Quantitative autoradiography of angiotensin II receptors in the brain and kidney

The rennin-angiotensin system is an important component in the regulation of systemic blood pressure. Angiotensin II is the principle effector peptide of this system. Interaction of angiotensin II with specific receptors can produce is several organ systems. When administered into the brain this octa-peptide produces a variety of responses including a stimulation of drinking, increased systemic blood pressure and several neuroendocrine responses. Its effects on the kidney include alterations in arteriolar resistance, mesangial cell contraction and a feedback inhibition of the release of renin. Since this peptide produces profound effects on homeostasis by an interaction with specific receptors, the quantitative technique of in vitro autoradiography was applied to localize receptor populations for angiotensin II. Specific binding sites for a radiolabeled form angiotensin II were localized in various brain and kidney regions. In the rat brain high densities of angiotensin II receptors were observed in the paraventricular and suprachiasmatic nuclei of the hypothalamus, supraoptic nucleus and the posterior lobe of the pituitary, brain areas in which angiotensin II modifies neuroendocrine functions. In addition, receptor populations were detected in several brainstem cardiovascular regions including the locus coeruleus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus. Binding was also detected in several circumventricular organs including the subfornical organ and the organum vasculosum of the lamina terminalis, regions where angiotensin II is believed to produce a dipsogenic effect. Angiotensin II receptors were also detected in several brain regions involved in mediating a variety of somatic and visceral sensory functions. In the kidney angiotensin II receptors have been localized to the glomerulus, vasa recta and ureter. Additional sites were also detected outside the vasa recta in the renal medulla. These localization indicate regions of the kidney involved in angiotensin II receptor mediated alterations in kidney functions. These result supply addition support for many of the postulated roles for angiotensin II in homeostasis by specific actions in the brain and kidney. Additionally, several new potential actions of the renin-angiotensin system in modulating central nervous system sensory input have been indicated.

QUANTITATIVE AUTORADIOGRAPHY OF ANGIOTENSIN II RECEPTORS IN THE BRAIN AND KIDNEY by Donald Richard Gehlert A dissertation submitted to the faculty of The University of Utah in partial fullfillment of the requirements for the degree Doctor of Philosophy Department of Pharmacology The University of Utah March 1985 THE UNIVERSITY OF UTAH GRADCATE SCHOOL SUPERVISORY COM~IITTEE APPROVAL of a dissertation submitted bv DONALD RICHARD GEHLERT This dissertation has been read by each member of the following supervisory committee and bv majority vote has been found to be satisfactory. 8 February 1985 8 February 1985 ~rFid01le 8 February 1985 Donald N. Franz 8 February 1985 ~~,~ Glen R. Hanson 8 February 1985 C. Dean Withrow THE l:NIVERSITY OF CTAH GRADUATE SCHOOL FIr\AL READING AI)PR()V AI~ To the Graduate Council of The l'llivcrsity of Utah: I ha\'e read the dissertation of Donald Richard Gehlert In ItS final form and have found that (1) 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 Super\'is­ory Committee and is ready for submission 10 the Graduate 'chon!. 8 February 1985 Date A pproved for the M<1:jor Department Donal • Reed Chairman I Dean Appro\'ed for Ihe (~radll~llt' COllllcil ---=----Ja-m-e-s----L--. ~lc;/::(~-·- D('an of '1'11(' Graduah' SdlOol Copyright @ Donald Richard Gehlert 1985 All Rights Reserved ABSTRACT The renin-angiotensin system is an important component in the regulation of systemic blood pressure. Angiotensin II is the principle effector peptide of this system. Interaction of angiotensin II with specific receptors can produce in several organ systems. When administered into the brain this octa-peptide produces a variety of responses including a stimulation of drinking, increased systemic blood pressure and several neuroendocrine responses. Its effects on the kidney include alterations in arteriolar resistance, mesangial cell contraction and a feedback inhibition of the release of renin. Since this peptide produces profound effects on homeostasis by an interaction with specific receptors, the quantitative technique of in vitro autoradiography was applied to localize receptor populations for angiotensin II. Specific binding sites for a radiolabeled form of angiotensin II were localized in various brain and kidney regions. In the rat brain high densities of angiotensin II receptors were observed in the paraventricular and suprachiasmatic nuclei of the hypothalamus, supraoptic nucleus and the posterior lobe of the pituitary, brain areas in which angiotensin II modifies neuroendocrine functions. In addition, receptor populations were detected in several brainstem cardiovascular regions including the locus coeruleus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus. Binding was also detected in several circumventricular organs including the subfornical organ and the organum vasculosum of the lamina terminalis, regions where angiotensin II is believed to produce a dipsogenic effect. Angiotensin II receptors were also detected in several brain regions involved in ,mediating a variety of somatic and visceral sensory functions. In the kidney angiotensin II receptors have been localized to the glomerulus, vasa recta and ureter. Additional sites were also detected outside the vasa recta in the renal medulla. These localizations indicate regions of the kidney involved in angiotensin II receptor mediated alterations in kidney function. These results supply additional support for many of the postulated roles for angiotensin II in homeostasis by specific actions in the brain and kidney. Additionally, several new potential actions of the renin-angiotensin system in modulating central nervous system sensory input have been indicated. v TABLE OF CONTENTS ABSTRACT • • • • LIST OF FIGURES ACKNOWLEDGMENTS PART I: DISTRIBUTION OF [125I )-ANGIOTENSIN II BINDING SITES IN THE RAT BRAIN: A QUANTITATIVE AUTORADIOGRAPHIC STUDY INTRODUCTION • • MATERIALS AND METHODS Tissue Preparation • • • • • • • • • Biochemical Studies • • • • • Quantitation of t~5oradiograms • • Preparation of [ I)-Angiotensin II RESULTS Biochemical Experiments • Autoradiographic Localization Studies Spinal cord • • • • • • • • • • Myelencephalic and metencephalic structures Mesencephalic structures • Diencephalic structures • • • • • • Telencephalic structures • Miscellaneous • • • • DISCUSSION • • • Correlation of Receptor Density with Immunohistochemical Evidence • • • • • • • • • • • • • • • • • Correlation with Physiological Data • Dipsogenic response Cardiovascular responses • Neuroendocrine responses • • Electrophysiological data Sensory function REFERENCES • • • • • • • • iv •• viii ix 2 4 4 4 6 7 8 8 17 19 26 26 26 31 36 39 40 42 42 43 46 47 47 49 PART II: IN VITRO AUTORADIOGRAPHIC LOCALIZATION OF r125I]-ANGIOTENSIN II BINDING SITES IN RAT AND DOG KIDNEY INTRODUCTION • • • • • • • • • • • • • • • • • • 57 MATERIALS AND METHODS RESULTS DISCUSSION • REFERENCES CURRICULUM VITAE vii 59 62 73 78 81 LIST OF FIGURES Figure PART I: 1. Dissociation of [125I ]-angiotensin II from the lateral septum 9 2. Association of [ 125 I]-angiotensin II to slide-mounted tissue sections . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3. Saturation analysis of [ 125 I)-angiotensin II binding to the lateral septum • • • • • • • • • 14 4. Distribution of [125I ]-angiotensin II binding sites in the rat thoracic spinal cord • • • • • • • • • • • • • • • 22 5. Distribution of angiotensin II receptors in the rat cervical spinal cord and brainstem • • • • • • • • • 24 6. Angiotensin II receptors in the rat cerebellum 27 7. [125I ]-Angiotensin II binding sites in the rat forebrain. 29 8. Autoradiographic localization of angiotensin II receptors in the rat hypothalamus • • • • • • • • • • 32 9. Localization of angiotensin II receptors in the rat olfactory bulb and frontal cortex • • • • • • 34 10. Distribution of angiotensin binding sites in the septal region 37 11. Autoradiographic images on LKB Ull2~film after apposition to kidney sections labeled with [ I]-angiotensin II • • 63 12. r125I]-Angiotensin II binding in the ureter 65 13. Localization of angiotensin II receptors in the rat renal cortex using photographic emulsion-coated coverslip technique • • • • • • • • • • • • • • • • • • • • • 68 14. Scatchard analysis of r125I]-angiotensin II binding to the rat kidney . . . . . . . . . . . . . . . . . . . . . . . . . .. 