­osmotic incubation. This result is explained. by the proposed. existence of independent pathways for fluid phase pinocytosis and receptor mediated. endocytosis. These studies represent the first report of RVD in alveolar macrophages. The process of RVD in these cells is dependent on the loss of K +, Cl-, and osmotically obliged water. The characterization of ion loss by ion sUbstitution experiments as well as pharmacological assays indicate that the ion pathways operating during RVD in these cells are most similar to the independent cation and anion conductance pathways reported. to operate in human peripheral blood lynphocytes and platelets. Kinetic analysis of cation loss demonstrated. that cation loss was rapid and preceded both the changes in surface receptor number and the initiation of RVD. The loss of K + was first order and extrapolated back to the time of media dilution indicating that ion transporters were already present on the cell surface before receptor redistribution and were activated :irrmediately upon dilution. v Preliminary investigations into the cause of inhibition of receptor intemalization indicate that although cell volume/moz:phology changes correlate with changes in surface receptor number, the changes induced by forces of swelling may not be the direct cause of inhibition. Alterations of intracellular pH as a result of hypo­osmotic incubation may be involved. in the inhibition of intemalization processes. Further investigations are required to determine this. Hypo-osmotic incubation of cells provides a unique "tool" for further study into the mechanism of recycling and membrane movement. It is the first reported procedure that affects the intemalization "limb" of the recycling pathway without affecting the exteriorization "limb. " In addition, hypo-osmotic incubation provides and experimental means of separating the processes of fluid phase pinocytosis and receptor mediated endocytosis. vi I would like dedicate this work to my family and friends, especially my mother Ruth, for her support and inspiration, my niece Krissy, my sister :Mary, and my close friend Cindy. TABLE OF CONTENTS .AB:S~ ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• i v LIST OF FIQJRES................................................. • x LIST OF T.AB:LES ••••••••••••••••••••••••••••••••••••••••••••••••••• xiii LIST OF .ABBREV'IA.TIONS ••••••••••••••••••••••••••••••••••••••••••••• xiv s .................................................... ~ Chapter 1. IN'I'RODOCTION' •••••••••••••••••••••••••••••••••••••••••••••••••• 1 Transporters Involved. in Intracellular pH Horneost.asis.......... • . • • • • • • • • • • • • • • • • • • • • • • . • • . • • • . • • . . . . 2 Cell Volume is Dependent on Ion Transporter Activity ••••••••••.••••..••.••••••••..•••.••.•.... 5 Regulatory Volume Increase •••••••••••••••••••••••••.••••..••.. 5 Regulatory Volume Decrease •••••••.•••••.••.••••.••..••...•.... 8 Regulation of Transporter Activity •••.•••••..•••..•.•••.•.••. 17 Re-fereIlces •••••••••••••••.•..•••.•••••.•••••••...•••..••..•.. 23 2. EFFECT OF HYPQ-OSM:>TIC INCUBATION 3. ON ~ RECYCL~ •••••••••••••••••••••••••••••••••••••••• 34 .Abstract.. • . . • • • • • . • • • • . • • . • • • . • • • . . • . • • . • . • • • . • . . . . • • . • • . • . . . 34 Introdu.ct.ion. • • . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • . . • • . • • • • . . • ••. 35 ~terials and ~thods •••••••••••••••••••••••••••••••••••••••• 36 Re-sul ts. • • • • • • • • • . • • . • • • • . • • • . . • • • • • • • • . • . . . • • . . . • • . • . • . . • • . . 39 DisCllssion .•••••••••••••••.•••••••.••••..••..•••.•••..•••..•. 75 Re-ferences .•...••..•••••.•.•••..•••....•....••..•........•... 85 THE MECHANISM OF REGUIATORY VOLUME DECREASE IN RABBIT ALVEOLAR ~PHAGES ••••...•.•..•.•.•....• .••••• 89 .Abst.ract.. • • • • • . • • • . . • • • • . • • • • • • • . • • • • . . • • • . • • • • . • . . • • • • . • . • . . 8 9 Introdllct.ion. • • • . • • • • . . . • • • . . • • • . . . • • . • • • . . . • • . . • • . • • . . • • . .. 90 ~terials and ~thods •••••••••••••••••••••••••••••••••••••••• 92 Re-sults ••...•••..••.•..••••..••....•....•.•.•.•.••........... 97 DisCllssion ....•.•..••..•.•••.••.•.•.........•......••.•••... 125 Re-ferences •...•.••••.••.••....•...••...••..•...•....•....... 132 4. PRELIMNARY' EXPERIMENTS INVESTIGATING THE MECHANISM OF INHIBITION OR RECEPTOR ~IZA.TION ••••••••••••••••••••••••••••••••••••••••••••• 135 .Abstract.. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • . • 135 Introdtlct.ion ••••••••••••••••••••••••••••••••••••••••••••••.• 136 ~terials and ~thods ••••••••••••••••••••••••••••••••••••••• 138 Results ••••••••••••••••••••••••••••••••••••••••••••••••••••• 139 DisCllssion •.•••••••••••••••••••••••••••••••••••••••••••••.•• 153 Fu.t'Ure Aims ••••••••••••••••••••••••••••••••••••••••••••••••• 157 Stmlla.ry • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 161 RefereI1ces • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 162 5. S~ AND FUTURE AIMB •••••••••••••••••••.••••••••••••••••. 163 Future .Ainls....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 165 APPENDIX. EFFECTS OF FIXATIVE 05M)IARITY ON CELL VOLUME: UTILIZATION OF THE COULTER COUNTER PARTICLE ~Yzm. ••••••••••••••••••••••••••••••••••••••••••••••••• 167 .Abstract.. • • • • • • • • • • • • • . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • . . • • . • • • 167 Introdtlct.ion. • • • • • . • • • • • • • • • • • • . • • • . • • • • • • . . . • • • • • • • • • • • . • • • 167 ~terials and ~thods ••••••••••••••••••••••••••••••••••••••• 169 Results ••••••••••••••••••••••••••••••••••••••••••••••••••••• 174 DiSCllssion ••••••••••••••••••••••••••••••.•••••••••••..•••.•• 178 RefereI1ces •••••••••••••••••••••••••••••••••••••••••••••••••• 182 ix LIST OF FIGURES Figure 2-1. E~f~ of hypo-£~tic incubation on surface bmdin.g for aM- I -T ..•.....................•............. 40 2-2. Concentration dependence of ligand binding •...•••••....•••. 43 2-3. Effect of different media osmolarities on surface receptor ntmlber ....•.•..•...•••.•.••.••••.•••....•. 46 2-4. Time course of surface binding at different media osmolarities...................................... ••• 47 2-5. Loss of surface bound ligand during hypo-osmotic incubation ................................................. 50 2-6. Rate of loss of surface bound ligand during iso-osmotic and gypo-osmotic incubation at 37 C •••....•.•••.•••....•..••.•.••..•...••••. 52 2-7 • ~ffect o~ hYJ?O-osmotic incubatio~2gn the specific mternal~zat~on rate (Ke) of aM- I-T .•••.•..••...•.•..... 54 2-8. Rate of recovery of surface binding activity in iso-osmotic or hypo-osmotic media after cell surface trypsinization .••..•..•..•••..••........ 57 2-9. Effect of repeated exposure of cells to hypo-osmotic media on surface receptor ntmlber..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 2-10. Effect of extended incubation in hypo-osmotic media on specific internalization rate (Ke) ••...•••........ 63 2-11. Effect of hypo-osmotic incubation on HRP aCClmllllation ............................................... 65 2-12. Effect of hypo-osmotic incubation on uptake of HRP during short exposures to the fluid phase ma.rker .•........•.........•.....•.......... 67 2-13. Simultaneous uptake of HRP and loss of prebound surface ligand by cells incubated in iso-osmotic or hypo-osmotic media •....••....•..•..•........ 69 2-14. HRP accumulation by cells after 60 minutes in hypo-osmotic .media ••••........•......•......•........... 73 2-15. Proposed model of effects of hypo-osmotic incubation on receptor mediated endocytosis and fluid phase pinocytosis ..••...•..............•...•.••.•••••.........•.. 78 3-1. Effect of hypo-osmotic incubation on alveolar macrophage cell volumes as measured by the Coulter Counter •••••...•.....••••...•.•..•.•..••....... 98 3-2. Time gourse 00 regulatory volume decrease at 37 C and 0 C .•.•.•••••••.•..••..••••...•••....•••....•.. 99 3-3. Scanning electron micrographs of rabbit alveolar macrophages after various times in hypo-osmotic media ••.•••••..••••..•.....•.•..•... 102 3-4. Equilibration of intrgcellular K+ and Na+ 1.1pOn incubation at 37 C ••••..•.••.••••••.....•..•.••...... 106 3-5. Effects of media ion composition on RVD in alveolar macrophages ..••••..••....••••..•.......•••••..... 108 3-6. Inhibition of RVD in the presence of Oligomycin C ......... 111 3-7 • Effect of gramicidin on cell volume during hypo-osmotic incubation •••••••••..•..•..••••..••.......... 113 3-8. 8~ efflux from alveolar macrophages during RVD ..•••.•••••...••••...•••......•...•.•.•.•.•....• 117 3-9. Effect of hypo-osmotic incubation on the absolute cation content of alveolar macrophages ..••••...••......... 119 3-10. Rate of cation loss from macrophages during RVD ........... 121 + 3-11. The percen~ loss o~ K from gells ~sed to hypo-osmot~c solut~ons at 37 C and 25 C ....••....•........ 123 3-12. Effect of hypo-osmotic incubation on the ~tracell¥lar concentration of K and Na ..•.•••..•••....••.•..•......•..•..•••.......... 124 4-1. Effects of incubation in high K+ media on surface receptor n~r......... . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . 140 xi 4-2. Effects of sucrose addition to hypo-osmotic KCI on cell vol~ and surface receptor ntnnber .••....••.•.•...•••.•..•.......••.......... 143 4-3 • Effect of incubation in Na-propionate on surface receptor ntnnber and cell vol~ •.•.••...•.....•.•......... 146 4-4. Effect of sucrose addition on surface receptor number of cells in Na-propionate •........•••...•.......... 149 4-5. Effect of sucrose addition on cell volure of cells incubated in Na-propionate •....•....••.................... 151 4-6. Effect of extracellular pH on surface recep.tor binding .••••..•.••••..••...••.••.....•......... .. 152 A-1. Effect of fixative and wash buffers of various osmolarities on cell volure .....•.......•..•.............. 177 A-2. Vol~ of fixed cells after incubation at aOc ••••••••••••• 179 xii LIST OF TABLES Table 2-1. Effect of Hm-osmotic Incubation on Surface Bin~g of I -MAN-BSA and I -Tf (Fe) 2 ..•••.•••••.••••.•••••.•••.•.••••••••...•. 41 3-1. Comparison Sf cell Volume M=asured by the Coulter Counter or ~O ..••.•.••••..••••.•..••.•••...•••.•••.•.•. 104 A-I. Scanning Electron ~croscopy Buffers ...•...•.•.•.••.••... 170 A-2. Scanning Electron ~croscopy Fixatives •••...••...•••..... 171 A-3. Scheme of Various Fixative and Wash Conditions .••.•...••• 176 LIST OF ABBREVIATIONS aMt25I-T •••••••..••••••••••••• aMaCroglobulin-125Iodinated-trypsin CHO ••••••.•••..•••••••.•...••••••.•••.•.•••• Chinese hamster ovary DIDS •...•••..••• 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid D:r.1S0 •.•••••••••••.••••••••••.•••••••••••.•••••• DiInethy 1 sulfoxide EDTA ..••••••••...•••••.•••••••. Ethylene-diamine-tetra-acetic acid EGTA •••..••••••••••••••• Ethyleneglycol-bis-N,N'-tetra-acetic acid HBSS ••..••••••••••.•••••••••...••••• Hank's balanced salt solution fIR.P •••••••••••••••••••••••••••••••••••••••• Horseradish }?eroxidase 125I-MAN-BSA •••••••••••• 125Iodinated-mannose-bovine serum albumin 125I-Tf(Fe)2 .••••••.••••.••••••• 125Iodinated diferric transferrin MDCK .••••••••••.••••••••••••••••.•••••• MBrdin-Darby canine kidney ~H/H ••.••..••••••••••• M[nimal essential media - Hank's / He}?es PBS •••..••...••••••••••••••••••••..••••• Phosphate buffered saline P~ •.••.•••••••••••••••••••••••.•••••••• Phorbol Iny'ristate acetate RVD ••••••••••..•••••••••...••••••••••.• Regulatory volume decrease RVI •••.••••••••••••••••..••••••••...••• Regulatory volume increase SITS .•••••••••••• 4-acetamido-4'-isothiocyano-2,2' disulfonic acid ACKNOWLEDGMENTS I wish to thank the members of my corrmittee for direction and constructive criticism. I would like to thank my fellow graduate students for their support, critical evaluations, and friendships. My appreciation is also extended to the postdocs, research fellows, and technicians in the laboratory, with whom I have shared numerous scientific discussions. I would like to thank Steve Wiley for help with calculations of the internalization data. I would also like to thank Diane :M::-Vey Ward and Stephanie Hamilton for their experimental contributions. I would like to acknowledge and thank Sandy Kaplan for the scanning electron microscopy. I wish to thank Don MOrse for his artistic assistance. I am especially grateful to Saundra Buys for her continued support and invaluable scientific (as well as grammatical) advice. Last, but most inportantly I would like to thank my mentor Jerry Kaplan, for his unending support and invaluable discussions. CHAPTER I INTRODUCTION The plasma membrane plays an irrportant role in maintaining cell volume (1,2), intracellular pH (3), and intracellular ion concentrations (4). As a semipenneable barrier, the plasma membrane allows the free diffusion of only water and some small molecules. The ability of cells to carry on vital cell functions and elicit appropriate responses, regardless of variations in the extracellular environment, is ensured by this selective barrier. The plasma membrane is capable of maintaining the intracellular concentration of ions at values different than would be predicted if ions were passively distributed between the inside and outside of cells. For example, most cells maintain a high intracellular K+ and low Na + concentration, whereas nonnal extracellular fluids are high Na +, low K+. It is through the selective partitioning of cations that this difference can be maintained. The mechanism by which cells regulate intracellular ion concentration has been the focus of intense investigation for many years. In 1957, the membrane protein which is primarily responsible for the maintenance of the K + INa + gradient was identified and characterized (5). The protein was found to be an integral membrane ATPase which maintains high K+ (135 roM) and low Na + (15 roM) by actively transporting 3 Na+ ions out of the cell for every 2 K+ ions 2 ptllTped into the cell. Hydrolysis of one ATP provides the energy for each 3 Na + 12K+ cycle. Thus, even as Na + passively moves down its electrochemical gradient from the outside to the inside of the cell, the activity of this plasma membrane ptlIl'p maintains K + and Na + homeostasis. Transporters Involved in Intracellular E!! Homeostasis Intracellular H+ concentration is maintained primarily through the activity of transporters located at the cell surface. Intracellular pH appears to be inportant in regulating the rate of many intracellular processes. For example, the activities of two glycolytic enzymes are pH sensitive. A reduction of only 0.1 pH unit results in a reduction of phosphfructokinase activity by 10- to 20- fold (6). As demonstrated both in situ and in vitro, conversion of phosphorylase b to phosphorylase a by the enzyme phosphorylase b kinase is dramatically decreased upon reduction of pH (7). The mechanisms regulating intracellular pH are not completely understood. It was assumed for years that H+ and K+ were distributed between the inside and the outside of the cell according to Donnan equilibrium (8), and that regulation of intracellular pH could occur merely by changes in K+ and H+ distribution. In 1934, the work of Fenn and Cobb (9) showed that intracellular pH was Imlch higher than predicted from simple Donnan equilibrium. The first proposal that active ion transport might be required for pH regulation was made in 1955 when Hill suggested that H+ distribution across the plasma membrane involved H+ extrusion by a mechanism analogous to the sodium 3 punp (10). Direct evidence for a transport system regulating intracellular pH appeared in 1971. Mssseter and Siesjo studied the effects of exposure to high pC02 on the intracellular pH of whole rat brain (11). This exposure induced the expected decrease of intracellular pH (0.13 units over 15 minutes). Interesting'ly, pH began to recover starting at 45 minutes, and by 3 hours pH had returned to control values. The authors concluded that the recovery of pH must be due to the active transport of H+ out of the cell or HC03 - into the cell, since, once no.rmal values were attained, no further pH changes were noted, and because transport of either H+ out or HC03- into the cell would require movement of ions against their concentration gradients. Until the late 1970s, most investigations of intracellular pH were :perfonned in whole tissue or in invertebrate giant nerve and muscle cells. Regulation of pH in squid axons (12), barnacle muscle (13), and snail neurons (14,15) results from H+ extrusion which was dependent on the presence of extracellular HC03 - • Inhibitors such as 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and 4- acetamido-4'-isothiocyano-2,2'disulfonic acid (SITS), known inhibitors of the anion exchanger of erythrocytes, completely blocked the ability of these cells to recover pH after an induced acid load. The H+ transport mechanism was also shown to be dependent on the presence of intracellular CI - and extracellular Na+. Based on these observations, it was concluded that pH regulation in these invertebrate cells was mediated by a Na + I H+ exchanger with the simultaneous operation of a CI- I HC0 3 - exchanger. 4 In vertebrate cells, pH homeostasis is primarily accomplished by the independent activity of an amiloride sensitive Na+ I H+ exchanger. Moolenaar and co-workers provide direct evidence for the existence of this exchanger in neuroblastoma cells (16). The activity of this exchanger is not dependent on extracellular HC03 -, suggesting that CI­I HC03- exchange is not O}?erating during pH regulation in these cells. Intracellular pH regulation by human fibroblasts (17) and rat thymic lymphocytes (18) is also regulated by the independent O}?eration of this electroneutral Na+ I H+ exchanger. Over the last five years, work on the identification and characterization of this seemingly ubiquitous Na + I H + transporter has continued. A principal reason for the intense interest in this transporter has been the proposed role of intracellular H + as a signal for cell activation (19). For example, a rapid rise in intracellular pH mediated by the Na + I H + transporter occurs during fertilization of sea urchin eggs (20). One of the first effects on cells of mitogenic hormones is the rise in intracellular pH resulting from activation of the transporter (21-25, ref 26 for review) • There is evidence to suggest that the activity of this transporter is regulated by intracellular H+ concentration. It has been proposed that an allosteric H+ - modifier site on the cytoplasmic domain of the transporter responds to increases in intracellular H + concentration and activates Na + I H+ exchange (27-30). Growth factors may activate the Na + I H+ exchanger by inducing a confonnational change in the transporter resulting in a change in the H+ - modifier site affinity for H+. Thus, tranporters have a major role in the 5 maintenance of intracellular pH during no:nnal as well as activated growth states. Cell Volume is Dependent on Ion Transporter Activity Another cell response that is highly dependent on the activity of transporters located at the cell surface is cell volume homeostasis (1,2). Regulation of cell volume is defined as the adjustment of cell volume back towards steady-state values during continuous exposure to aniso-osmotic conditions. Investigations by Roti Roti and Rothstein on mouse leukemia cells indicated that if volume regulatory mechanisms were intact, cells cquld survive, and indeed. divide, in a hypo-osmotic environment for at least 90 passages (31). Exposure of cells to hypo-osmotic rredia results in passive cell swelling. TIle active decrease of volume to nonnal values is termed Regulatory Volume Decrease (RVD). If cells are exposed to hyper-osmotic solutions, shrinking occurs and the increase in volume that occurs to restore cells to near nonnal values is termed Regulatory Volume Increase (RVI). Both of these processes have been reported to occur in a number of different cell types. Depending on the cell type studied, there are several mechanisms by which either RVI or RVD can occur. Regulatory Volume Increase To date, RVI appears to be acconplished by one of two mechanisms, first, either the operation of a Na+ / K+ / 2CI- co-transport system (32) or second, by the coupled activation of Na + / H+ and CI- / HC03- exchangers (33,34). 6 Some of the first studies of volume regulation in vertebrate cells were done by K:regenow in 1973, utilizing duck. red blood cells (35). In this cell type, cell shrinkage in response to hyper-osmotic solutions activates a Na+ / K+ / 2CI- cotransport system. This system effects the cellular accumulation of ions and osmotically obliged water resulting in RVI. K:regenow found that volume regulation did not occur if any of the three ions were excluded from the extracellular media (36). RVI was inhibited by the epithelial loop diuretic, furosemide, which inhibits CI- reabsorption from the thick. ascending limb of the loop of Henle (37). Based on these observations, K:regenow proposed that a furosemide-sensitive cotransport system was activated (38) • In 1979, K:regenow provided evidence for the cotransport of all three ions during RVI in duck. red blood cells. After inhibition of the red cell anion exchanger with the disulfonic stilbene derivative SITS, measurement of 36CI- fluxes upon hyper-osmotic incubation demonstrated that a separate CI- movement acconpanied the cation influx (39). Although the precise mechanism of regulation of this transport system is not known, both the activation and inactivation of this cotransport system in these cells is volume sensitive. As cells approach their normal steady-state volume during RVI, the rate of ion flux declines to basal levels. Other cell types which utilize a Na + / K+ / 2CI- cotransport system to acconplish RVI include human fibroblasts (40), Ehrlich Ascites tumor cells (41), frog epidennis (42), the medullary thick ascending loop of Henle in the rabbit and mouse (43,44) and avian and sheep red blood cells (45-47). 7 The activation of an entirely different RVI system was first described by Gala in 1980 (48). Amphiuma (salamander) red blood cells regulate their volume after exposure to hyper-osmotic solution by activation of separate cation and anion exchangers. Upon hyper­osmotic shrinking, these cells regain their normal volume by a net intracellular gain of Na+, CI-, and ~O. It was noted that during RVI in these cells, the extracellular media became more acidic, and that Na + influx was dependent on the presence of extracellular HC03- . It was also noted that the Na + influx during RVI was completely inhibited by the diuretic amiloride, an inhibitor of Na + / H+ exchange. Direct measurement of cell membrane potential in volume static versus volume regulating cells revealed no difference. Thus, it appeared that RVI in these cells proceeded. via an electrically-silent pathway. From these results, Gala proposed a model for coupled transport of cations and anions in which a Na + / H+ exchanger was working in parallel with a CI- / HC03 - exchanger. These exchangers appeared to be separate and distinct pathways, functionally coupled through the net flux of H+. Operation of this proposed exchange system would result in net Na +, CI - , and water flux into the cell, with H+ and HC03- cycled between the intracellular and the extracellular space (see ref 33 for schematic) • The activation of parallel anion and cation exchangers similar to those described by Gala have been described for a number of different cell types. Rothstein and co-workers have characterized the RVI response in human peripheral blood lynphocytes (49,50), thymic lymphocytes (51), and Chinese Hamster Ovary cells (52). RVI in these 8 cells results from the activation of a Na+ / H+ exchanger working in parallel with a CI- / HC03 - exchanger. Interestingly, lymphocytes and rno cells exhibit RVI only if they have previously undergone swelling in hypo-osmotic solutions and subsequent RVD. When these cell are then retumed to iso-osmotic media, they rapidly shrink and the RVI mechanism is activated. Cells can then reswell to nonnal volumes by 25 minutes. There is no experimental explanation for why these cells require a round of osmotic swelling and RVD in order to activate the RVI mechanisn. RVI can also be induced by exposure of cells to phorbol esters (53), weak. acids, or ionophores which result in the acidification of the cytoplasm (49). Studies in lymphocytes have shown that the Na+ / H+ exchanger activated in response to cytoplasmic acidification is the same exchanger that results in RVI when operating in parallel with the CI- / HC0 3 - exchanger (54). Thus, RVI induced by cytoplasmic acidification can be used to detennine whether various cell types express this Na+ / H+ exchanger. Swelling as a result of exposure of cells to weak. acids and subsequent activation of the Na + / H+ exchanger can easily be assayed by electronic cell sizing (55). Activation of the Na + / H + exchanger by osmotic means or by cytoplasmic acidification exhibits arniloride sensitivity and dependence on extracellular Na + • Regulatory Volume Decrease Regulatory volume decrease (RVD) is the process by which cells recover to nonnal steady-state volume after exposure to hypo-osmotic conditions. Studies of volume regulation in invertebrate cells showed that regulation of volume was the result of changes in the 9 intracellular content of amino acids (ninhydrin-positve substances) . The coupled loss of amino acids and intracellular water resulted in volune decrease after hypo-osmotic exposure (56). Volume regulation in vertebrate cells occurs by a different mechanism than volume regulation in invertebrate cells. Fugelli observed. that red blood. cells of the European flounder had the ability to decrease their volune after osmotic swelling but the decrease in volume could not be correlated with intracellular ninhydrin-positive substances (57). In all vertebrate cell types studied to date, RVD involves the loss of K + and Cl- and osmotically obliged water resulting in cell shrinking (2). Three transport mechanisms have been characterized and are involved in the volume induced loss of K + and Cl- in various cell types. The first involves the coupled exchange of cations and anions. The second is activation of a cotransport system of cations and ions. The third is the parallel activation of cation and anion conductance pathways. In Amphiuma, the coupled exchange system which is activated in response to hypo-osmotic exposure is similar to that activated by hyper-osmotic exposure (33,34). upon hypo-osmotic