70 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. James K. Wamsley, for providing the opportunity to conduct my thesis project in his laboratory. His encouragement, guidance and support have allowed me to obtain seemingly distant goals in a very short period of time. I also wish to express my appreciation to the other members of my supervisory committee, Drs. Donald N. Franz, Salvatore J. Fidone, Glen R. Hanson and C. Dean Withrow for their helpful advice. This thesis work could not have been accomplished without the generous assistance of Dr. Robert Speth at the University of Washington. I also wish to thank Dr. Henry Yamamura at the University of Arizona for his valuable contributions to my career. Thanks are also extended to Margaret Long, Jane Stout and Rosalyn Fowles for their assistance in preparing. this manuscript. Portions of this manuscript have been submitted for publication in Peptides and The Journal of Neuroscience. Finally, I wish to acknowledge the continuous support of my wife, Sue. Without her patience and confidence , this task would have been far more difficult. She can only hope that someday the endless years of training will end and I will finally get a job. PART I: DISTRIBUTION OF [125I ]-ANGIOTENSIN II BIh~ING SITES IN THE RAT BRAIN: A QUANTITATIVE AUTORADIOGRAPHIC STUDY INTRODUCTION A substantial body of evidence indicates that a distinctly separate renin-angiotensin system exists within the brain. All the enzymes and precursors required for both the synthesis and degradation of angiotensin II, the major effector peptide for this system, have been detected within the brain (Ganten and Speck, 1978; Phillips et al. , 1979) • Central administration of angiotensin II produces a stimulation of thirst (Epstein et al., 1970; Fitzsimons, 1972; Severs and Summy-Long, 1976; Severs et al., 1978) and sodium appetite (Buggy and Fisher, 1974; Avrith and Fitzsimons, 1980; Bryant et al., 1980), release of vasopressin (Bonjour and Malvin, 1970; Severs et al., 1970; Keil et al., 1975), secretion of adrenocorticotrophic hormone (Maran and Yates, 1977; Ramsay et al., 1978) and a marked pressor response (Bickerton and Buckley, 1961; Severs and Daniels - Severs, 1973). Receptors for this peptide have also been detected by a number of investigators utilizing brain homogenate preparations (Bennett and Snyder, 1976; Cole et al., 1978; Harding et al., 1981; Tonnaer et al., 1983). The displacement characteristics of [125IJ-angiotensin II by related peptide analogues indicate that this ligand is binding to a physiologically functional central receptor population (Mann et a1., 1981). In order to define the potential locus for the central actions of angiotensin II within the brain, we have utilized an in-vitro procedure to label slide-mounted tissue sections with [1251] -angiotensin II for the detection of receptor populations by 3 quantitative autoradiography (Young and Kuhar, 1979; Palacios et al., 1981; Wamsley and Palacios, 1983). Preliminary reports of this work have appeared elsewhere (Gehlert et al., 1984a,b). MATERIALS AND METHODS Tissue Preparation Male Sprague-Dawley rats (200-300g) were sacrificed by intracardial perfusion with ice-cold isotonic saline without added fixative (the inclusion of even low concentrations of fixative was found to inhibit binding. particularly to the circumventricular organs). Brains were then rapidly removed and frozen onto brass cryostat chucks using plastic embedding media (Histoprep; Fisher Scientific Corp •• Pittsburgh. PA) and kept frozen at -70°C until use. When ready for analysis, the tissues were allowed to come to cryostat temperature and 10 micron tissue sections were cut and thaw-mounted onto cold chrome-alum/gelatin coated microscope slides. These slides were allowed to desiccate at -10°C for 2 hours after which they were stored at -25°C. Biochemical Studies The purpose of the initial studies was to establish binding conditions which provide the highest specific to nonspecific (signal-to-noise) ratio. To determine these conditions, the dissociation, association and saturation properties of the receptor population were analyzed. Usually the measurement of the radioactivity bound to the tissue in experiments of this type involves wiping the section from the slide and subsequently counting the radioactivity by gamma counting techniques. However. the relatively low specific binding of (12SI ]-angiotensin II to rat forebrain 5 observed in preliminary studies made accurate use of this technique impractical. Therefore, these experiments were analyzed by using the quantitative properties of the autoradiographic images produced on LKB Ultrofilm (LKB Instruments, Rockville, MD). In these experiments, sections through the rat forebrain were preincubated for 30 minutes in a buffer containing 0.4% bovine serum albumin (Pentex; Miles Laboratories, Elkhart, IN), 10mM MgCI 2 , 150mM NaCI, 5mM DTT, 5mM EGTA and 50mM NaHP0 4 (pH 7.2) at room temperature. This preincubation allowed for the dissociation of any endogenous ligand present in the tissue sections which could interfere with the subsequent binding. The slides were then transferred to an identical incubation media containing 0.5 nM [ 125 I}-angiotensin II for 45 minutes at room temperature. Sections representing nonspecific binding were incubated in the additional presence of 3 micromolar unlabeled va15-angiotensin II (Bachem; Torrance, CA). Incubations were terminated by a dip in media followed by various rinsing periods in fresh, ice-cold media. Following the rinse, sections were dipped twice in distilled water and rapidly dried using a cool, dry stream of air. After overnight storage in a self-defrosting freezer, the slides were affixed to photographic mounting boards and apposed to sheets of LKB Ultrofilm (LKB Instruments, Rockville, MD) in X-ray cassettes. The films were removed and developed after a 7-21 day exposure period. To evaluate the association of [ 125 I]-angiotensin to slide-mounted tissue sections, similar conditions were utilized. Following a 30-minute preincubation, various incubation periods were employed. The slides were then rinsed for 4 minutes in ice cold media, after which they were treated as previously described. The 6 saturation properties of the angiotensin II receptors were evaluated by incubating sections from various areas of rat brain in presence of O.I-l.SnM rI25 I]-angiotensin II using the optimal conditions determined in the dissociation and association experiments. The displacement of angiotensin II was evaluated by including a 1 micromolar concentration of captopril (Squibb; Princeton, NJ), met-enkephalin, substance P, angiotensin I, angiotensin II I (Sigma; St. Louis, MO), Sar 1-lIe5 -G8ly - angiotensin II and Sar1 -TI5e -A8la - angiotensin II (Bachem; Torrance, CA) in the incubation media. Quantitation of Autoradiograms The latent autoradiographic images, representing total or nonspecific binding of rI25I]-angiotensin II, were quantitated by use of computer-assisted microdensitometry (Unnerstall et al., 1982; Rainbow et al., 1982; Wamsley and Palacios, 1983). Grain densities were read on a Leitz (West Germany) Orthoplan microscope equipped with a DADS model 560 computerized microdensitometry system (Stahl Research Laboratories; Rochester, NY). Optimal binding conditions were determined by positioning a 100-250 square micron window over the appropriate area of the autoradiogram and reading the densi ty as a percent extinction of the incident light. In the mapping, saturation and displacement experiments, quantitation was accomplished by comparing the grain density produced by [125I ]-angiotensin II bound to the tissue sections to that produced by [1251 ] brain paste standards. These standards were prepared and utilized by the method of Unnerstall 7 et ale (1982) with the substitution of di-[125 I ]-angiotensin II as the source of radioactivity. Receptor localizations were determined by staining the tissue sections with cresyl violet or ACEB (alkaline-cyclohexylaminopropane sulfonic acid-ethanol-butanol) stains and comparing them to the grain distribution on the autoradiograms. Localizations were confirmed by two independent investigators using the atlas of Paxinos and Watson (1982). Autoradiograms and stained tissue sections were photographed using a Leitz Orthomat camera system. Preparation of [125JJ-Angiotensin II [ 125 I] -lIe 5 -angiotensin (SA 1700-2000 Ci/mmol) was synthesized according to the method of Speth and Husain (1984). Briefly, iodinations were carri~d out in the presence of chloramine T at a 10:1 ratio of angiotensin II to [1251 ] (Amersham Corp., Arlington Heights, IL). Mono-[125I ]-angiotensin II was separated from uniodinated angiotensin II, free [ 125 I] and di-[ 125 I]-angiotensin II by a s~. ngle chromatographic step on a cation exchange column (CM-32, Whatman, Inc., Clifton, NJ). Purity of the final product was verified by thin layer chromatography and high performance liquid chromatography as described elsewhere (Speth and Husain, 1984). Unlabeled Asp-lIe 5 -angiotensin II was generously supplied by Dr. M. Khosla. RESULTS Biochemical Experiments A highly discrete distribution of autoradiographic grains, representing specifically bound [ 125 I] -angiotensin II, were observed in the areas of autoradiograms from sections of rat forebrain corresponding to the lateral septum and piriform cortex. For these initial studies the grain densities corresponding with the lateral septum were utilized. The results of the dissociation analysis can be seen in Figure 1. Grain densities representing [ 125 I]-angiotensin II nonspecifically bound to the lateral septum were rapidly reduced during the first 10 minutes of rinsing with little change seen after 10 minutes. Specific binding, as determined by the difference between the grain densities associated with the images representing total and nonspecific binding, demonstrated little change during the first 10 minutes of rinsing whereupon a slow dissociation became apparent. The highest specific to nonspecific ratios were obtained at a rinse time of 10 minutes where approximately 35% of the binding was specific. Similar information was obtained when examining grain densities associated with the piriform cortex (data not shown). The association rate of [ 125 I]-angiotensin II with receptors in the lateral septum was then examined. Slide-mounted tissue sections were preincubated for 30 minutes followed by an incubation with 0.5 nM [1251 ] -angiotensin II for various intervals. The binding was then quantitated as described in Methods. These results are shown in 9 Figure 1. Dissociation of r12SI]-angiotensin II from the lateral septum. Following preincubation, sli11~ounted sections of rat forebrain were incubated with O.SnM [ I]-angiotensin II for 60 minutes at room temperature, followed by various rinsing times. Non~pecific binding was determined by adding 3 micromolar unlabeled val -angiotensin II to the incubation media. The labeled tissue sections were then apposed to LKB Ultrofilm for 1 week. The latent images were then quantitated using computerized microdensitometry (see text for details). A slow reduction in grain densities, representing nonspecific binding, was observed during the first 10 minutes of rinsing with little change in specific binding indicated by the difference in grain densities between the images representing total and nonspecific binding. Each data point represents the average of 8 determinations which differed by less than 10%. .U- ~u 0..