incubation, Amphiuma red blood. cells lose intracellular K+ and Cl- via electroneutral exchange of K+ / H+ and Cl- / HC03- . Because H+ acts as the counter-cation for K+ during RVD, as well as the counter-cation for Na + during RVI, a feature of the coupled exchange system is an alteration in extracellular pH. RVI results in the acidification of the media due to exchange of intracellular H + for extracellular Na + . During RVD, there is alkalinization of the extracellular media, consistant with the cellular uptake of H+ in exchange for 10 intracellular K+. In order to determine the mechanism of regulation of the coupled exchange system involved in RVD, cala investigated. the role of ca2+ as a modulator of the response (58). ca2+ depletion by EGTA resulted. in a 60% inhibition of the RVD response in cells exposed. to hypo-osmotic media. To determine if increases in intracellular free ca2+ activated. K+ / H+ exchange, the calcium ionophore A23187 was added to cells in iso-osmotic, ca2+ -containing media. Under these conditions, the cells lost K+ , CI-, and water with the same kinetics as cells undergoing RVD as a result of decreased. osmolarity. cala proposed. that ca2+ acted. through a modifier site to activate the K+ / H+ exchanger. In a later study, cala and co-workers found that hypo­osmotic dilution resulted. in increases of intracellular free ca2+ measured. by calcium-sensitive dyes (59). Increasing concentrations of extracellular ca2+ resulted. in increased. K+ / H+ exchange activity during RVD. Hypo-osmotic swelling apparently increased. the affinity of the modifier site for ca2+. Interestingly, whereas ca2+ appears to stimulate K+ / H+ exchange in swollen cells, ca2+ was inhibitory for Na + / H + exchange in shrunken cells. Based. on phannacological studies, cala hypothesized. that K+ / H+ and Na+ / H+ exchange was mediated. by the same membrane rnoeity and that the ion specificity was confered. by confonnational change . The operation of a volume dependent K+ / H+ exchanger coupled with a CI- / HC03 - exchanger has so far been observed only in the Amphiuma red. blood cell. In other red blood cells which have the capacity to volume regulate, RVD is accomplished. by activation of K+ 11 and Cl- cotransport. Duck red blood cells lose K+ and Cl- and osmotically obliged water after swelling in hypo-osmotic media (1). The K+ loss is Cl- dependent and can be inhibited by addition of the loop diuretic , bumetanide. The K+ flux is passive, with the RVD response dependent on an outwardly directed K+ electrochemical gradient. The movement of K+ is absolutely dependent on the presence of Cl. upon hypo-osmotic swelling, K+ and Cl- are lost from the cell in equal amounts resulting in the electroneutral loss of salt and osmotically obliged water. Cl- then reenters the cell through the Cl­I HC03 - exchanger which returns intracellular Cl- to apparently normal concentrations and results in media alkalinization. If SITS, an anion exchange blocker, is added to these cells during hypo-osmotic incubation, RVD proceeds nonnally but media alkalinization does not occur. The third ion transport system shown to effect RVD is the simultaneous operation of K+ and Cl- conductances. This mechanism has been demonstrated in a variety of cells including frog and toad urinary bladder epithelitml (60,61), human lymphocytes (62-64), and cultured cells including Ehrlich Ascites ttmlor cells (65), HeLa cells (66), Chinese Hamster Ovary cells (52), MOCK cells (67), and most recently, human platelets (68). In 1973, Roti Roti and Rothstein reported that RVD in mouse leukemic cells (L5178Y) was characterized by a decrease of intracellular K+ as the result of an increased membrane permeability for K+ (69). In a series of papers in the early 1980s, Rothstein, Grinstein, and co-workers continued the study of volume regulation in 12 lymphocytes, the results of which constitute the most complete characterization of RVD mediated by K + conductance and Cl- conductance (64). In 1982, Grinstein, Dupre, and Rothstein found that a K+ conductance was activated in human peripheral lymphocytes during hypo­osmotic incubation (70). K+ movement through the charmel was passive and net movement in or out of the cells was dependent on the K+ electrochemical gradient. In normal media (high Na +), K + moved out of the cell (rtdown" its electrochemical gradient). If extracellular Na + was replaced by K+, K+ could not leave the cell against the gradient, and RVD did not occur. This charmel is electrogenic because loss of K + through this charmel results in a net charge difference between the inside and the outside of the cell. The K + loss through this charmel results in osmotic activity only when there is net salt loss from the cell; thus anion loss must also occur during RVD. Grinstein and co-workers demonstrated that hypo-osmotic exposure also increased the conductive penreability of Cl- in lymphocytes (62). This charmel mediated the bi -directional movement of Cl - which, like K+ conductance, was dependent on an electrochemical gradient. By ion substitution experiments, it was detennined that anions and cations move independently through the volume activated pathways (62). Upon hypo-osmotic incubation, both pathways were activated to effect the net loss of KCl and osmotically Obliged water resulting in RVD. Further investigations into the operation of these pathways led to the findings that 1) during no:rmal iso-osmotic incubation, Cl- conductance was less than K+ conductance, with K+ conductance operating at barely detectable levels, 2) upon hypo-osmotic exposure, both K+ and Cl- 13 permeabilities increased, with Cl- conductance increasing orders of magnitude over iso-osmotic levels and exceeding the activated K+ permeability, and 3) Cl- conductance is the limiting factor in net salt loss (71,72). The Cl- conductance is activated only when cell swelling reachs a threshold volume of 1.15 over the iso-osmotic value. The operation of the Cl- conductance is time dependent and after a certain period of time, is inactivated regardless of the relative volume recovery or ion change (72). In contrast, the K+ pathway appears to remain open as long as the cells are swollen. Thus, these pathways are independently activated but are functionally linked. The operation of K+ and Cl- conductance pathways can be differentiated from either cotransport or coupled exchange on the basis of pharmacologic specificity. RVD in lyrcphocytes is not inhibited by ouabain, an inhibitor of the Na + / K+ ATPase, or furosemide, an inhibitor of Na + / K + / 2Cl- cotransport. However quinine, an inhibitor of the ca2+ activated K+ conductance in red cells described by Gardos (73,74), inhibits K+ efflux and hence, RVD in these cells (70, 71). RVD is only partially inhibited by high concentrations of DIDS, the inhibitor of the Cl- / HC03- exchanger. The Cl- conductance is however sensitve to oligomycin C (71). In addition, the Cl- pathway carmot transport divalent anions or large anions like gluconate, but can transport other small ions such as N03- (61). In this sense, the Cl- conductance exhibits much less specificity than the Cl- pathway operating in the cotransport system. Little is known about the regulation of these channels other than they are activated upon increased cell volume. The operation of the 14 K + conductance is ca2+ dependent, and studies to investigate the regulatory role of ca2+ in RVD have been pursued. Depletion of extracellular ca2+ by long incUbation of cells with chelators results in the inhibition of RVD (71). ca2+ depletion in this manner appears to inhibit K+ efflux but has no effect on CI- transport. In addition, in iso-osmotic solutions, some cell shrinking can be induced by iso­osmotic solutions in the presence of extracellular ca2+ and the calcium ionophore A23187 (61). However, no changes in intracellular ca2+ have been observed either during passive swelling or RVD (75). Thus, the specific role of ca2+ in the activation and regulation of this process remains unclear. Maintenance of cell volume appears to be important in cell sw:vival, since the response can be found throughout the phylogenetic tree. Only a few cell ty:pes do not have the ability to volume regulate. One such example is the human red blood cell (76). Most cell types that have been tested (from bacteria up to various marnnalian cell types) exhibit the ability to volume regulate. The osmotic changes to which cells are exposed in vivo are generally much smaller than those used in the laboratory to induce volume regulation. There are some situations in vivo which may, however, require volume regulation. For example, epithelial cells lining the stomach and ileum are routinely exposed to ani so-osmotic solutions (77). The ability of cells to withstand these changes in fluid osmolarity requires that cells posses volume regulatory mechanisms (78) Another exanple of an osmotically challenging environment is the 15 mammalian kidney (79). The concentrating mechanism in the renal papilla results in osmotic gradients several fold above isotonicity. Interstitial fluid and tubular fluid from the corticamedullary boundary to the papilla is increasingly hyper-osmotic. In addition, changes in the state of hydration of the animal drastically affects the osmolarity of the medullary and papillary tubule and interstitial fluids. Hence, operation of volume regulatory mechanisms may be important in cells in this environrrent. In vitro studies have demonstrated that cells from the mammalian kidney exhibit volume regulatory capabilities (80-90). Although there appear to be circumstances under which volume regulation is physiologically significant, there is an alternative explanation for the conservation of transporters involved in volume regulation. These transporters also function in other ion regulatory processes. The Na + / H + exchanger which is involved. in RVI also has a major role in the regulation of intracellular pH (18~ 27). It has been shown that the transport systems responsible for volume regulation in renal epithelial cells are the same transporters which effect net transepithelial salt transport (86,87). It is not known which function is of primary importance, but it appears that ion transporters have several roles. One demonstration of this is the human peripheral blood lyrrphocyte which undergoes RVI upon hyper­osmotic incUbation only under specific conditions. These cells must first be exposed to hypo-osmotic solutions and undergo one round of RVD before the Na+ / H+ exchanger can be activated in hyper-osmotic conditions (50). If the principle function of the Na + / H + 16 transporter is cell volume regulation, it would follow that osmotic shrinking would be the only signal required to activate this exchanger. That is not the case and suggests that some other function, such as pH regulation, may be the primary physiologic function of the Na + / H+ transporter in this cell type (50). Transporters with characteristics similar to those utilized during RVD and RVI have been described in other cell processes. Increases in a quinine sensitive potassium conductance have been associated with activation and proliferation of lymphocytes (91,92) and pancreatic B-cells (93-95). Neurons (96) and muscle fibers (97) exhibit a ca2+ dependent K+ conductance with characteristics similar to those described for the K+ conductance operating during RVD in lymphocytes. These K+ conductances share characteristics with the ca2+ dependent K+ conductance described by Gardos for human red blood cells as early as 1958 (73). Chromaffin granule exocytosis is associated with changes in granule osmolarity. These changes in osmolarity result from activation of ion transporters in the granule menibrane as well as activation of ion transporters in the plasma menibrane (98-103). Finally, neutrophil chemotaxis, superoxide radical generation, and granule release are associated with alterations in intracellular H+, cation, and anion concentrations (104-106). These changes, which are thought to be involved in affecting the various responses, are the result of activation of ion transporters. 17 Regulation of Transporter Activity The mechanism by which ion transporters are regulated is not conpletely elucidated. Intracellular pH plays a role in regulation of coupled cation and anion exchangers such as the Na + / H+ and CI- / HC03 - transporters. As mentioned, H+ acts at a modifier site on the cytoplasmic side of the Na + / H+ exchanger (53). Intracellular ca2+ modulates K+ efflux during RVD in the Amphiuma system (58). Many ion transporter systems, however, do not appear to be modified by ca2+ or pH and the mechanism of regulation of these transporters is unknown. Studies on the regulation of other transporters have provided information that may be applicable to ion transporters. Glucose transport in adipocytes is controlled by the number of glucose transporters present on the surface of the cell (107-112). Kono demonstrated that glucose transporters were also present within intracellular vesicles (110). Use of radiolabelled cytochalasin-B, which binds to the glucose transporter has been used to directly quantitate the number of transporters on the surface and within vesicles before and after stimulation (110). Addition of insulin to cells induced the reversible fusion of these transporter-containing vesicles with the plasma membrane, resulting in an increased number of transporters at the cell surface and thus, increased glucose transport. Upon decay of the stimulatory signal (insulin binding), increase in transport activity could be reversed by internalization of the transporters into the intracellular vesicles. Thus, glucose transport appears to be regulated by the rapid and reversible insertion of intracellular transporters into the plasma membrane. 18 A number of other surface transport activities have been demonstrated to be regulated in a similar fashion. Al-aqwati and co­workers presented morphological data demonstrating that upon honnonal stimulation, the loss of intracellular vesicles from the cytosol of the toad bladder epithelium correlated with an increase in HC03- transport (113-117). Similar observations have been made in the parietal cell of the stomach in which H+ secretion by the gastric parietal cell is associated with the depletion of intracellular vesicles (118-121). Na+ channels in the mammalian bladder are also thought to be contained within intracellular vesicles which can fuse with the plasma membrane upon the appropriate stimulation (122-124). Thus, it has been proposed that fusion of intracellular transporter­containing vesicles with the cell surface results in increased transport activity at the cell surface (125-127). The ability to alter surface transport activities by changing the distribution of transporters between the cell surface and the intracellular space offers some attractive features as a regulatory mechanism. Because the total number of transporters in the cell is unaltered, protein synthesis is not required. TIlls mechanism of regulation provides a rapid (within seconds) response to changes in the extracellular environment and thus, might be expected to be involved in other cell stimulation/response processes as well as homeostatic processes. TIlls mechanism, referred to as translocation by Lienhard (126), has been best demonstrated for glucose transport activity. Even in this system, however, the mechanism of translocation is not completely 19 understood. Characterization of the intracellular transporter­containing vesicle is still lacking. The signal (s) coupling insulin binding with vesicle fusion is not known. The cytosolic machinery involved in fusion and retrieval has yet to be detennined. Translocation has also been observed as a mechanism of altering cellular distribution of membrane receptors. The receptors for low density lipoproteins, transferrin, and amacroglobulin-protease complexes are recycling membrane proteins (128). Upon binding of ligand by these receptors, the receptor-ligand complex is internalized into intracellular vesicles (129,130). Although these vesicles are not yet completely characterized biochemically or mOlphologically, it has been shown that they are distinct from other cytosolic organelles such as the Golgi, endoplasmic reticulum, and lysosomes (131). This membrane vesicle system has been given a variety of names, including, endosome, endocytic apparatus, and CURL (Compartment for Uncoupling of Receptor-Ligand complexes) (132). Once within the endocytic apparatus, the fate of the ligand-receptor complex is determined. For the complexes discussed here, ligand is uncoupled from the receptor and directed to the lysosome, while the unoccupied receptor is returned to the cell surface (133). Once at the surface, receptors can undergo futher rounds of binding, internalization, uncoupling, and exteriorization, referred to as recycling. Unoccupied receptors may also recycle (134,135). In addition, the majority of receptors are not on the cell surface but are contained within the endocytic apparatus. The steady-state distribution of receptors is the result of a balance between the rate of internalization and exteriorization. 20 As long as both of these rates remain constant, the number of receptors on the cell surface or within the endocytic apparatus will remain unchanged. Wiley and Kaplan (136) and Buys and co-workers (137) provided a model for the study of reversible fusion of receptor­containing vesicles with the cell surface which may aid in the characterization of other transport systems. They observed that a variety of honnones and secretagogues were capable of increasing the number of surface receptors in a rapid and reversible manner. The increases in surface receptors could be directly correlated with the depletion of receptors from intracellular vesicles (pools). These agents increased the rates of exteriorization of receptors, resulting in an increase in the number of receptors at the cell surface (136,138). The increase in surface receptor number is analogous to the increase in glucose transporters seen following insulin stimulation, and suggests that receptor translocation may provide a model for regulation of glucose transporters and ion channels. Increases in a variety of surface molecules could be the result of increasing the exteriorization rate, decreasing the internalization rate, or a combination of both. The studies within this dissertation were undertaken to examine the hypothesis that transporter/receptor recycling rates were involved in the regulation of cell responses. Based on the initial observation that exposure of rabbit alveolar macrophages to hypo-osmotic solutions resulted both in a volume regulatory response (RVD) and a reversible increase in surface receptor number, these studies focused on two points. First, was the regulation of transporters involved in RVD due 21 to changes in the nurriber of transporters at the surface? Although direct quantification of transporters was not possible, other membrane components (receptors) could be assayed and were used as "markers" for movement of membrane between the surface and the inside of the cell. It seemed. likely that if translocation was occurring, transporter redistribution would follow the membrane movement "marked" by recycling receptors. Second, what part of the recycling pathway was affected by hypo-osmotic exposure? A variety of conditions can differentially affect the internalization or exteriorization "limbs" of the recycling pathway. For example, ATP levels or lowered tercperature affects exteriorization to a much greater degree than internalization (139). IncUbation of cells with alkyl amines affects uncoupling of receptor-ligand complexes within the endocytic apparatus and causes the inhibition of exteriorization of unoccupied receptors without affecting internalization rates (140). By experimentally dissecting the recycling pathway, the mechanisms regulating membrane component movement through the cell can be elucidated. The alveolar macrophage is a valuable system for the study of membrane component recycling and was used in the following studies because 1) this cell type can be obtained in high yield and purity, 2) this cell expresses a nurriber of different receptors in assayable quantities, and 3) a great deal of infonnation about membrane dynamics in this cell type has already been collected. These studies represent the first investigations into the mechanism of RVD in alveolar macrophages. These cells effect RVD by the loss of K+ and Cl- through independent pathways. The activity of 22 these transporters is not dependent on translocation from intracellular pools, rather K+ and Cl- pathways are already present on the cell surface and are activated immediately upon hypo-osmotic swelling. Hypo-osmotic exposure of these cells also results in a transitory inhibition of internalization without affecting exteriorization. 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Effect of hypotonic media on K and Na content of proximal renal tubules. Am. ~ Physiol. 232:F42-F49. 90. Welling, P.A., M.A. Linshaw, and L.P. Sullivan. 1985. Effect of barium on cell volume regulation in rabbit proximal straight tubules. Am. ~ Physiol. 249:F20-F27. 91. Lee, S.C., D.E. Sabath, C. Deutsch, and M.B. prystowsky. 1986. Increased voltage-gated potassium conductance during interleukin 2- stimulated proliferation of a mouse helper T lymphocyte clone. J. Cell BioI. 102:1200-1208. 30 92. Decoursey, T.E., K.G. Chandy, S. Gupta, and M.D. cahalan. 1987 . Two types of potassium channels in murine T lyrrphocytes. J. Gen. Physiol. 89:379-404. 93. lebnm, P., W.J. Malaisse, and. A. Herchuely. 1982. Paradoxical activation by glucose of quinine-sensitive potassium channels in the pancreatic B-cell. Biochem. Biophys. Res. Comm. 107:350-356. 94. Pace, C.S., anda J.T. Tarvin. 1983. pH modulation of glucose-induced electrical activity in B-cells: Involvement of Na/H and HC03/CI antiporters. ~ M=mbr. BioI. 73:39-49. 95. Pace, C.S., and. J. T. Tarvin. 1982. Influence of anion transport on glucose-induced electrical activity in the B-cell. Diabetes. 31:653-655. 96. Reichardt, L.F., and RB. Kelly. 1983. A molecular description of nerve tenninal function. Ann. Rev. Biochem. 52:871- 926. 98. Burgoyne, R.D. 1984. Machanisms of secretion from adrenal chromaffin cells. Biochim. Biophys. Acta. 779:201-216. 99. Angeletti, R.H., J.A. Nolan, and S. Zaremba. 1985. Catecholamine storage vesicles: Topography and function. Trends BioI. Sci. 10:240-243. 100. Yel*en, G. 1984. Ionic penneation and blockade in Ca2+_ activated K channels of bovine chromaffin cells. J. Gen. Physiol. 84:157-186. --- 101. Pazoles, C.J. 1982. Anion and proton transport in chromaffin granules. Fed. Prac. 41:2769-2774. 102. Pollard, H.B., C.J. Pazoles, C.E. Creuty, anda o. Zinder. 1979. The chromaffin granule and possible mechanisms of exocytosis .. Int. Rev. Cytol. 58:159-197. 103. Pollard, H.B., C.J. Pazoles, C.E. Creuty, J.H. Scott, O. Zinder, and A. Hotchkiss. 1984. An osmotic mechanism for exocytosis from dissociated. chromaffin cells. J. BioI. Chern. 259:1114-1121. 104. Naccache, P.H., H.J. Showell, E.L. Becker, and R .. I. Sha'afi. 1977. Transport of sodium, potassium, and calcium across rabbit polymoIphonuclear leukocyte membranes. Effect of chemotactic factor. J. Cell BioI. 73:428-444. 105. Showell, H.J., P.H. Naccach.e,+R.I.+Sha'afi,+~d E.L. Becker. 1977. The effects of extracellular K , Na , and ca on lysosomal enzyme secretion from polymoIphonuclear leukocytes. J. Immunol. 119:804-811. 31 106. Naccache, P.H., H.J. Showell, E.L. Becker, and R.I. Sha'afi. 1977 • Changes in ionic movements across rabbit polymorphonuclear leukocyte rrenbranes during lysosomal rrenbrane release. Possible ionic basis for lysosomal enzyme release. ~ Cell BioI. 75:635-649. 107. Cushman, S.W., and L.J. Wardzala. 1980. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma rrenbrane. ~ BioI. Chern. 255:4758-4762. 108. Simpson, I.A., and S.W. Cushman. 1985. Regulation of glucose transporter and honnone receptor cycling by insulin in the rat adipose cell. Curr. ~ :M=mbr. Transp. 24:459-503. 109. Kono, T., F.W. Robinson, T.L. Blevins, and o. Ezaki. 1982. Evidence that translocation of the glucose transport activity is the major mechanism of insulin action on glucose transport in fat cells. J. BioI. Chern. 257:10942-10947. 110. Kono, T., F.W. Robinson, T.L. Blevins, and O. Ezaki. 1982. Evidence that translocation of the glucose transport activity is the major mechanism of insulin on glucose transport in fat cells. J. BioI. Chern. 257:10942-10947. -- 111. Ezaki, 0., M. Kasuga, Y. Akanauma, K. Takata, H. Hirano, Y. Fujita-Yamaguchi, and M. Kasahara. 1986. Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents. ~ BioI. Chern. 261:3295-3305. 112. Toyoda, N., F.W. Robinson, M.M. Smith, J.E. Flanagan, and T. Kono. 1986. Apparent translocation of glucose transport activity in rat' epididymal adipocytes by insulin-like effects of high pH or hyperosrnolarity. ~ BioI. Chern. 261:2117-2122. 113. Al-Awqati, Q. 1978. H+ transport in urinary epithelia. Am. ~Physiol. 4:F77-F88. 114. Gluck, S., C. cannon, and Q. Al-Awqati. 1982. Exocytosis regu*ates urinary acidification in turtle bladder by rapid insertion of H punps into the luminal rrenbrane. Proc. Natl. Acad. Sci. 79:4327-4331. -- 115. Masur, S.K., E. Holtzman, and R. Walter. 1972. Honnone­stimulated exocytosis in the toad urinary bladder. Some possible implication for turnover of surface rrenbranes. J. Cell BioI. 52:211- 219. ---- 116. Masur, S.K., J. Gruenberg, and K.E. Howell. 1987. Endosomal compartment of toad bladder epithelium. Am. ~ Physiol. 252:C115- C120 . . 117. Masur, S.K., E. Holtzman, and R. Walter. 1972. Honnone- 32 stimulated exocytosis in the toad urinaray bladder. Some possible implication for turnover of surface membranes. J. Cell BioI. 52:211- 219. --- 118. Forte, T .M., T .E. Machen, and J .G. Forte. 1977. Ultrastructural changes in oxyntic cells associated with secretory function: A membrane-recycling hypothesis. Gastroenterology 73:941- 955. 119. Wolosin, J.M.+and ¥".G. Forte. 1981. Changes in the membrane environment of the (K + H ) -ATPase following stimulation of the gastric oxyntic cell. J. BioI. Chern. 256:3149-3152. 120. Wolos in , J.M., and J.G. Forte. 1981. Isolation of the secreting oxyntic cell apical membrane - identification of an electroneutral KCI syrrport. In: Membrane Biophysics: Structure and Function in Epithelia. New York, NY: Alan R. Liss, Inc. 189-204. 121. Forte, J.G., J.A. Black, T.M. Forte, T.E. Machen, and J.M. wolosin. 