- ...J(f)~ «IU t-ZW o o'a.. t-Z(f) o 0 <l o CD o ~ o o (\J NO 1 1.~ N 11. X3 1.N3J~3d o o f() - 11 Figure 2. Nonspecific binding was similar at all time points while specific binding, as represented by the difference in grain densities between the total and nonspecific images, slowly equilibrated. Because binding appeared to reach equilibrium by 45 minutes, a 60-minute incubation time was utilized for all remaining experiments to insure equilibrium. Similar sections through the rat forebrain were used to determine the saturation kinetics of [ 125 I]-angiotensin II binding to the lateral septum. The results of these experiments are shown in Figure 3. The specific binding of ( 125 I]-angiotensin II, represented by the difference in grain densities between the autoradiographic images produced by sections labeled in the presence of [ 125 I]-angiotensin II alone and those incubated with the addition of 3 micromolar va15-angiotensin, began to asymptote between 1.0 and 1.5 nH [ 125 I]-angiotensin II (Figure 3). The highest specific to nonspecific ratios were obtained at a concentration of 0.7 nM [ 125 I]-angiotensin II and thi8 concentration was subsequently utilized for the localization experiments. Subsequently, saturation experiments were conducted in a variety of brain regions and the binding quantitated using radioactive standards (see Methods). The results of Scatchard analysis of the saturation data from several brain regions can be seen in Table 1. Dissociation constants (Kd) appeared to cluster around two values. A high affinity population (Kd = 0.6 nM) which was seen in the lateral septum, median eminence, piriform cortex, and superior colliculus, and a lower affinity population (Kd = 2 nM) which was 12 Figure 2. Association of [ 125 I]-angiotensin II to slide-mounted tissue sections. Ten micron tissue sections were preincubated for 30 minutes, followed by various incubation periods. After a 4-minute rinse, the sections were dried and apposed to LKB Ultrofilrn for 1 week. Quantitation of the generated grain densities indicated that no increase in specific binding occurred after 45 minutes; therefore, a 60-minute incubation was utilized in subsequent experiments. The points are the average of 8 determinations. U lL. -U wU 0..- -I(f)~ <tIU I-ZLLI OOf:l. J-Z(f) o 0 <J C-\.I z o I­< t <DO:: .1- Z LLI U Z o U ~--------~------~------~~O o to o v o C\J NOIJ.~NIJ.X3 J.N3~tJ3d o 14 Figure 3. Saturation analysis of [ 125 I]-angiotensin II binding to the lateral septum. Serial sections through the rat septum were preincubated for 30 minute'~5followed by a 60-minute incubation with various concentrations of [ I]-angiotensin II. This was followed by two 5-minute rinses in ice-cold media. The labeled sections were apposed to LKB Ultrofilm for 1 week and the latent autoradiographic images quantitated. High signal-to-noise (specific to nonspecific) rf~~os were obtained at a concentration range of 0.6-0.9 nM [ I]-angiotensin II. o U -LL 0 WU u_ Q..LL --lU')- <tI U I-ZW OOQ.. I-ZU') o 0 <J 0 o CD ~ Z 0- v~ o C\J ~ w :E ~ ~~~----~~--~~~~--~~o o lO o v o C\J NOI.1~ N I.1X3 .1N3~~3d o 16 Table 1. Scatchard analysis of [125I ]-angiotensin II binding to several brain nuclei. Values were determined by Scatchard analysis of quantitative autoradiography using serial sections f1.~ several brain regions incubated in media containing 0.1 to 1.5 nM [ I]-angiotensin II. Six data points, each representing 4 to 6 determinations, were analyzed by linear regression on a Hewlett-Packard 9845C computer. Abbreviations: K~ (dissociation constant), B (maximal binding capacity) and r ( inear correlation coefficient)m. ax Area Bm ax (fmoles/mg tissue) r Lateral Septum 0.53 2.55 0.95 Medial Geniculate 1. 98 6.13 0.93 Median Eminence 0.70 5.40 0.90 Piriform Cortex 0.66 3.74 0.95 Subiculum 2.04 7.47 0.94 Subthalamic Nucleus 1.88 10.66 0.96 Superior Colliculus 0.63 4.82 0.88 17 observed in the other areas examined. The receptor number (Bmax) differed by a factor of 4 in the areas studied. The displacement characteristics of several peptides and the angiotensin converting enzyme inhibitor captopril were examined against the binding of [1251 ] -angiotensin II. Table 2 summarizes these results. Substance P and captopril exhibited little displacement of specifically bound [125 I ]-angiotensin II, while angiotensin III and the angiotensin II receptor antagonists Sar1 -lIe5 -G8ly - angiotensin II and Sar1 -lIe5 -A8la - angiotensin II respectively, were potent displacers. Angiotensin I, and to a much lesser extent, met-enkephalin, exhibited some displacement at a 1 micromolar concentration. Autoradiographic Localization Studies Using the conditions determined in the biochemical experiments, the following binding protocol was established. Ten micron thick coronal or sagittal sections were preincubated in media for 30 minutes followed by a 60-minute incubation in the same media containing 0.7 nM [ 125 I]-angiotensin II. Sections representing nonspecific binding were incubated in the additional presence of 3 micromolar valine5-angiotensin II. Incubations were terminated by a dip in buffer followed by two 5-minute rinses in fresh, ice-cold buffer. The rinse media was then removed from the slide by two dips each in two changes of distilled water followed by rapid drying of the sections with a stream of forced air. This binding procedure produced 50-90% specific binding, depending on the nucleus examined. 18 Table 2. Displacement of [ 125 I] -angiotensin II by various compounds expressed as a percent of tota\ 2~pecific binding. Serial tissue sections were incubated in 0.7nM [ I]-angiotensin II with or without the presence of a 1 micromolar concentration of the test compound. The subsequent autoradiograms were quantitated using standards and the displacement calculated as a percent of total specific binding. Non~pecific binding was defined by the addition of 1 micromolar val -angiotensin II to the incubation media. Abbreviations are SC (superior colliculus), NTS (nucleus of the solitary tract), PC (piriform cortex), SFO (subfornical organ), SgV (substantia gelatinosa of the trigeminal nucleus) and APit (anterior pituitary). Values are the average of 4 determinations which differed by less than 10%. Percent Total Specific Binding Compounds Brain Areas SC NTS PC SFO SgV APit Captopril 94.0 100.2 96.3 100.7 108.0 101.0 Met-Enkephalin 90.4 77.2 76.7 89.5 81.0 87.0 Substance P 102.8 94.1 101.9 100.9 94.3 100.2 Angiotensin I 46.3 8.4 18.6 21.8 13.8 78.3 Angioten~in 181 0.0 0.0 0.0 0.0 0.0 2.2 Sar -lIe -Gly - Aygiot~nsin8II 11.5 4.5 5.6 0.4 8.1 1.5 Sar -lIe -Ala - Angiotensin II 0.0 0.0 0.0 0.0 0.0 0.0 19 Autoradiographic grain densities were quantitated using computerized microdensitometry. The grain densities produced by the labeled tissue sections were compared to those produced by 10 micron thick r 125I ]_brain paste standards which were apposed to the films along with the tissue sections. An extensive listing of the quantitation of [125I ]-angiotensin II binding can be found in Table 3. In general, binding was confined to highly discrete areas of the brain containing principally gray matter, whereas few grain densities were found associated with white matter regions. In addition, there was a high density of autoradiographic grains (representing specifically bound r125I]-angiotensin II) over the circumventricular organs, cerebral blood vessels, choroid plexus and in the pia matter surrounding the brain. The inclusion of even low concentrations of formaldehyde in the perfusate utilized during tissue preparation resulted in a dramatic reduction in binding to the latter four areas. Spinal cord. The grain densities in autoradiograms representing specific [125I ]-angiotensin II binding to the rat cervical, lumbar and thoracic spinal cord were very discretely localized (Figures 4,5). In the cervical region, binding was detected primarily in the substantia ge1atinosa of the dorsal horn (laminae II and III). Lower specific grain densities were associated with the lamina X, while even lower levels of specific binding were observed in lamina IV and the ventral horn. An analogous distribution was seen in the lumbar region. In the thoracic spinal cord, a similar distribution was also observed with the addition of a high density of autoradiographic grains associated with the intermedio-1ateral cell column (lamina IX). 20 Table 3. Quantitation of [125 I ]-angiotensin II binding. Autoradiographic grain densities were quantitated by positioning a microphotometer beam over a region of the autoradiogram corresponding to 100-250 square microns of tissue area. These values were then converted to fmoles/~ tissue by comparing these grain densities to those produced by [ I]-brain paste standards. Values represent the average of 6-12 determinations from 2-4 animals (mean ± SEM). Area Amygdala Medial Nucleus Area Postrema Caudate-Putamen Cerebellum Molecular Layer Cortex Cingulate Frontal Piriform Temporal Superficial (Laminae I-III) Lamina IV Lamina VI Choroid Plexus Dorsal Motor Nucleus of the Vagus Habenula Lateral Medial Hippocampus CAl Hypothalamus Median Eminence Paraventricular Nucleus Suprachiasmatic Nucleus Inferior Olive Medial Nucleus Lateral Olfactory Tract Lateral Septum Locus Coeruleus Medial Geniculate Body Nucleus of the Solitary Tract 125 I-Angiotensin II fmoles/Ing tissue 3.96±0.41 O.IO±O.Ol O.09±O.05 O.68±O.09 O.28±O.14 O.54±O.05 1. 