1981. Ultrastructural changes related to functional activity in gastric oxyntic cells. Am ~ Physiol. 241:G349-G358. 122. Lewis, S .A., and J .L.C. deM:>ura. 1982. Incox:poration of cytoplasmic vesicles into apical membrane of manrnalian urinary bladder epithelium. Nature. 297:685-690. 123. Minsky, B.D., and F .J. Chlapowski. 1978. Mo:r:phometric analysis of the translocation of lumenal membrane between cytoplasm and cell surface of transitional epithelial cells during the expansion-contraction cycles of marrrnalian urinary bladder. ~ Cell BioI. 77:685-697. 124. Lewis, S.A., and J.L.C. de Moura. 1982. Incox:poration of cytoplasmic vesicles into apical membrane of marrrnalian urinary bladder epithelium. Nature. 297: 685-687 . 125. Stanton, B.A. 1984. Regulation of ion transport in epithelia: Role of membrane recruitment from cytoplasmic vesicles. Lab. Invert. 51:255-257. 126. Lienhard, G.E. 1983. Regulation of cellular membrane transport by the exocytic insertion and endocytic insertion and endocytic retrieval of transporters. Trends BioI. Sci. 8: 125-127 . 127. Stanton, B.A. 1984. Regulation of ion transport in epithelia: Role of membrane recruitment from cytoplasmic vesicles. Lab. Invert. 51 :255-257 . 128. Kaplan, J. 1981. Polypeptide-binding membrane receptors: Analysis and classification. Science. 212:14-20. 129. :Mellman, I.S., H. Plutner, and P. Ukkonen. 1984. 33 Internalization and rapid recycling of macrophage Fc receptors tagged with monovalent antireceptor antibody: Possible role of a prelysosomal conpartrnent. ~ Cell BioI. 98:1163-1169. 130. :M;llman, I. S. 1982. Endocytosis, membrane recycling and Fc receptor function. In: M:!nbrane Recycling. D. Evered and G.M. Collins, editors. Pitman Books Ltd. London. 35-58. 131. Mlller, W.A., R.M. Steinman, and Z.A. Cohn. 1983. Membrane proteins of the vacuolar system. III. Further studies on the conposition and recycling of endocytic vacuole membrane in cultured macrophages . ~ Cell BioI. 96: 29-36. 132. Helenius, A., I.S. :M;llman, D. Wall, and A. Hubbard. 1983. Endosorres. Trends BioI. Sci. 8: 245-250. 133. Brown, M. S ., R. G. W. Anderson, and J. L. Goldstein. 1983. Recycling receptors: the round trip itinerary of migrant membrane proteins. Cell. 32:663-667. 134. Watts, C. 1985. Rapid endocytosis of the transferrin receptor in the absence of bound transferrin. J. Cell BioI. 100: 633- 637. --- 135. Ajioka, R.S., and J. Kaplan. 1986. Intracellular pools of transferring receptors result from constitutive internalization of unoccupied receptors. Proc. Natl. Acad. Sci. 83: 6445-6449. 136. Wiley, H.S., and J. Kaplan. 1984. Epidermal growth factor rapidly induces a redistribution of transferrin receptor pools in human fibroblasts. Proc. Natl. Acad. Sci. 81:7456-7460. 137. Buys, S.S., E.A. Keogh, and J. Kaplan. 1984. Fusion of intracellular membrane pools with cell surfaces of macrophages stimulated by phorbol esters and calcium ionophores. Cell. 38:569- 576. 138. McVey-Ward, D., and J. Kaplan. 1986. Mitogenic agents induce redistribution of transferrin receptors from internal pools to the cell surface. Biochem. J. 238:721-728. 139. Clarke, B.L., and P .H. Weigel. 1985. Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis. J. BioI. Chern. 260:128-133. 140. Kaplan, J., and E.A. Keogh. 1981. Analysis of the effect of amine on inhibition of receptor-mediated and fluid phase pinocytosis in rabbit alveolar macrophages. Cell. 24: 925-932 . CllAPTER II EFFECT OF HYPQ-OSMJTIC INO.JBATION ON MEMBRANE RECYCLING Abstract Incubation of alveolar macrophages in hypo-osmotic media causes a time and terrperature dependent increase in the number of surface receptors for three different ligands. Exposure of cells to solutions of 210 mOsM or less at 370C but not at OOC, resulted in an increase in the number of surface receptors for diferric transferrin, aMacroglobulin-protease complex, and mannose-terrninated glycoproteins. Upon media dilution at 37oC, surface receptor number reached a maximum within 5 minutes and returned to near-nomal values by 30 minutes. A series of independent experiments demonstrated that the increase in surface receptor number was the result of a decrease in the rate of internalization of receptors, either occupied or unoccupied. The rate of receptor exteriorization was unaltered by hypo-osmotic incubation of cells. The rate of fluid-phase pinocytosis was also inhibited upon incubation in hypo-osmotic solution. In experiments in which both receptor-mediated endocytosis and fluid phase pinocytosis were measured on the same samples, inhibition of both processes occurred with the same kinetics and to a similar extent. The rate of receptor mediated endocytosis recovered to nomal rates after 60 minutes in 35 hypo-osmotic solutions, Whereas, the rate of fluid phase pinocytosis did not recover to the same extent. Introduction It is now well-accepted that plasma membrane receptors for a number of different ligands are recycled after ligand internalization (1,2). Surface receptors bind ligand and receptor-ligand complexes are internalized into intracellular vesicles, tenned. the endocytic apparatus (3). Within the endocytic apparatus ligand and receptor are uncoupled, and ligand is directed to the lysosome for processing. Unoccupied receptors are returned, or exteriorized, to the plasma membrane, and undergo additional rounds of internalization and exteriorization, the complete process referred to as recycling (4,5). Constitutively recycling, that is, in the absence of ligand, also occurs (6). The rate of recycling of unoccupied receptors is subject to modulation by a variety of honnones, or agents Which mimic "second messengers" such as phorbol esters and calcium ionophores (7-10). Little is known, however, about the cellular components or mechanics of recycling through the endocytic apparatus. One well­established approach by which mechanisms underlying cellular processes have been examined is to define metabolic and/or ionic requirements of the processes. Recent studies indicate that different steps in the recycling pathway exhibit differential requirements for ions and ATP. For exa:rrple, ATP depletion or lowered tenperatures affects the exteriorization "limb" of recycling to a greater extent than the internalization "limb" (11). Alterations in extracellular ion concentrations also differentially affects internalization processes. 36 Exposure of neutrophils to hyper-osmotic solutions results in the inhibition of receptor-mediated endocytosis but not fluid phase pinocytosis (12). These Observations demonstrate that processes involving membrane movement are not obligatorily linked. These results suggest that various experimental conditions can be used to study specific steps in the recycling pathway. This study describes the effect of hypo-osmotic fluids on macrophage endocytic activities. Exposure of cells to hypo-osmotic solutions gives rise to an increase in the number of at least three different surface receptors. This increase is the result of an inhibition of internalization of unoccupied receptors which appears to be a consequence of a "general" inhibition of internalization. Yet while the internalization "limb" of the recycling pathway is inhibited, the exteriorization of receptors to the surface is unaffected. Thus hypo-osmotic solutions serves as an experimental "tool" to specifically separate these two processes. M'a.terials and M=thods Cells Rabbit alveolar macrophages were obtained by bronchial lavage as described by Myrvik (13). Cells were maintained in HBSS containing 5mM glucose at 40C until the start of an experiment. Cells were incubated at 370C for 60 minutes before the start of all experiments in which ligand binding would be assayed (14). Ligand Preparation and Measurement of Binding Activity 37 For the experiments described in this manuscript we employed the following radioligandsi alpha-macroglobulin-125-I -trypsin complex (aM- 125I _T), 125-I-diferric transferrin (125I - Tf (Fe)2)' and 125-I-mannose tenninated bovine serum albumin (125 - I -M'AN-BSA). Procedures for the preparation and measurement of radioligand binding to cells have been described previously (15-17). To distinguish between surface bound and internalized aM-125I_T, a modification of the EDTA stripping procedure described elsewhere was used (18). Cells were washed three times with ice-cold HBSS to remove unbound ligand and 2.0 rnls of the strip solution (10 rrM Na 4 EDTA in ca2+, M;;2+ free HBSS at pH 6.6) was added. Cells were then allowed to incubate at OOC for 3 minutes, pelleted, and the supernatant saved. This procedure was repeated two more times for a total of three EDTA washes. Radioactivity in the washes was detennined. The cell pellet was solubilized with 1.0% SDS and radioactivity detemined. Radioactivity removed by "stripping" represents surface bound ligand while the radioactivity in the cell pellet represents internalized ligand. Stripping by this method routinely resulted in removal of 85% to 95% of the surface bound ligand. Solutions The osmolarities of media and buffers were detemined by freezing point depression using an Osmette A automatic osmometer. The osmolarities of iso-osmotic solutions ranged from 303 to 325 mOSM. Hypo-osmotic (50%) solutions were prepared by diluting iso-osmotic 38 solutions with equal amounts of distilled ~O. The final osmolarity (ranging from 148 n()sM to 158 n()sM) of hypo-osmotic (50%) solutions was verified on the Osmette A. For preparation of solutions of varying hypo-osmolarity, appropriate volumes of water were added to iso-osmotic sanples to attain the final osmolarity desired. In dilution dependent experiments, the osmolarities ranged from 310 mOsM to 100 n()sM. Trypsin Treatment of Cells Bovine pancreas trypsin (0.01 %) from Worthington Inc. was prepared in MEM-H/H. Soybean trypsin inhibitor (Sigma) was dissolved in MEM-H/H to result in a 0.1% solution. Trypsin was added to cells at oOe for 30 minutes. The cells were washed twice with ice-cold HBSS and trypsin inhibitor added for 10 minutes. M3asurement of Horseradish Peroxidase Uptake and Loss of Pre-bound Surface Ligand Cells (2.0 x 106) were allowed to adhere to 35rrro dishes for 60 minutes at 37oe. Plates were transferred to oOe and washed once with cold HBSS and 1.0 ml of cold media containing bovine serum albumin (BSA) (final concentration 8.0 mg/ml) was added to each plate. Cells were incubated with ligand at a final concentration of 1.0 x 10-9 M for 90 minutes. At the end of the incubation, cells were washed 4 times with 1.5 mls of cold HBSS. Cells were transferred to 37°C and 2.0 mls of warm iso-osmotic or hypo-osmotic media containing horseradish peroxidase (HRP), mannan, and BSA (at a final concentration of 4.0 mg/ml HRP, 4.0 mg/ml rnannan, and 2.0 mg/ml BSA) was added to each plate. Cells were incubated for specified times, 39 transferred. back to OOC, and washed. 12 times with 2.0 mls cold HBSS. Surface bound ligand was distinguished. from internalized. ligand by the EDTA wash technique described above. Cell rnonolayers were solubilized in 0.1% Triton X-100 and radioactivity and HRP activity determined. (19) • Additional Procedures Protein detenninations were performed. using as described by Lowry et al (20) using Bovine Serum Albumin fraction V (Sigma. Chemical Co. St.Louis Me) as a standard. Results Hypo-osmotic Solutions Induce an Increase in Surface Receptor Number Exposure of rabbit alveolar macrophages to hypo-osmotic solutions (150 rnOsM) resulted. in a time and temperature dependent change in surface ligand binding activity. Incubation of cells in hypo-osmotic solutions at 370C resulted. in a rapid increase in the ability of cells to bind aM-125 - I -T (Figure 2-1). Surface binding increased. approximately twofold over control levels, with the maximum increase occurring 1 to 5 minutes after dilution of the incubation media. Binding activity returned. to near normal values within 30-40 minutes of continuous incubation in hypo-osmotic media. No increase in ligand binding activity was observed on cells exposed. to hypo-osmotic fluids at OOC. This reversible increase in binding activity was not unique to aM-125 - I -T and similar changes were observed for 125_ I -MAN-BSA and 125_I _Tf (Fe) (Table 2-1). In each instance, the kinetics and the 2 relative magnitude of the increase, as well as the time course of 40 0.30 .C=) 0.25 "C .C- m u ....- .-.. 0.20 u (1) Q. en 0.15 0.10 -t--,--r--........ --.----,..--.....---.--..--....-........ -......-.....--....I o 5 10 15 20 25 30 Time (minutes) Figure 2-1. Effect of hypo-osmotic incubation on surface binding for aM-125-I-T. Celbs (2.0 x 106 / 1.5 mls) were incubated in MEM-H/H for 60 minutes at 37 c. An :9Hal vol~ of water (0) or media (.) was added to each tube at 37 C or 0 C. At the times indicated cells were pelleted., washed twice with ice-cold HBSS and resuspended. in cold ~a containing BSA for 90 minutes in the presence of 5.0 x 10 M aM-125- I-T. The data are plotted as amount of specifically bound ligand (fmoles/ug protein). Error bars represent the standard error of the mean. 41 Table 2-1 Effect of ~~50smotic IncUba!