14±O. 25 O.21±O.OS O.Ol±O.02 O.03±O.OI O.84±O.OS 4.SS±O.28 1.21±O.06 1.19±O.14 O.43±O.06 2.09±O.21 3.80±O.29 4.29±O.07 1. IO±O.IS O.OS±O.04 O.61±O.19 2.75±O.18 1.27±O.20 4.31±O.13 Olfactory Bulb Glomerular Layer External Plexiform Layer Olfactory Tubercle Organum Vasculosum of the Lamina Terminalis Periaqueductal Gray Pituitary Anterior Lobe Reticular Formation Spinal Cord Cervical Lamina IV Lamina X Substantia Gelatinosa Ventral Horn Lumbar Lamina IV Lamina X Substantia Gelatinosa Ventral Horn Thoracic Lamina IV Lamina IX Lamina X Substantia Gelatinosa Ventral Horn Subfornical Organ Subiculum Subthalamic Nucleus Superior Colliculus Supraoptic Nucleus Thalamus Dorsal Medial Nucleus Gelatinosus Nucleus Reuniens Nucleus Rhomboidius Ventral Posterior Trigeminal Nucleus Substantia Gelatinosa 0.S8±0.09 0.67±0.lS 0.20±0.07 3.S7±0.21 0.73±0.09 6.02±0.30 0.31±0.OS 0.20±0.02 0.29±0.03 0.89±0.06 0.19±0.02 0.19±0.01 0.30±0.01 0.S6±0.OS O.lS±O.Ol 0.19±0.01 0.82±0.08 0.30±0.01 0.56±0.06 0.1S±0.01 3.49±0.18 1. 65±0.18 2.65±0.24 2.40±0.14 4.60±0.18 1. 66±0.11 1.87±0.19 0.60±0.11 1.41±0.31 0.39±0.08 0.35±0.07 21 22 Figure 4. Distribution of [ 125 I]-angiotensin II binding sites in the rat thoracic spinal cord. A125 A section of rat upper thoracic spinal cord was incubated with [ I] -angiotensin II and apposed to LKB Ultrofilm to produce the autoradiogram seen in the photomicrograph. The grains appear dark against a light background. A high grain density is associated with the dorsal horn (DR), intermedio-Iateral cell column (IML) and the pia matter (P). B. A serial section from that which produced the autoradiogram seen in ! was labeled in the additional presence of 3 micromolar unlabeled val -angiotensin to produce the autoradiogram seen in this photomicrograph. The diffuse grain distribution seen here represents nonspecific binding. c. This photomicrograph shows the distribution of autoradiographic grains produced by a labeled section of lower thoracic spinal cord. Note the grain densities associated with the dorsal horn (DH). Since several exposure periods and ligand specific activities were used, only a qualitative representation of the receptor density is presented. Bar = 250 microns. B .-. 24 Figure 5. Distribution of angiotensin II receptors in rat cervical spinal cord and brainstem. A. An autoradiogram showing the distribution of autoradiographic grains in the lower cervical spinal cord is seen in this photomicrograph. Note the grains associated with the dorsal horn (dh) • B. Depicted in this photomicrograph is an autoradiogram demonstrating the grains produced by a labeled section of upper cervical spinal cord. Note the grain densities associated with the substantia gelatinosa (sg). C. The distribution of autoradiographic grains corresponding to a section of medulla is seen here. A high grain density was associated with the nucleus of the solitary tract (nts) and the medial nucleus of the inferior olive (io). Bar = 500 microns. 26 ~elencephalic and metancephalic structures. In the medulla, high grain densities were associated with the nucleus of the solitary tract (Figure 5), dorsal motor nucleus of the vagus, nucleus intercalatus, nucleus commissuralis, and the medial nucleus of the inferior olive. Lower grain densities were seen in regions corresponding to the dorsal nucleus of the inferior olive and the substantia gelatinosa of the spinal trigeminal nucleus. A very low diffuse distribution of specific binding was observed throughout the remainder of the medulla including the reticular formation. In the pons, very few nuclei exhibited specific binding of [ 125 I]-angiotensin II. The exceptions were the high grain densities found associated with the locus coeruleus and the low grain density seen over the dorsal tegmental nucleus. Moderate grain densities found in the cerebellum (Figure 6) were associated with the molecular layer. Few grains were seen in the granular cell layer or white matter. Mesencephalic structures. The binding in the midbrain was confined to a few discrete nuclei. A very high density of grains was seen over the superior colliculus (Figure 7) while a much lower grain density was seen in regions of the film corresponding to the periaqueductal gray matter. Very few specific grains were observed in the areas of other midbrain structures. Diencephalic structures. A number of structures within the thalamus had low to moderate grain densities indicating the presence of angiotensin II receptors. Moderate grain densities were seen in the habenula, medial geniculate body, and several midline thalamic nuclei including nucleus rhomboideus, nucleus gelatinosus (Figure 7) 27 Figure 6. Angiotensin II receptors in the rat cerebellum. A. A stained section of rat cerebellum is seen in this photomicrograph. Note the location of the molecular layer (m), granule cell layer (gr) and the white matter (w). B. The autoradiogram produced by the labeled tissue sections seen in A is depicted here. Note the high grain density corresponding to the molecular layer, with little specific grain densities associated with the white matter or granule cell layer. c. 5 A serial section was incubated with the addition of 3 micromolar val -angiotensin II to produce this autoradiogram. The diffuse distribution of grains seen here represents nonspecific binding. Bar = 500 microns. 29 Figure 7. [125 I ]-angiotensin II binding sites in the rat forebrain. A. A high density of autoradiographic grains can be seen in regions corresponding to the organum vasculosum of the lamina terminalis (OVLT) and piriform cortex (PC) while a very low density corresponds to the white matter of the lateral olfactory tract. B. Discrete grain densities localized in the thalamus, specifically in the nucleus rhomboidius (RR) and nucleus gelatinosus (GE). C. The grain densities associated with the superior colliculus (SC) and medial geniculate body (MG) are depicted here. D. An autoradiogram representing nonspecific binding (in a section adjacent to the one labeled in panel C). Bar = 500 microns. 31 and nucleus reuniens. A diffuse distribution of low density was found in several of the lateral thalamic nuclei and in the lateral geniculate body. Dense concentrations of angiotensin II receptor binding could only be found in the subthalamic nucleus. In the hypothalamus, very high grain densities were found corresponding to the paraventricular and suprachiasmatic nuclei (Figure 8). A dense band of grains was also observed over the median eminence. A more moderate level was produced by the labeling in the ventromedial hypothalamus and, in addition, a high density of grains was seen associated with the supraoptic nucleus and anterior pituitary. Little specific binding was seen in the intermediate and posterior lobe of the pituitary. Telencephalic structures. A very low density of autoradiographic grains was detected in most laminae of the cerebral cortex. A low density of specific binding was found in the superficial laminae of the cingulate and temporal cortex and in regions of sagittal sections which corresponded to the frontal pole (Figure 9). A moderate density of autoradiographic grains was also observed in lamina I and III of the piriform cortex surrounding the lateral olfactory tract (Figure 7). Specific grain densities, indicating the presence of angiotensin II receptors, were also discretely localized in the olfactory and rostral limbic regions. In the olfactory bulb, specific binding was confined to the glomerular and external plexiform layers (Figure 9) where a moderate grain density was observed. A very low grain density was seen throughout the remaining components of this structure. 32 Figure 8. Autoradiographic localization of angiotensin receptors in the rat hypothalamus. A. A high density of autoradiographic grains is seen in this photomicrograph corresponding to the location of the lateral ventricle choroid plexus (LV), subfornical organ (SFO) and the paraventricular nucleus of the hypothalamus (PV). B. A serial se~tion from that seen in A was labeled in the presence of excess val -angiotensin II to generate this autoradiogram representing nonspecific binding. C. Autoradiographic grains corresponding to the periventricular (PE) and suprachiasmatic (SC) nucleus of the hypothalamus. D. High densities of autoradiographic grains corresponding to the median eminence (ME). Bar = 500 microns. .1" 34 Figure 9. Localization of angiotensin II receptors in the rat olfactory bulb and frontal cortex. A. A labeled sagittal section of rat brain generated this autoradiogram. Note the grain densities associated with the frontal pole (fp) and the glomerular (lg) and external plexiform (EPL) layers of the olfactory bulb. B. An autoradiogram showing the distribution of nonspecific binding in these regions. Bar = 500 microns. 