~gn on Surface Binding of I-~-BSA and I-Tf(Fe) 2 Time 125I - Tf (Fe) * 125I_~_BSA* (minutes) (fmoles/ug protein) (fmoles/ug protein) Control 0.024 (+) 0.003 0.045 (+) 0.006 1 0.043 0.004 0.082 0.001 3 0.062 0.006 0.115 0.010 5 0.067 0.009 0.120 0.010 7 NO NO 0.117 0.006 10 0.055 0.006 0.084 0.004 15 0.048 0.008 0.057 0.004 30 0.035 0.006 0.056 0.002 * Specific binding at OOC was det§~ed as desI2~ in the text Ligand concentration was 1.0 X 10 M for both I -Tf (Fe) 2 and LSI - MAN-BSA. Error represents the standard error of the mean. (NO, not determined) ECCLES Tn SCIEt~CES LIBRARY 42 recovery were similar. In addition, similar results were seen whether cells were in suspension or plated. The increase in binding activity could result from either an increase in ligand-receptor affinity or an increase in receptor number. To distinguish between these possibilities, the following e.x}?eriment was :Performed. Cells were incubated in either iso-osmotic or hypo-osmotic media at 370e for 5 minutes, placed in iso-osmotic media at oOe, and surface binding of aM-125I_T determined as a function of ligand concentration. Cells exposed to hypo-osmotic fluids at 370e bound more aM-125_I_T at saturating ligand concentrations than cells maintained in iso-osmotic media (Figure 2- 2a). The concentration of ligand which gave rise to half-maximal saturation of binding was 2.0 x 10-9 M in iso-osmotic solutions and 2.2 x 10-9 M in hypo-osmotic solutions. These results demonstrate that the increase in surface binding that occurred as a result of hypo-osmotic incubation was due to an increase in receptor number rather than an increase in ligand-receptor affinity. Similar results were Obtained when 125I - Tf (Fe)2 was employed as the ligand. Unlike aM-125I_T, the binding of 125I - Tf (Fe)2 to its receptor is readily reversible, allowing binding data to be analyzed by the method of Scatchard (20). Cells were incubated in hypo­osmotic or iso-osmotic media for 5 minutes at 37oe, placed at aOe, and binding measured (Figure 2-2b). In cells incubated in hypo-osmotic media the Kd was 2.3 x 10 -8 M, and 2.8 x 10-8 M for cells maintained in iso-osmotic media. Thus, for at least two different ligands increased binding resulted from an increase in surface receptor number 43 Figure 2-2. Concentration ~dence of ligand binding. Cells were incubated at 37 C for 60 minutes at which point an equal volume of water (6.0) or media ( .. )-) was added to each sample and the incubation continued for 5 minutes. The cells were then incubated. at OOC with different concentrations of (A) aM-125-I -T (A •• ) or (B) 125- I-Tf1~5) 2 (0.-) and specific binding determined. The binding data for I-Tf(Fe)2 are in the for.m of a Scatchard plot. 0.8 0.6 Q c =a c iuii 0.4 ;;::: ·u G) a. tn 0.2 0.0 0 2.5 ...- .. 2.0 ,0.. . ....... G) 1.5 e u. ........ ~ 1.0 ::::I 0 £D 0.5 0.0 /I-J ;~!-J //t If 5 10 15 Concentration (10·') .~ • 0.1 0.2 0.3 Bound 0.4 44 A I ~I 20 B 0.5 0.6 rather than an increase in the ligand/receptor affinity. Increase in Surface Receptor Number is Dilution Dependent 45 To detennine the relationship between increased receptor number and media osmolarity, the following ex,periments were :performed. Cells were incubated in iso-osmotic media at 370C and varying amounts of water was added in order to give resultant osmolarities ranging from 310 n()sM to 100 n()sM. Cells were exposed to these solutions for 5 minutes, the time of maximum receptor increase upon hypo-osmotic incubation. After incubation at 37oC, cells were washed and incubated with aM-125I_T at OOC and specific binding detennined. Alterations in media osmolarity from 310 n()sM to 235 mOsM did not result in a significant change in surface receptor number (Figure 2-3). From approximately 210 mOsM down to 110 mOsM, the increase in surface binding was linear and approximately proportional to the media dilution. In this ex,periment, a 2.0-fold increase in surface binding was observed. at 150 mOsM and an approximate 3.1-fold increase in binding occurred at 118 mOsM. ~a dilutions to osmolarities below 110 mOsM resulted in cell lysis. Increases in surface receptor number during hypo-osmotic incubation at 370C appeared to be inversely correlated to media osmolarity. Although incubation in media of different osmolarities affected the magnitude of the surface receptor increase, the time course of the response was similar at different osmolarities (Figure 2-4) . At 170 mOsM, a surface receptor number increase of 1.75 fold occurred at 3 minutes. The maximum increase in surface binding at 120 mOsM occurred 46 0.50......--------------------, 0.40 C) _5 "-c- 0.30 al (.) ;: -0 0.20 (1) c. en 0.10 0.00 -i---.------..---..--.,...-.-......---.-----r----I o 100 200 300 400 Media Osmolarity (mOsM) Figure 2-3. Effect of different media osmolarities on surface receptor number. Cells were incubated in iso-osmotic media at 370C for 60 minutes. Specific volumes of water were added to tubes resulting in the final media osmolarity indicated on the abscissa. The cells were incubated for 5 minutes at which time surface binding for aM-125-I-T was determined as described in Figure 2-1. 47 0.30 -r-----------------........ 0.25 C) .5 "C 0.20 .5 m (.) :;:: ·u 0.15 CD Q. U) 0.10 0.05 ........ -.....-...--....-...... -...---.... ........ ...--.....----....-...-........ -..----1 o 5 10 15 20 25 30 Time (minutes) Figure 2-4. Time course of surface binding at different media osmolarities. Cells were incubated. in MEM-H/H at 170 mOsM (C) or 120 mOsM (A) for the times indicated. and specific binding detennined. as in Figure 2-1. Error bars represent the standard error of the mean. 48 between 3 and 5 minutes and was 2.7-fold over control values. Recovery of surface receptor number at both dilutions occurred by 30 ,minutes. Increased Surface Receptor Number Results from an Inhibition of InternaIIzation The receptors assayed in this study are typical of a class of receptors primarily involved in ligand internalization (Class II receptors; Kaplan 1981) (21). One hallmark of these receptors is that after internalization of the ligand/receptor complex, ligand is released from the receptor within the endocytic apparatus, and the unoccupied receptor is returned (exteriorized) to the cell surface. The complete process of internalization and exteriorization of the receptor is referred to as recycling. In alveolar macrophages, the majority of receptors for ~125I_T, 125I - Tf (Fe) 2' and 125I -MAN- BSA are found not on the cell surface but within the endocytic apparatus. Under normal iso-osmotic conditions approximately 80% of these receptors are found inside the cell and only 20% are found on the cell surface. Studies in this laboratory demonstrated that the presence of intracellular "pools" of receptors is the result of constitutive recycling of unoccupied receptors (6). Rapid changes in surface receptor number are due to changes in the distribution of cellular receptors between the endocytic apparatus and the cell surface. These redistributions could occur either by an acceleration of receptor movement fran internal "pools" to the cell surface, by a decrease in the rate of internalization of receptors, or by changing both internalization and exteriorization rates. The following experiments 49 were perfonned to distinguish between these possibilities. The initial experiments were designed. to test the affects of hypo-osmotic incubation on receptor internalization. Cells were incubated. at oOe in the presence of aM-125_I_T. The cells were then washed. and incubated. at 370e in either hypo-osmotic or iso-osmotic media. At specified. times, cells were placed. at oOe and the amount of surface and internalized. ligand detennined. by the EDTA wash procedure. The amount of radioactivity in the supernatant reflectes surface bound ligand while the amount of radioactivity associated. with the cell pellet after the EDTA wash represents internalized. ligand (18). This type of experiment was possible because the ligand used., aM-125I_T, binds essentially in an irreversible manner to its receptor. Therefore, the loss of ligand from the cell surface is due to internalization, not dissociation. There was a decrease in the ability of cells to internalize prebound ligand within 1 minute after dilution (Figure 2-5a). The increase in surface receptor number in the absence of ligand was measured. in a parallel sample (Figure 2-5b) • A 2.2-fold increase in binding was observed at 3 minutes after media dilution. Cells in hypo-osmotic media internalized. ligand at half the rate of cells in iso-osmotic media (Figure 2-6). These results suggest that the increase in surface receptor number during hypo-osmotic incubation is due to an inhibition of receptor internalization. These results also suggest that internalization of both occupied. and unoccupied. receptors is affected. to the same extent by hypo-osmotic incubation. Another method used. to verify that hypo-osmotic incubation 50 Figure 2-5. Loss of surface bound ligand during hypo-osmotic incUbation. 125 0 (A) Cells were incUbated with aM- - I -T at 0 C for 90 minutes at which point cells were washed to remove unbound ligand and placed in either iso-osmotic (&) or hypo-osmotic (A) media at 37oC. At specified times, cells were washed with ice-cold HBSS followed by 3 washes with ice-cold EDTA strip solution. Specific surface binding (fmoles/ug protein) was dete:r:mined from EDTA sensitive radioactivity and adjusted '00r cell protein. (B) Cells were incUbated for 60 minutes at 37 C in iso-osmotiS media, washed once with ice-cold HBSS, and allowed to incUbated at 0 C for an 15 minutes. Cells were then placed in either iso-osmotic (.) or hypo-osmotic (C) media at 370C for the times indicated. The cells were washed. with ice-cold HBSS and specific binding detennined as described in Figure 2-1. 51 0.30 0.25 cen 0.20 :e; c m (.) 0.15 :;: Oil eaCnD. 0.10 0.05 0.00 0 5 10 15 20 25 30 Time (minutes) 0.50 0.40 en c :e; c 0.30 m (.) :;: til 0.20 CD ean. 0.10 o 5 10 15 20 25 30 Time (minutes) 52 ~c -------c -------c .~ en en o • ....J. C (1) Co) ~ (1) a. 0.0 1.0 2.0 3.0 4.0 5.0 Time (minutes) Figure 2-6. Rate of loss of surf8ce bound ligand during iso-osmotic and hypo-osmotic incUbation at 37 c. Specific surface binding determined in Figure 2-5a was expressed as percent of control surface binding at time zero. The (.) represent values from iso-osmotic samples and the (0) represent hypo-osmotic samples. 53 inhibited receptor internalization was measurement of the specific receptor internalization rate, or endocytic rate constant (Ke ). As described by Wiley et al., the Ke is the probability of a receptor being internalized in one minute and is a measurement of the internalization rate at steady-state conditions (23,24). To measure the Ke , cells are incubated in the presence of ligand, and at specified times the amount of surface bound and internalized ligand is detennined. The amount of ligand internalized is plotted versus the integral of surface bound ligand (25) _ The slope of the line yields the Ke- The "In/Sur" method of analysis, a previous approach for detennining the Ke carmot be used in these experiments because during hypo-osmotic incubation surface binding is not constant (24). Cells were incubated at 370e for 60 minutes and ~25I_T in water or media was added to yield a final osmolarity of either 300 rrOsM or 150 rrOsM. At specified times cells were placed at oOe, washed, and surface bound ligand distinguished. from internalized ligand by EDTA washing. Inmediately upon hypo-osmotic dilution the amount of surface bound ligand on cells incubated in hypo-osmotic media increased 2.0- fold over surface bound ligand on cells in iso-osmotic media, similar to the increase of surface receptor number measured by all previous methods. Analysis of ke yielded a value of 0.76 min for cells incubated in hypo-osmotic media and 1. 70 min-I for cells in iso­osmotic media, a difference of 2. O-fold (Figure 2-7). These results further suggest that the increase in surface binding can be accounted for by the decreased rate of internalization upon hypo-osmotic incubation. 800 ........ U) .9o:! 600 ..E.. ........ "'C CI) .~ (ij .c. oS -c 400 200 54 o 200 400 600 800 Integral Surface (fmoles x min) Figure 2-7. Effect of hypo-osmo!~g incubation on the specific internalization rate (K ) of aM- I -T . Cells were incubated !n iso-osmotic media at 370C for 60 minute§8at which point an equal volume of water or media containing 1.0 x 10 M aM-125-I -T was added. to cells for the indicated times. Cells were placed at OoC and washed with EDTA stripping solution as described in Figure 2-5. Data were plotted as internalized ligand (finoles/cell) versus the integral of specific surface binding (finoles/cell) for cells incubated in iso-osmotic (.) media or hypo-osmotic (0). 55 The previous experiments, provide evidence for decreased rates of internalization resulting from exposure of cells to hypo-osmotic solutions. To determine whether hypo-osmotic incubation had any effect on the rate of receptor exteriorization, the value was determined in cells incubated in iso-osmotic and hypo-osmotic media. In order to increase the sensitivity of the measurement of receptor appearance, cells were treated with proteases to inactivate receptors already present at the surface (i.e., to decrease background). The increase in surface binding under these conditions would directly reflect the movement of receptors from internal "pools" to the cell surface. (The rate of receptor reappearance is orders of magnitude faster than can be accounted for by de novo synthesis.) One potential problem with this approach is that treatment of cells with proteolytic enzymes could affect the hypo-osmotic response. To test this possibility, the following experiment was performed. While receptors for 125I -MAN-BSA are trypsin sensitive, receptors for aMr125I_T are not. Cells were exposed to trypsin and assayed for their ability to respond to hypo-osmotic media by measuring changes in surface binding for ~25I_T. Cells were incubated with 0.01% trypsin at OOC for 30 minutes, followed by a 10 minute incubation in 0.1% Soybean trypsin inhibitor to inactivate the protease. After washing the samples several times at aOe, cells were placed at 2SoC in either iso-osmotic or hypo-osmotic media and surface binding of aMr125I_T was determined. The lower temperature was chosen in order to slow the response and provide better comparison. Exposure of cells to trypsin did not significantly reduce surface binding for aMr125I_T (less than 15% loss in binding activity after protease treatment) (Figure 2-8a) . Incubation of trypsin treated cells resulted in changes in surface receptor number to the same magnitude as cells which had not been 56 trypsin treated. As expected, the changes in surface receptor number were slower at this terrperature, with the maximum receptor increase occurring between 10 and 15 minutes after media dilution. In a parallel experiment, cells were treated as above but surface binding for 1251 -MAN-BSA was assayed. Exposure of cells to trypsin at OOC dramatically reduced surface binding of 125I -MAN-BSA to only 15% of untreated samples, confirming the protease sensitivity of this receptor. The rate of appearance of receptors was then determined in cells incubated at 2SoC (Figure 2-Sb). Over the first 4 minutes, the rate of 125I -MAN-BSA receptor appearance was similar for cells in iso­osmotic and hypo-osmotic media. Although the initial rate of receptor appearance was similar for 125I -MAN- BSA, surface binding in hypo­osmotic media increased 2.0-fold over control levels by 10 minutes. This increase was probably due to the inhibition of internalization as a result of hypo-osmot.l.C exposure. Receptors for aM-125 I-T and 1251 - MAN-BSA in hypo-osmotically treated cells recovered to near control values by 30 minutes after media dilution. These results demonstrate that hypo-osmotic incubation of cells does not affect the rate of receptor exteriorization. Taken together, these data demonstrate that the hypo-osmotically induced increase in surface receptor number is the result of a decreased rate of receptor internalization while the rate of receptor exteriorization remains constant. 57 Figure 2-8. Rate of recovery of surface binding activity in iso­osmotic or hypo-osmotic medig after cell surface trypsinization. Cells were incubated at 37 C for 60 minutes and then shifted to OoC in the presence of 0.01% trypsin for 30 minutes. The cells were washed, and Ft:en incubated with 0.1% Soybean Trypsin Inhibitor for 10 minutes at 0 C. The cells we'be resuspended in iso-osmotic (. ) or h~-osmotic (0) media at 28 C and at the indicated times, placed at o C and spec!gic binmg was determined. (~8 Cells were incubated with 5.0 x 12 M aMr I-T or (B) 1.0 x 10 M 125-I-MAN-BSA for 90 minutes at 0 C. 58 A 1.00 tn c 0.75 :a c m (J ;;:: 0.50 '(3 Q) Q. en 0.25 0.00 -+-----,~-_.,_--....._-___r--_r_--.._- o 5 10 15 20 25 30 Time (minutes) B 1.50 tn C :a ,5 1.00 al (J ;;:: '(3 Q) Q. en 0.50 0.00 -O----.~-...... --.....--___r--_r_--..._ ..... o 5 10 15 20 25 30 Time (minutes) Decline of Surface Receptor Number Reflects a Recovery of Internalization Rates The exposure of cells to hypo-osmotic fluids results in a 59 reversible increase in the number of surface receptors which with continued incubation of cells declined to near nonnal values. The decrease in surface binding activity could be due to either a decrease in the number of surface receptors, or a decreased ligand/receptor affinity. Scatchard analysis revealed that the Kd for 125-I -Tf (Fe) 2 binding to cells incubated in hypo-osmotic media (Kd = 9.4 x 10-9 M) for 30 minutes was similar to that detennined for cells incubated in iso-osmotic media (Kd = 8.7 x 10-9 M) for 30 minutes. The decline of surface receptor number could result from either an irreversible loss of receptors from the cells, or internalization of receptors into the endocytic apparatus. The following experiments were perfonned to distinguish between these hypotheses. If the decrease in surface receptor number was due to a recovery of internalization a subsequent exposure to hypo-osmotic media would result in a surface receptor increase similar to the original response. Cells were incubated in hypo-osmotic solutions at 370C for 30 minutes, at which time, surface receptor number had returned to normal values. The cells were then allowed to re-equilibrate in iso-osmotic solutions and after 30 minutes, again subjected to hypo­osmotic incubation at 370C. Figure 2-9a represents the change in surface receptor number induced upon hypo-osmotic incubation at 370C and Figure 2-9b illustrates surface receptor number alterations upon a second hypo-osmotic exposure of these cells. The magnitude and the kinetics of the second response were similar to the first. 60 Figure 2-9. Effect of repeated exposure of cells to hypo-osmotic media on surface receptor number. (A) Cells were incubated for 60 minutes at 370e in iso-osmotic media at which point an equal volume of water was added and cells allowed to incubate for specified times. The cells were placed at aOe and surface binding of aM-125-I -T was detennined as in Figure 2-1. (B) Cells were incubated in hypo-osmotic media for 30 minutes after which time an aliquot (110 uls / 2.0 mls) of lOX PBS was added to restore iso-osmolarit~ (300 mOsM). Cells were incubated for an additional 30 minutes at 37 e at which point an equal volume of water was added for the indicated times. Cells were washed. at oOe, and surface binding detennined . 61 0.40 c0 ) 0.30 =s c til ;u: 0.20 'u CD c. en 0.10 0.00 ....-...... -..--....... -..-................... -..--.---.-...................... o 5 10 15 20 25 30 Time (minutes) 0.40 0c ) 0.30 =s c til ;u: 0.20 °u CD c. en 0.10 0.00 -1--.._....-....... -..-...-............. -..--....... ......,.-......-...,... ...... o 5 10 15 20 25 30 Time (minutes) 62 In order to detennine whether receptors were irreversibly lost from cells, steady-state binding of 1251 -Tf (Fe) 2 was detennined. Because both receptor and ligand are recycled, 1251 -Tf (Fe) 2 serves as a label for all available receptors in the recycling pathway at steady-state conditions. Thus, if there was a net irreversible loss of receptors from the cells, steady-state binding of 125I - Tf (Fe)2 would be reduced. Steady-state binding of transferrin was similar for cells incubated either in iso-osmotic media or hypo-osmotic media. Cells were incubated in iso-osmotic or hypo-osmotic media for 30 minutes, washed at OOC, and returned to iso-osmotic media at 370C in the presence of ligand. After 60 minutes, total cell associated ligand was detennined. Cells in iso-osmotic media bound 0.731 + 0.002 fmoles/ug cell protein and cells in hypo-osmotic media bound 0.687 + 0.006 fmoles/ug cell protein. This 6.0% difference was within background values of binding and suggested that there is not a significant decrease in cellular receptors as a. result of hypo-osmotic incubation. Taken together, these results suggest that the affects of hypo-osmotic incubation on receptor movement was reversible and re-inducible. Finally, if receptor recovery was due to recovery of the rate of internalization, then Ke at times after receptor recovery should be similar to iso-osmotic rates. This hypothesis was tested in the following experiment. Cells were incubated in hypo-osmotic or iso-osmotic media for 60 minutes and the rate of internalization detennined. Both groups of cells exhibited similar rates (Ke = 1.68 min-I) (Figure 2-10). This value compared well with the control rate 63 800 ........ x: 600 (5 -E... "-" "CD ~ as .c.:. .C.D-- c: 400 200 o ~~--~---¥----~--~----~--~----~--~ o 200 400 600 800 Integral Surface (fmoles x min) Figure 2-10. Effect of extended incubation in hypo-osmotic media on specific internalization rate (K ) . Cells were incubated in iso-osffiOtic media for 60 minutes at 37oC. An equal volume of water or media was added and the sarrples were incubated for an additional 60 minutes. Cells were then pelleted and the hypo-osmotically treated cells were resuspended in hypo-osmotic ( Il) media containing ligand and iso-osmotically treated cells were resuspended in iso-osmotic (A) media containing ligand. At specified times, cells were washed with HBSS and then EDTA stripping solution. Specific binding and internalization rate was determined as in Figure 2-7. 64 determined in Figure 2-7 (done in parallel). This result supports the hypothesis that the transient increase in surface receptor number during hypo-osmotic incubation is due to a reversible inhibition of the rate of receptor internalization. Hypo-osmotic Incubation Induces Inhibition of Fluid Phase Pinocytosis Fluid phase pinocytosis was assayed in order to further characterize the effects of hypo-osmotic incubation on plasma membrane internalization. Fluid phase pinocytosis is the nonspecific uptake of extracellular molecules and fluid by cells. Provided that certain criteria are met, horseradish peroxidase (HRP) can serve as a convenient tool to measure fluid phase uptake. The major criterion is that incubation of cells with HRP must be under conditions which preclude binding of the marker to the cell surface. Addition of excess amounts of mannan to the incubation media binds to receptors for mannose terminated glycoproteins and prevents any binding of HRP (a mannose terminated glycoprotein) to these receptors (26). upon hypo-osmotic exposure of cells the rate of accumulation of HRP was decreased as corrpared to cells in iso-osmotic media (Figure 2- 11). At the earliest time assayed (1 minute after dilution) the rate of accumulation of HRP in hypo-osmotically treated cells was approximately 50% that of control cells. Interpretation of these experiments may be obscured by the fact that some percentage of the fluid marker initially internalized may not accumulate within the cell but may be released into the extracellular media, a process referred to as diacytosis (27). The decrease in accumulation of HRP by cells oc ;: .S!! ::s E ::s (,) ~ a.. a: :z: 65 0.35 ...,---------------------. 0.30 0.25 0.20 0.15 0.10 0.05 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Time (minutes) Figure 2-11. Effect of hypo-~smotic incubation on HRP accumulation. 8:11s were plated (2.0 x 10 ) on 35 TIm dishes for 60 minutes at 37 C. M:dia was replaced with 0.9 mls wann media and the cells were incubated for 10 minutes. To each plate, 1.1 mls of media (.&) or water (4) containing HRP, rnannan, and BSA was added to result in a final concentration of HRP and rnannan of 4 mg/ml. At specified times, plates were transferred to OoC and washed 10 times with 2.0 mls of ice-cold HBSS. The cells were solubilized with 0.1% Triton X-100 and HRP activity and cell protein determined. Data are expressed as amount of HRP accumulated (ng HRP lug cell protein) . 66 in hypo-osmotic media may therefore reflect changes in the rate of diacytosis rather than changes in the rate of internalization. The half time for diacytosis in macrophages has been reported to be on the order of 5 minutes (27). By measuring HRP accumulation during the first 3 minutes of exposure to the marker, accumulation represents only the rate of internalization, since at these early times diacytosis is minimal. Thus, short "pulses" of HRP exposure can be used to measure HRP uptake at various times during hypo-osmotic incubation. Results from these experiments indicate that hypo-osmotic incubation decreased the rate of uptake of HRP by half (Figure 2-12) . In addition, experiments to directly measure the rate of diacytosis in cells indicate that hypo-osmotic incubation has no effect on the rate of diacytosis in these cells (data not shown). It should be noted however, that the amount of diacytosis occurring in these cells under control conditions was barely detectable. These results suggest that hypo-osmotic incubation not only inhibits receptor mediated endocytosis but also inhibits other membrane internalization processes in a similar manner. Simultaneous M:asurement of Ligand Internalization and Fluid Phase Pinocytosis Hypo-osmotic incubation appeared to cause a general inhibition of cellular internalization processes, as indicated by similar degrees of inhibition of both receptor mediated and fluid phase endocytosis. In order to detennine whether both of these processes were affected in an identical manner, measurement of ligand internalization and fluid phase pinocytosis were performed on the same samples simultaneously. 125 100 .c... 75 G) Co) .. G) D.. 50 25 o 10 20 30 40 Time (minutes) Figure 2-12. Effect of hypo-osmotic incubation on uptake of HRP during short exposures to the fluid phase marker. 