36 Autoradiograms from labeled tissue sections through the forebrain exhibited very high densities of specific .grains associated with the subfornical organ (Figures 8, 10) and the organum vasculosum of the lamina terminalis (Figure 7), while moderate grain densities were seen over the lateral septum (Figure 10) and the median preoptic area. The hippocampal formation revealed little specific binding except for the subiculum which showed a high grain density and CAl which had a low grain density. In the amygdaloid nuclear complex, a moderate density of specific grains was found confined to the medial nucleus. Miscellaneous. Along with labeling of areas within the blood-brain and CSF-brain barriers, several additional structures evidenced specific [ 125 I]-angiotensin II binding sites. A high density of autoradiographic grains was observed corresponding to several circumventricular organs, including the organum vasculosum of the lamina terminalis (Figure 7), median eminence (Figure 8), pineal gland and subfornical organ (figure 8), with lower grain densities associated with the ventral, but not dorsal, portion of the area postrema. The choroid plexus was associated with a moderate grain density as were the cerebral blood vessels and the pia matter surrounding both the brain and spinal cord. 37 Figure 10. Distribution of angiotensin binding sites in the septal region. A. Specific labeling is seen in this autoradiogram corresponding to the location of the lateral septum (sl), subfornical organ (SFO) and the preoptic division of the suprachiasmatic nucleus (posc). B. A serial tissue sectio~ was incubated with the addition of 3 micromolar unlabeled val -angiotensin II to produce this autoradiogram. Note the diffuse distribution of grains. Bar = 500 microns. DISCUSSION In this study, the in vitro technique of receptor autoradiography has been applied to map the distribution of angiotensin II receptors in the rat CNS. This quantitative technique allows one to selectively study a receptor population in an individual brain nucleus unencumbered by other areas which demonstrate only nonspecific binding. Autoradiograms generated by apposing LKB Ultrofilm to tissue sections labeled with [ 125 I]-angiotensin II indicate that only a few highly discrete brain nuclei contain angiotensin II receptors. The binding characteristics of [125 I ]-angiotensin II to several brain nuclei were evaluated using the quantitative properties of LKB Ultrofilm. These receptor populations had similar binding properties to those described by several investigators using homogenate preparations (Bennett and Snyder, 1976; Cole et al., 1978; Harding et al., 1981; Mann et al., 1981; Tonnaer et al., 1983). In addition, receptor affinity differed approximately three-fold when comparing populations in different brain nuclei. The functional significance of these findings, however, remains to be seen. The gross regional distribution of [125I ]-angiotensin II binding sites in the rat brain was also quite similar to that observed in homogenate preparations (Sirett et al., 1977; Harding et al., 1981). A high density of sites was observed in the septum, thalamus, hypothalamus and the medulla. In this and other studies, the light microscopic resolution of receptor autoradiography has allowed the 40 qualitative confirmation of specific binding in a few very highly discrete nuclei within these brain regions (Gehlert et al., 1984 a,b; Mendelsohn et a1., 1984). Using the improved conditions as described in the results, we have been able to conduct an extensive quantitative mapping of these highly discrete receptor populations throughout the rat brain and spinal cord. Grain densities representing specifically bound [125I ]-angiotensin II have been localized not only to the circumventricular organs where well known physiological effects emanate, but also to several brain nuclei where no known function of the central renin-angiotensin system can be assigned. Correlation of Receptor Density with Immunohistochemical Evidence Immunoreactive angiotensin II-containing nerve terminals have been reported in many of the areas demonstrated in this study to contain angiotensin II receptors. Angiotensin-like immunoreactiv:f.ty has been consistently reported in the substantia gelatinosa of the spinal cord, substantia gelatinosa of the spinal trigeminal nucleus, median eminence, intermedio- lateral cell column, periaqueductal grey, paraventricular and supraoptic nuclei of the hypothalamus, nucleus of the solitary tract, locus coeruleus, dorsal motor nucleus of the vagus and the lateral septum (Fuxe et al., 1976; Changaris et al., 1978; Ganten et al., 1978; Brownfield et al., 1982; Weyhenmeyer and Phillips, 1982). Several studies have also reported immunocytochemical detection in the frontal cortex, superior colliculus (Brownfield et al., 1982; Weyhenmeyer and Phillips, 1982), lateral olfactory tract (Changaris et al., 1978; Wehenmeyer and Phillips, 1982), thalamus (Fuxe et al., 1976; Weyhenmeyer and 41 Phillips, 1982), subthalamic nucleus (Fuxe et al., 1976), piriform cortex, pineal (Changaris et al., 1978), suprachiasmatic nucleus and the organum vasculosum of the lamina terminalis (Weyhenmeyer and Phillips, 1982). The presence of angiotensin immunoreactive nerve terminals in regions of the brain containing angiotensin II receptors presents strong evidence that these receptor populations could be acted upon by neuronally released angiotensin II. One must consider, however, the possibility that the immunoreactive material represents angiotensin II which has been taken up by neurons using a receptor­mediated endocytotic process. The activity of angiotensin converting enzyme, the synthetic enzyme for angiotensin II, shows a similar distribution when compared to angiotensin II receptors. In the brainstem, converting enzyme activity has been observed in the nucleus of the solitary tract, area postrema, locus coeruleus, choroid plexus, inferior olive and the cerebellar cortex (Chevillard and Saavedra, 1982b). Forebrain areas such as the medial habenula, anterior pituitary, supraoptic and paraventricular nuclei of the hypothalamus and the subfornical organ all contained a relatively high activity (Saavedra et al. ,I 1982). Interestingly, the immunoreactivity for angiotensin converting enzyme, has been reported to exhibit a somewhat different distribution from that of angiotensin-like immunoreactivity in the brain (Brownfield et al., 1982) as do the binding sites for [3H]-captopril (Strittmater et al., 1984), an angiotensin converting enzyme inhibitor. Although the predominant localization of r3H]-captopril binding sites was to structures in the striatonigral pathway, a number of additional areas containing these sites overlapped with regions showing the presence of ECCLES HEALTH SClfNr.~~ I, 42 angiotensin II receptors (e.g., the locus coeruleus, subfornical organ, median eminence and supraoptic nucleus). Biochemical studies have yielded a variety of contradictory results in supporting a separate central role for the renin-angiotensin system (Reid, 1977; Ganten, 1978; Meyer et al., 1982). Recently, Ganten and coworkers (1983 a,b) have reported that inhibition of brain angi.otensin converting enzyme leads to an accumulation of angiotensin I in spontaneously hypertensive rats, indicating that active angiotensin II synthesis occurs within the brain. Correlation with Physiological Data A number of physiological actions have been attributed to the interactions of angiotensin II with specific receptors within the brain. Central administration of angiotensin II has been demonstrated to produce both behavioral and neuroendocrine actions. Dispogenic response. The induction of thirst by centrally administered angiotensin II is believed to be mediated by two circumventricular organs, the subfornical organ (Simpson and Routtenberg, 1973; Simpson et al., 1978; Mangiapane and Simpson, 1980) and the organum vasculosum of the lamina terminalis (Hoffman and Phillips, 1976). The detection of specific angiotensin II binding in these regions indicates that interaction of angiotensin II with receptors in these structures could be responsible for the dipsogenic action. However, some evidence exists that the preoptic area may also be involved in mediating this response (Mogenson and Kucharczyk, 1978). The moderate density of autoradiographic grains seen in the median preoptic area, as well as the high density of grains seen in 43 adjacent areas including the supraoptic nucleus and the preoptic division of the suprachiasmatic nucleus, support the hypothesis that neurons in these nuclei also participate in the dipsogenic response to angiotensin II. Cardiovascular responses. The pressor response to centrally administered angiotensin II is perhaps the most studied effect of the proposed brain renin-angiotensin system. The area postrema, a circumventricular organ located in the medulla, is believed to mediate this effect in several species (Joy and Lowe, 1970; Ferrario et al., 1972). However, in rats (Brody et al., 1978) interaction with angiotensin II receptors in the subfornical organ appears to initiate the pressor response (Mangiapane and Simpson, 1980). Specific autoradiographic grains, indicating the presence of specifically bound [ 125 I] -angiotensin II, have been detected in both the subfornical organ and ventral portion of the area postrema in the present study; thus, both these structures could be involved in the pressor responses. However, the unusual distribution of autoradiographic grains observed over the area postrema may indicate ligand diffusion from the nucleus of the solitary tract. In addition to the circumventricular organs, a high density of autoradiographic grains has been localized in several cardiovascular centers within the blood-brain barrier. Two principal cardiovascular nuclei in the medulla, the nucleus of the solitary tract and dorsal motor nucleus of the vagus, were associated with a high specific grain density after labeling sections with [ 125 I] -angiotensin II. The labeling extended caudally to the nucleus commissuralis and the nucleus intercalatus. The nucleus of the solitary tract receives baroreceptor, chemoreceptor 44 and a variety of other afferent inputs via the ninth and tenth nerves (Lipski et al., 1975; Lipski et al., 1977; Spyer et al., 1984). An electrolytic lesion of this area produces a fulminating neurogenic hypertens:f_on (Doba and Reis, 1973). The close proximity of the nucleus of the solitary tract to the area postrema has led to the suggestion that the two may interact in the regulation of systemic blood pressure (Barnes et al., 1977; Palkovits, 1980). The dorsal motor nucleus of the vagus is innervated by efferent fibers from the nucleus of the solitary tract (Palkovits and Zaborszky, 1977). This area of the brainstem is involved in the modulation of a variety of visceral sensory afferents including cardioinhibitory vagal functions. Stimulation of the neurons in the locus coeruleus, a region where we detected a high density of angiotensin II receptors, also produces a pressor response (Ward and Gunn, 1976 a,b). Thus, the presence of angiotensin II receptors in