67 50 Cells were plated and incubated in either iso-osmotic rred.ia or hypo­osmotic media at 37oC. During the last 3 minutes of incubation, HRP, rnarman, and BSA (final concentrations of 6.0 rng/ml HRP, mannan, and 3.0 rng/ml BSA) were added to plates and the incubation continued to the times indicated. Plates were washed and HRP activity and cell protein detennined. The data represent a composite of three separate experiments. Error bars represent the standard error of the rrean. The amount of HRP taken up by cells incubated in iso-osmotic rred.ia at the start of the experiment is defined as 100% and the data are expressed as percentage of that value. 68 Figure 2-13 demonstrates that the rate of ligand loss from the cell surface and the rate of HRP accumulation are affected to the same extent. The rate of internalization in iso-osmotic media was 8% /minute and 20% /minute for cells in hypo-osmotic media, representing a 2.50 fold decrease (Figure 2-13d) . The rate of HRP accumulation by the same cells in hypo-osmotic media was 0.0045 ng HRP/ug cell protein and 0.0115 ng HRP/ug cell protein for cells in iso-osmotic media, a 2.56 fold decrease (Figure 2-13b). The close correlation between the extent of inhibition of these two processes suggests that hypo-osmotic incubation inhibits receptor mediated endocytosis by a general inhibition of cellular internalization processes. In a parallel experiment with the same batch of cells, the increase in unoccupied surface receptor number was 2. 6-fold over control values (Figure 2-13a). This correlates well with the extent of both the inhibition of fluid phase pinocytosis and the rate of loss of prebound ligand. Interestingly, in all assays perfonned on this batch of cells, responses to hypo-osmotic incubation exhibited a 1 minute lag before either internalization was inhibited or surface receptor number increase occurred. In all other experiments perfonned (n=3 for simultaneous measures; n=5 for parallel measures), inhibition and surface receptor increase occurred immediately. Nonetheless, this experiment lends even further support for the hypothesis that surface receptor number increase is directly correlated with inhibition of receptor mediated endocytosis, and that inhibition of receptor mediated endocytosis reflects a general inhibition of all cellular internalization processes. 69 Figure 2-13. Simultaneous uptake of HRP and loss of pre-bound surface ligand by cells incubated in iso-osmotig or hypo-osmotic media. Cells were plated and in~ted at 37 C as in Figure 2-11. Cells were washed and placed at 0 C for 90 mingtes. (A) Sanples were brought back up to 37 C by the addition of 1.5 mls of iso-osmotic (.) or hypo-osmotic media (CJ). At the indicated times, plates were transferred to OOC, washed, and surface binding determined as in Figure 2-1. (B) Cells were placed at OOC in the presence of 1.0 x 10-8 M aM-125- I-T for 90 minutes and then were washed three times with ice-cold HBSS. Plates were transferred to 370C and 1.5 mls of either iso­osmotic (closed) or hypo-osmotic (open) media containing 6.0 mg/ml HRP, 6.0 mg/ml mannan, and 3.0 mg/ml BSA were added. At the indicated times, cells were transferred back to OOC, washed 12 times, and EDTA stripped as described in Figure 2-5. Amount of HRP accumulated (ng HRP lug cell protein) by the cells was determined and plotted (c._). (C) EDTA sensitive counts from iso-osmotic (&) and hypo-osmotic (A) sanples in (B) were determined and plotted as specific binding. (D) The loss ligand from the cell surface in (C) was plotted as percent of control at time zero. 70 0.80 =Qca 0.60 c m u ='u 0.40 G) a. CJ) 0.20 0.00 0 5 10 15 20 25 30 Time (minutes) 0.06 B 0.05 c ..0.-. C\1 0.04 :; E ::a uu 0.03 C aQ:. 0.02 ::t: 0.01 0.00 0.0 1.0 2.0 3.0 4.0 5.0 Time (minutes) 71 0.20 15 Time (minutes) L--'---'-"'-"-2-. 1 0 3.0 0.0 4.0 5.0 Time (minutes) The Rate of Fluid Phase Endocytosis Does not Recover During incubation of cells in hypo-osmotic media, the rate of 72 receptor internalization recovers to control values with time. The following experiment was perfo:rmed. to d.eteDlline whether the rate of fluid phase pinocytosis also recovered to control values. .HRP accumulation was assayed in cells immediately upon media dilution, or 60 minutes after continuous incubation in hypo-osmotic media. At this time the rate of .HRP accumulation was almost three times less in hypo-osmotic media than in iso-osmotic media (Figure 2-14). The rate of fluid phase pinocytosis was 0.312 ng HRP/minute in control cells and 0.113 ng HRP /min for cells in hypo-osmotic media. After 60 minutes at 37oC, the rates of HRP uptake in both control and hypo-osmotically treated cells were similar to the rates measured at early times (Figure 2-15b). The rate of uptake for control cells was 0.352 ng .HRP /minute and 0.080 ng .HRP /minute for cells incubated in hypo-osmotic media. Unlike the rate of receptor internalization, the rate of fluid phase pinocytosis did not return to iso-osmotic values. Thus, although hypo-osmotic exposure affects both fluid phase pinocytosis and receptor mediated endocytosis in an identical manner early in the incubation, the effects on these processes can be separated by the differential sensitivity of these processes to prolonged hypo-osmotic incUbation. Figure 2-14. HRP accumulation by cells after 60 minutes in hypo­osmotic media. 73 Cells were plated. and incubated. as in Figure 2-10. (A) At time zero media was aspirated. and 1.5 mls of iso-osmotic ( .. ) or hypo-osmotic media (to) containing 4.0 mg/ml HRP, 4.0 mg/ml, and 2.0 mg/ml BSA was added. back to the plates for the indicated. times. (B) Plates were incubated. for 60 minutes in iso-osmotic (.&) or hypo-osmotic (6) at which time media was replaced. with iso-osmotic media (6) or iso­osmotic media (A) containing HRP, mannan, and BSA as in (A). At specified. times, plates in (A) and (B) were washed. at OOC and HRP activity and cell protein determined.. 74 3.0 A 2.5 c 0 :is 2.0 E (::,s) 1.5 ill: (,) / :cIc:X.ct. 1.0 ;.,1 / !/*_ __ i 0.5 ".",a ....... a a 0.0 ,...1""" a . 0.0 2.5 5.0 7.5 1 0.0 12.5 15.0 Time (minutes) 3.0 B 2.5 c 0 =a=s 2.0 :; E (::,s) 1.5 (,) /1 cc.t. 1.0 i/• 6 IX ::c 0.5 / --a L"II. ...... a....-a 0.0 ,6".",6 0.0 2.5 5.0 7.5 10.0 12.5 15.0 TIme (minutes) 75 Discussion Surface receptor number for several different ligands is a function of the distribution of receptors between the cell surface and the endocytic apparatus (6). The rate of entry of receptors into the endocytic apparatus is a function of the internalization rate of unoccupied receptors, while movement of receptors to the cell surface is due to the rate of exteriorization. Receptor Redistribution Addition of mitogenic hormones, or of agents which mimic intracellular messengers, alters the distribution of receptors between the cell surface and intracellular "pools." The initial effects of these agents is acceleration of the rate of exteriorization of unoccupied receptors (7-10). Exposure of cells to hypo-osmotic solutions effected an increase in surface receptor number; however, this increase was accomplished by a completely different mechanism. Cells exposed to hypo-osmotic solutions exhibited a transient reduction in endocytosis of both occupied and unoccupied receptors. ~asurement of the affects of hypo-osmotic incubation on movement of occupied receptors is shown in Figure 2-6 in which prebound ligand internalization is decreased by 50%. The effects of hypo-osmotic fluids on internalization of unoccgpied receptors are demonstrated in Figure 2-8. In this experiment the rate of receptor exteriorization was unaltered, yet surface receptor number increased. Taken together, these data suggest that the increase in surface receptor number is due to hypo-osmotically induced "block" of internalization of both occupied and unoccupied receptors. 76 An inhibition of fluid phase pinocytosis was also observed upon hypo-osmotic incubation of cells. In the majority of experiments perfonred, increased receptor number were observed at the earliest time (1 minute) measured after media dilution. In Figure 2-13, data fran 1 experiment were presented in which there was a lag of one minute before increases in receptor number or decreases in pinocytosis were observed. The reason for a lag in the response in this one experiment is not known but it provides further evidence for a direct correlation between surface receptor number increase and inhibition of receptor mediated internalization, as well as confi:rnti..ng a parallel inhibition of both receptor mediated and fluid phase pinocytosis. Relationship Between Receptor M::diated Endocytosis and Fluid Phase Pinocytosis Previous reports in the literature have suggested that receptor mediated endocytosis and fluid phase pinocytosis occur by the same pathway (28,29). The studies in this chapter provide evidence for a parallel inhibition of both fluid and receptor mediated endocytic activities, and suggest that hypo-osmotic incubation affected both in a similar manner. Interestingly, the rate of receptor mediated endocytosis of cells in hypo-osmotic solutions was shown to recover to normal levels after 60 minutes while fluid phase pinocytic activity remained inhibited. Thus, although hypo-osmotic incubation initially affected the rates of both receptor mediated endocytosis and fluid phase pinocytosis to the same extent, these processes were not necessarily linked and could be differentiated on the basis of recovery. 77 These results are most readily explained by the existence of two separate pathways for fluid phase uptake and receptor mediated endocytosis. The model outlined in Figure 2-15 proposes that two separate pathways exist, and outlines the proposed affects of hypo­osmotic incubation on each pathway. It is assumed that the majority of fluid uptake (90%) occurs via smooth vesicle pathway, and internalization by coated pits accounts for only a fraction (10%) of total fluid uptake. A 50% decrease in internalization via the receptor mediated pathway would only decrease uptake of the fluid phase marker by 5%. A parallel decrease in the smooth vesicle pathway would decrease net fluid uptake by 45%. (Note: At this time it carmot be distinguished whether fewer vesicles are being internalized or whether the vesicles that are internalized are smaller. Both conditions could give rise to the same results.) Relative to a decrease of 90% to 45% upon inhibition of the fluid phase pathway, a 5% decrease is small and not within the limits of detection. The independent recovery of the receptor mediated endocytosis pathway would result in the recovery of fluid phase uptake by this pathway of 5% of total cellular fluid phase activity. In Figure 2-15, fluid uptake is not shown to occur by the coated pit pathway to emphasize its negligible contribution to fluid phase uptake in this proposed model but uptake of sorre fluid is expected. There are several other models that could explain these results. The proposed model that fluid phase pinocytosis occurs via a different pathway than receptor mediated endocytosis is part of a growing body of investigations that have found that these two 78 Figure 2-15. Proposed model of effects of hypo-osmotic incubation on receptor mediated endocytosis and fluid phase pinocytosis. (A) Internalization during iso-osmotic incubation. (B) Parallel inhibition of fluid phase pinocytosis and receptor mediated endocytosis irrmediately after dilution of the incubation media to 150 rrOsM. (C) Recovery of receptor mediated endocytosis but not fluid phase pinocytosis after 30 minutes in hypo-osmotic media. ( • ) represent fluid phase molecules and (.) represent ligand. A • ~ B , .4 • 8 c 79 • . ... • • • • , Cd , , (0 80 processes can be functionally separated. Buys et al. found that in J774 cells, phorbol esters increased fluid phase uptake while the calcium ionophore, A23187 decreased this process. At the same time both agents induced a decrease in receptor mediated internalization (10). Hypotonic shock and K+ depletion in Hep 2 cells resulted in inhibition of internalization of poliovirus (presumably via coated pits), while uptake of human rhinovirus type 2 via smooth vesicles was unaltered (31). Sandvig et al. have reported that "pinching off" of coated vesicles is inhibited by cytoplasmic acidification, but that fluid phase uptake is only negligibly affected (32). Finally, morphological studies of the suckling rat ileum demonstrate two separate pathways for the uptake of receptor bound molecules and the fluid phase markers (33). As early as 3 minutes after labeling, the two markers could not be found in the same corrpartment. The authors did not discount the possibility that both receptor mediated and fluid phase uptake occur by the same vesicle and rapid sorting resulted in segregation into two intracellular pathways. Inhibition of Internalization and Normal EXteriorization While hypo-osmotic incubation reduced receptor internalization by 50%, the rate of movement of receptors from intracellular "pools" to the cell surface was not altered. In fact, the increase in surface receptor number can be explained solely on the basis of inhibition of internalization. This observation is consistent with the notion that internalization and exteriorization are not Obligatorily linked and that independent regulation of these rates occurs. The separation of 81 these two "limbs" of the recycling pathway has been reported previously. Epidermal growth factor