these cardiovascular areas indicates that angiotensin II may play a modulatory role in the medullary control of blood pressure. Additionally, there is evidence for involvement of the intermedio-lateral cell column of the thoracic spinal cord in hypertension (Loewy and Kellar, 1980). While only a low density of sites were seen in this area of the rat, we have recently reported a high density of angiotensin II receptors in the intermedio-lateral cell column of the cat (Gehlert et al., in press). Thus, the presence of angiotensin II receptors in these cardiovascular areas indicates that angiotensin II may play a modulatory role in the central control of blood pressure. The limbic system is also believed to influence systemic blood pressure via projections to and from the nucleus of the solitary tract 45 and dorsal motor nucleus of the vagus. Several of these areas, including the paraventricular and supraoptic nuclei, exhibited a high density of autoradiographic grains when labeled with 1?5 [ - I]-angiotensin II. Electrical stimulation of either of these two brain areas produces an increase in blood pressure and inhibits reflex vagal bradycardia (Ciriello and Calaresu, 1980). The supraoptic and paraventricular nuclei are believed to produce these effects via connections with the nucleus of the solitary tract and dorsal motor nucleus of the vagus (Abboud, 1982). Perhaps the most striking correlation between angiotensin II receptors and cardiovascular control centers occurs in the areas believed to be involved in the emotional influences on blood pressure. In a recent report, Smith et ale (1984) outlined the afferent and efferent connections to the perifornical region of the hypothalamus which is believed to be responsible for the cardiovascular responses that accompany behavior (Smith et al., 1980). Afferents to this region originate in several brain areas which label with r125Il-angiotensin II including the lateral septum, paraventricular nucleus, supraoptic nucleus, organum vasculosum of the lamina terminalis, subfornical organ, subthalamic nucleus, locus coeruleus, periaqueductal gray, dorsal motor nucleus of the vagus and nucleus of the solitary tract (DeVito and Smith, 1982) • The distribution of efferent fibers is quite similar (Smith et al., 1984). The extensive overlap of angiotensin II receptors with, the areas involved in this physiological response indicate angiotensin II could be a modulator in these cardiovascular pathways. 46 Neuroendocrine responses. A number of hormones are released systemically after central administration of angiotensin II (Ganong et al., 1979). An intracerebroventricular injection of angiotensin II causes the release of vasopressin from the pituitary (Bonjour and Malvin, 1970; Severs et al., 1970; Keil et al., 1975; Padfield and Morton, 1977; Share, 1979) and has been shown to be capable of causing oxytocin release (Lang, 1981). Vasopressin is synthesized predominantly in the supraoptic nucleus while oxytocin is produced mostly from cell bodies residing in the paraventricular nucleus of the hypothalamus (Buijs et al., 1983). These are both transported via the median eminence to the posterior pituitary. Angiotensin II and vasopressin have been colocalized in fibers of this pathway (Kilcoyne et al., 1980). The neurons in this region have been reported to release vasopressin after addition of angiotensin II in vitro while a similar preparation of the posterior pituitary did not (Gregg and Malvin, 1978). Since high densities of angiotensin II receptors were localized in the paraventricular nucleus, supraoptic nucleus and the median eminence, while very few receptors were seen associated with the posterior pituitary, it follows that angiotensin II acts on the synthesis and/or transport of vasopressin and oxytocin rather than directly affecting the release of the neurohormones from terminals in the posterior pituitary. Angiotensin II also stimulates the secretion of adrenocorticotrophic hormone and prolactin from the anterior pituitary (Moran and Yates, 1977; Ramsay et al., 1978). The presence of a high density of receptors in the anterior pituitary indicates a potential site of action for these angiotensin-mediated neuroendocrine 47 response. The pineal and suprachiasmatic nucleus, which are involved in the regulation of diurnal rhythms (Preslock, 1984), also showed high receptor densities indicating a potential role for angiotensin II in influencing this important neuroendocrine cycle. Electrophysiological data. The location of angiotensin-sensitive neurons can be determined by observing single neuron discharges after the microiontophoretic application of angiotensin II (Nicoll and Barker, 1971; Wayner et al., 1973). Neurons which are rapidly excited by the iontophoresis of angiotensin II have been detected in the lateral septum (Huwyler and Felix, 1980; Simmonet et al., 1980), paraventricular and supraoptic nuclei of the hypothalamus (Akaishi et al., 1980) and the cat subfornical organ (Felix and Akert, 1974). The presence of a high density of angiotensin II receptors in these brain areas indicates that direct stimulation of receptors on the neurons is likely to be responsible for the excitatory response. Sensory function. The distribution of autoradiographic grains, indicating the presence of specifically bound [125IJ-angiotensin II, appears to suggest a role for angiotensin II in modulating sensory function. High grain densities were associated with several structures involved in mediating visceral sensory input. For instance, the nucleus of the solitary tract and dorsal motor nucleus of the vagus receive both peripheral cutaneous and visceral sensory fibers. As noted previously, both of these regions showed high densities of angiotensin II receptors. The substantia gelatinosa of the spinal cord, the trigeminal nucleus and the periaqueductal gray matter area are involved in nociception (Fields and Basbaum, 1978). All of these 48 regions also had significant densities of angiotensin II receptors associated with them. In addition, several somatic sensory functions are implied by the localization of angiotensin II receptors in regions of the brain involved in the processing of somatic sensory afferents. Grain densities were associated with the glomerular and external plexiform layers of the olfactory bulb, the superior colliculus and the medial geniculate body. These regions are involved in the processing of olfactory, visual and auditory information respectively. There are also direct retinal afferents to the suprachiasmatic nucleus, an area which contains angiotensin II receptors. In conclusion, the distribution of angiotensin II receptors within the rat brain correlates well with the previously reported sites of central action of angiotensin II and with the localization of angiotensin II containing nerve terminals. A large proportion of these receptor sites are located in discrete brain nuclei involved in somatic and visceral sensory input. 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Hoffman (1979) Evidence for an endogenous brain renin-angiotensin system. Federation Proc. 38:2260-2266. Preslock, J.P. (1984) clinical correlations. The pineal gland: Basic Endocrine Rev. 5(2):282-308. implications and Rainbow, T.C., W.V. Bleisch, A. Biegon, B.S. McEwen (1982) Quantitative densitometry of neurotransmitter receptors. J. Neurosci. Methods 5:127-138. Ramsay, D.J., L.C. Keil, M.C. Sharpe and J. Shinsako (1978) Angiotensin II infusion increases vasopressin, ACTH and 11-hydroxycorticosteroid secretion. Am. J. Physiol. 234:R66-R71. Reid, I.A. (1977) Is there a brain renin-angiotensin system? Circ. Res. 41:147-153. Saavedra, J.M., J. Fernandez-Pardal and C. Chevillard (1982) Angiotensin-converting enzymne in discrete areas of the rat forebrain and pituitary gland. Brain Res. 245:317-325. Severs, W.B., J. Summy-Long, J.S. Taylor and J.D. Connor (1970) A central effect of angiotensin: Release of pituitary pressor material. J. Pharmacol. Exp. Ther. 174:27-34. Severs, W.B. and A.E. Daniels-Severs (1973) Effects of angiotensin on the central nervous system. Pharmacol. Rev. 25:415-449. Severs, W.B. and J. Summy-Long (1976) The role of angiotensin in thirst. Life Sci. 17:1513-1526. Severs, W.B., D.G. Changaris, L.C. Keil, J.Y. Summy-Long, P.A. Klase and J.M. Kapsha (1978) The pharmacology of angiotensin-induced drinking behavior. Federation Proc. 37:2699-2703. Share, L. (1979) Interrelations between vasopressin and the renin-angiotensin system. Federation Proc. 38:2267-2271. Simmonet, G., B. Bioulac, F. Rodriquez, and J.D. Vincent (1980) Evidence for a direct action of angiotensin lIon neurons in the septum and in the medial preoptic area. Pharmacol. Biochem. Behav. 13:359-363. 54 Simpson, J. B. and A. Routtenberg (1973) Subfornical organ: site of drinking elicitation by angiotensin II. Science 181:1172-1174. Simpson, J.B., A.N. Epstein and J.S. Camardo (1978) The localization of dipsogenic receptors of angiotensin II in the subfornical organ. J. Compo Physiol. Psychol. ~:581-608. Sirett, N.E., A.S. McLean, J.J. Bray and J.I. Hubbard (1977) Distribution of angiotensin II receptors in rat brain. Brain Res. 122:299-312. Smith, O.A., C.A. Astley, J.L. DeVito, J.M. Stein and R.E. Walsh (1980) Functional analysis of hypothalamic control of the cardiovascular responses accompanying emotional behavior. Fed. Proc. 39:2487-2494. Smith O.A., J.L. DeVito and C.A. Astley (1984) Organization of centra.l nervous system pathways influencing blood pressure responses during emotional behavior. Clin. and Exper. Hyper. A6:185-204. Speth, R.c!2~nd A. Husain (1984) Preparation and one-step purification of mono-[ I}-angiotensin II for radioligand binding assays. J. Pharmacol. Meth. 11:137-151. Spyer, K.M., S. Donoghue, R.B. Felder and D. Jordan (1984) Processing of afferent inputs in cardiovascular control. Clin. and Exper. Hyper. A6:173-184. Strittmater, S.M., M.M.S. Lo, J.A. Javitch and S.H. Snyder (1984) Autoradiograp~ic visualization of angiotensin converting enzyme in rat brain with r H] -captopril: Localization to a striatonigral pathway. Proc. Natl. Acad. Sci. 81:1599-1603. Tonnaer, J.A.D.M., G.M.H. Engels, K. Voshart, V.M. Wiegant and W. Dejong (1983) Binding of angiotensins to rat brain tissue: Structure activity relationships. Brain Res. Bull. 10:295-300. Unnerstall, J.R., D.L. Niehoff, M.J. Kuhar and J.M. 3 Palacios (1982) Quantitative receptor autoradiography using [H] Ultrofilm: Application to mUltiple benzodiazepine receptors. J. Neurosci. Meth. 6:59-73. Wamsley, J.K. and J.M. Palacios (1983) Apposition technlques of autoradiography for microscopic receptor localization. In Current Methods in Cellular Neurobiology, J. Barker and J. McKelvy, eds., John Wiley and Sons, New York, pp 241-268. Ward, D.G. and C.G. Gunn (1976a) Locus coeruleus complex: Elicitation of a pressor response and a brain region necessary for its occurrence. Brain Res. 107:401-406. Ward, D.G. and C.G. Gunn (1976b) Locus coeruleus complex: Differential modulation depressor mechanisms. Brain Res. 179:255-270. 55 Wayner, M.J., T. Ono and D. Nolley (1973) Effects of angiotensin lIon central neurons. Pharmacol. Biochem. Behav. 1:679-691. Weyhenmeyer, J.A. and M.I. Phillips (1982) Angiotensin-like immunoreactivity in the brain of the spontaneously hypertensive rat. Hypertension ~:514-523. Young, W. s. III a~d M. J. Kuhar (1979) A new method for receptor autoradiography: [H] opioid receptors in rat brain. Brain Res. 179:255-270. PART II: IN VITRO AUTORADIOGRAPHIC LOCALIZATION OF [125I ]-ANGIOTENSIN II BINDING SITES IN THE RAT AND DOG KIDNEY INTRODUCTION The involvement of angiotensin II in hypertension is well documented (Malvin, 1979). Angiotensin II has a variety of physiologic roles, some of the most prominent being in the kidney. It produces alterations in glomerular filtration rate (GFR) by affecting arteriolar resistance (Freeman et al., 1973; Ploth and Navar, 1979; Hall et al., 1981) and may contract the mesangial cell layer to decrease glomerular capillary surface area (Hornyck et al., 1972; Sraer et al. , 1974). Angiotensin II also appears to be an intermediate in the actions of dibutyryl cAMP, parathyroid hormone, prostaglandin 12 and prostaglandin E2 resulting in a decrease of plasma flow rate and an increase total renal arteriolar resistance (Brenner et al., 1982). Using a variety of techniques, an increase in proximal tubular reabsorption was found to be directly mediated by angiotensin II (Johnson and Malvin, 1977; Harris, 1979; Ploth et al., 1979; Huang et al., 1982). There is also some suggestion that angiotensin II can alter reabsorption in the late proximal tubule, early distal tubule, and the loop of Henle as well (Ploth et al., 1979; Ploth and Navar, 1979). Angiotensin II also mediates a feedback inhibition on renin release by the juxtaglomerular apparatus (Sokabe, 1974; Freeman and Davis, 1979). The variety of alterations in kidney function produced by endogenous and exogenous angiotensin II implicate a potential role for kidney receptor populations in angiotensin induced hypertension. With 58 these possibilities in mind, the present study was undertaken to localize the potential sites of action of angiotensin II in the rat and dog kidney utilizing in vitro receptor autoradiography (Young and Kuhar, 1979; Palacios et al., 1981; Wamsley and Palacios, 1983). MATERIALS AND METHODS Male, Sprague-Dawley rats (200-250g) were sacrificed by intracardial perfusion with ice-cold sodium phosphate buffered saline at pH 7.4. Kidneys were removed and frozen onto brass cryostat chucks coated with OCT compound (Lab-Tek Products; Naperville, IL). Sections (10-16 microns) were cut on a Harris cryostat microtome (Harris; North Billerica, MA) at -15°C and thaw-mounted onto cold chrome/alum subbed slides. The kidneys from two mongrel dogs were treated in a similar manner. Serial sections were preincubated in a media containing 0.4% bovine serum albumin, 10mM MgC12, 150mM NaCI, 5mM EGTA, 5wl dithiothreitol, 30mM Na After 2 HP0 4 at pH 7.1 for 30 minutes. preincubation, the slides were incubated in the same media containing 0.5 nM r125I]-Ile-angiotensin II (SA = 1785 Ci/mmole) for 60 minutes. Sections representing nonspecific binding were generated by incubating sections in the presence of 3 micromolar unlabeled lIe 5 -angiotensin II or Val 5 -angiotensin II. Incubations were terminated by a dip and two 5-minute rinses in fresh media (without added radioactivity) at 4°C. The slides were then dipped twice in distilled water, placed on ice cold metal pans and dried with a stream of cool, dry air. Saturation studies using sections of rat kidney were performed by varying the concentration of [ 125 I] -angiotensin II in the incubation media from O.lnM to 1.3nM. After overnight desiccation, the slides were affixed to photographic mounting board and apposed to LKB UI trofilm (LKB 60 Instruments; Rockville, MD) in X-ray cassettes for 2-4 days, after which the film was removed and developed. To facilitate quantitation in the sa t ura t ~· on exper i ments, ( 12 ] - b ra~. n paste stand ar d s were prepared using di-iodo-angiotensin II in a manner similar to that for tritium brain paste standards as previously described in Part I (Unnerstall et al., 1982). An exposure of these standards was included on each sheet of film. Some slides were also positioned against emulsion coated coverslips (Young and Kuhar, 1979). This method entailed affixing emulsion-coated (NTB-3, Eastman Kodak; Rochester, ~~) coverslips over the slide mounted tissue sections. After a 6 day exposure, the coverslips were bent back, the latent images developed and the tissue subsequently stained. The coverslips were then permanently affixed to the slide, positioning the autoradiographic grains directly over the source of radioactivity in the tissue. Thus, a high resolution and an accurate localization of the autoradiographic grain distribution could be obtained. The latent images and stained tissue sections were photographed on a Leitz Orthoplan microscope equipped with an Orthomat Camera System (Leitz; West Germany). Grain density readings on the LKB U1 trofilm were made with a computer-assisted microphotometry system (DADS Model 560 Computer interfaced with an MPV Compact Photometer) attached to the Lei tz microscope. In the saturation experiments, these readings were converted to femtomoles ligand bound/mg tissue by comparing the grain densities over the tissue areas to those produced by [ 125 IJ brain paste standards (Unnerstall et al., 1982). 61 [ 125 I]-Ile 5 -angiotensin II was synthesized by a previously described method (Speth and Husain, 1984). Purity was confirmed by thin layer chromatography, high performance liquid chromatography and receptor binding techniques. RESULTS Incubation conditions, similar to those described here, have been used previously to define areas of high specific binding of [125 I ]-angiotensin II in the rat brainstem (Gehlert et al., 1984). The binding of [ 125 I] -angiotensin II to rat kidney was expected to take place in a similar fashion. The use of these conditions produced highly specific binding, represented by autoradiographic grains on LKB Ultrofilm, in several kidney areas shown in Figures 11A and lIB. The addition of either Va15-angiotensin II or Ile5-angiotensin II to the preincubation and incubation medias produced an image that had few autoradiographic grains in these areas (Figure lIe). Highly specific grain densities, representing [125I ]-angiotensin II binding to angiotensin II receptors, were seen in areas on LKB Ultrofilm that correspond to the glomeruli, cortex, medulla and the ureter (Figures 11 and 12, and Table 4). The use of the coverslip technique allowed further localization of these specific grains to the membranes of the glomeruli (Figure 13). Saturation studies performed on film gave evidence for two receptor affinities (Figure 14); a high affinity receptor which was associated with the vasa recta, and a lower affinity receptor seen in areas of the medulla outside the vasa recta. Receptor populations in the ureter had a dissociation contant (~) similar to that observed in the vasa recta, while the population in the glomerulus resembled the ~ measured in the medulla outside the vasa recta. Slides incubated 63 Figure 11. Autoradiographic imagf~son LKB Ultrofilm after apposition to kidney sections labeled with [ I]-angiotensin II. A. A photomicrograph of the autoradiographic grains on U1 trofilm a~~gsed over a kidney section incubated in the presence of 0.2 nM [ I]-angiotensin II. Note the high density of autoradiographic grains associated with the glomerulus (gl) and vasa recta (vr). B. The tissue section used to generate the autoradiogram depicted in ty~~ photomicrograph was incubated in the presence of 1.3nM [ I] -angiotensin II. Note the increased binding to areas outside the vasa recta in the medulla (M). C. The autoradiogram depicted in this f£gtomicrograph was produced from a section incubated with O. 2nM ~ I] -angiotensin II in the additional presence of 3 micromo1ar Val -angiotensin II. The diffuse autoradiographic grains seen here represents nonspecific binding. Bar = 500 microns. ... 1 " 65 Figure 12. [125 l ]-angiotensin II binding in the ureter. A. A tissue section stained with cresyl violet is seen in this photomicrograph. The arrow denotes the location of the rat ureter (u) • B. A portion of Ultrofilm apposed over the tissue section seen in A is seen in this photomicrograph. Note the high density of autoradiographic grains corresponding to the ureter. Bar = 500 microns. C. The labeled tissue section used to produce this autoradiogram was sectioned at a more medial aspect than those seen in A and B. Again, the high grain density associated with the ureter can be appreciated. I .}' ~.~~'. · ,.,:+t..;:~:. " 67 Table 4. Densitometer readings of grain densities on LKB Ultrofilm expressed as percent extinction. Readings were taken from a 625 square micron window positioned over the area on film corresponding to the appropriate kidney area. Readings from the cortex and medulla refer to areas outside the grain densities associated with the glomerulus and vasa recta, respectively. p < 0.05. Area Total Nonspecific % Specific Rat* Glomerulus 62.8±2.9 16.S±0.S 73.7 Cortex 24.7±1.1 18.2±0.7 23.5 Vasa Recta 60.6±2.6 6.2±1.1 89.9 Medulla 28.2±1.0 8.6±O.8 69.5 Ureter 66.7±1.5 6. S±1. 2 89.8 Dog** Glomerulus 74. 0±1. 8 20.8±O.9 71.9 Cortex 32.2±2.3 13.8±2.1 57.1 Medulla 34.2±1.6 10.3±0.8 69.9 Ureter 33.7±2.7 29.3±2.7 N. S. *N = 6 **N = 2 68 Figure 13. Localization of angiotensin II receptors in the rat renal cortex using the photographic emulsion-coated coverslip technique. A. After development of the latent autoradiographic image on the coverslip, the tissue section seen in this photomicrograph was stained with pyronine Y. The location of the glomerulus (gJ) corresponds to the arrow. The autoradiographic grains associated with the glomerulus can be seen directly as dark grains. B. The incident light used to take this photomicrograph makes the autoradiogram appear as white grains against a dark background. Again note the high grain density associated with the glomerulus. Bar = 500 microns. . • --';fo.~ :. ; '. . '. '- \'" ' . '" '. ,""'" - '-' .. ;\ .. ;; .. { .. ~ , ,"" .' 1 70 Figure 14. Scatchard analysis of [ 125 I]-angiotensin II binding to the rat kidney. Serial rat kidney sections were incubated in the presence of O.1-1.3nM concentrati~of iodinated angiotensin II and apposed to Ultrofilm for 6 daY\~5 [ I] Brain paste standards, containing known concentrations of [ IJ, were placed on the film with the labeled tf~~ue sections. Densitometric measurements were converted to fmoles [ I]-Angiotensin II bound/mg tissue by comparing the grain densities corresponding to the appropriate tissue areas to the grain densities produced by the standards. The Scatchard analysis of these data gave evidence for two binding affinities (see above). A high affinity receptor population (~=0.23nM, B =14.5-16.0 fmoles/mg tissue) and a low affinity populatton (~=0."~0.85nMJ B =16.5-17.5 fmoles/mg tissue) were localized to distinct kidney stru~f~res (see inset). 70-1 Kd -Bmax o GLOMERULUS 0.849 17. 5 o VASA RECTA 0.230 14.5 60-1 \. \. • URETER 0.232 16.0 II MEDULLA 0.772 16.5 I \.\. 50 ,. '" 0 lIJ \ lIJ ~ 40 "- • 0 z 0\. ::> 30 0 m 20~~ -\.\ 10 o I 'I ':>:>r o 5 10 15 20 25 BOUND 72 in the presence of 3 micromolar Ile5-angiotensin II or 3 micromolar Val 5 -angiotensin II had low amounts of autoradiographic grains in these areas. Binding to dog kidneys was localized to similar areas (Table 4); however the binding was more diffuse in the medulla and could not be localized specifically to any tubular or vascular populations. There was also no specific binding corresponding to the ureters in the sections of dog kidney we examined. DISCUSSION In vitro receptor autoradiographic techniques have been applied to localize angiotensin II receptors in the rat and dog kidney. Discrete grain densities, representing specific binding of [1251 ] -angiotensin II, have been detected in areas corresponding to the glomeruli, cortex, medulla and the ureter. The use of coverslip techniques has further localized this highly specific binding to the glomerular basement membrane, glomerular mesangial cells, smooth muscle lamina of the ureter, and the vasa recta in the medulla. In vitro receptor autoradiographic techniques are particularly advantageous for the localization of receptors for peptide neurotransmitters and hormones. The presence of peptidase inhibitors in the incubation media such as dithiothreitol, EGTA and bovine albumin provide metabolic stability so the bound peptide remains intact. This assures that the specific binding represents actual [ 125 I] -angiotensin II binding rather than labeled peptide fragments which can occur, due to metabolism, when using in vivo techniques. Nonspecific binding of labeled pept ide fragments is also reduced, allowing more precise measurement of the specific angiotensin II recognition sites in the tissues. Receptor labeling is thus performed under optimal conditions to produce a high degree of specific binding (Table 4). The renal renin-angiotensin system has been implicated in several types of hypertension, with the kidney being a source of angiotensin 74 II as well as one of the major sites of angiotensin's action (Kaplan and Sileh, 1964; Ferris, 1982). Physiologically, angiotensin II exerts a variety of effects on the kidney. It is a potent systemic vasoconstrictor and hypertensive agent, increasing both systemic and renal vascular resistance (Freeman et al., 1973; Zimmerman, 1973; Schweitzer, 1980). However, the specific effects of angiotensin on renal hemodynamics are presently ambiguous. It appears to cause an alteration in the muscle tone of the afferent or efferent arteriole which can produce an increase or decrease in glomerular filtration rate (Bock et al., 1968; Navar and Langford, 1974; Freeman and Davis, 1979; Frega et al., 1980; Hall et al., 1981; Huang et al., 1982). The presence of angiotensin II receptors in the renal vasculature provides additional evidence for the ability of angiotensin to alter renal vascular tone. Saturation studies indicate two receptor types of similar Bm ax , but different affinities exist in the kidney. A high affinity receptor population was found in the vasa recta and ureter. A lower affinity receptor population was localized to the glomerulus and medulla outside the vasa recta, presumably associated with tubular populations. The high affinity receptor may be associated with the smooth muscle in the vasculature and ureter while the lower affinity receptor could be associated with other types of tissue. Angiotensin II also appears to cause a reduct ion in glomerular size, an effect which may be mediated by glomerular mesangial cell contraction (Hornyck et al., 1972; Sraer et al., 1974»). Previous binding studies utilizing homogenate techniques have demonstrated specific binding of labeled angiotensin II in isolated glomeruli 75 (Sraer et al., 1974; Brown et al., 1980; Chansel, 1982) and these binding sites were further localized to the basement membrane (Sraer et al., 1977) • The use of in vivo autoradiography after systemic administration of labeled angiotensin II has also resulted in the localization of binding to the glomerular mesangial cells (Osborne et al., 1975). However, in this in vivo study, no binding to arterioles or tubular elements was reported. Our use of an in vitro technique demonstrates specific binding not only in the glomerular basement membrane and mesengial cells, but in the vasa recta and ureter as well. One can conclude that these additional receptor populations may either be inaccessible to circulating angiotensin II or that radiolabeled angiotensin II administered systemically is degraded either before reaching these sites or before the tissues could be fixed. Using the coverslip technique, a low density of autoradiographic grains was seen in the region of the proximal tubule and in the areas of the medulla containing the loop of Henle. These results indicate that angiotensin II receptors may be associated with these tubular elements. These findings support the physiological observations that angiotensin II and its analogs which can directly increase the proximal tubular sodium reabsorption rate (Johnson and ¥~lvin, 1977; Harris, 1979; Huang et al., 1982). Angiotensin II has also been implicated in alterations of reabsorption in the segment between the late proximal and early distal tubule (Ploth et al., 1979; Ploth and Navar, 1979; Ploth and Roy, 1982). The localization of autoradiographic grains in areas corresponding to the ascending loop of Henle suggest this segment may be the site of action. Since 76 specific angiotensin binding has been demonstrated in isolated rat renal brush border membranes (Brown and Douglas, 1982), our results indicate that these receptors are probably on the luminal side of the proximal tubule and are presumably inaccessible to circulating hormone. Immunoreactive angiotensin II has been detected in the kidney in several studies. Histochemical techniques have localized angiotensin-like immunoreactivity in the juxtaglomerular cells, afferent arteriole and mesangial cells (Taugner and Hackenthal, 1981; Naruse et al., 1982; Taugner et al., 1982a,b). These structures could release angiotensin II in order to activate the angiotensin II receptors we identified in each corresponding region. However, angiotensin II receptors have also been localized in the medulla and ureters. The functional significance of the binding to the ureter is unknown. However, since smooth muscle is a maj or component of the ureter, and since angiotensin II receptors have been demonstrated to exist in other nonvascular smooth muscles including the bladder (Aquilera and Catt., 1981). Because angiotensin II has been shown to contract smooth muscle in several tissues, it follows that angiotensin II may function to contract the ureter to move urine to the bladder. The use of in vitro receptor autoradiography in this study has uncovered the existence of angiotensin II receptors in areas where their existence was previously unidentified. The examination of the dissociation constants and receptor numbers as described here would be virtually impossible using any other technique currently available. 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Name: Birthdate Birthplace Universities 1976-1981 1981-1985 Degrees Publications: Abstracts CURRICULUM VITAE Donald Richard Gehlert June 27, 1958 Milwaukee, Wisconsin Purdue University Lafayette, Indiana University of Utah Salt Lake City, Utah B.S. in Pharmacy Purdue University Ph.D. in Pharmacology University of Utah Gehlert, D.R., K.G. Ferrell and D.E. Rollins. "Disposition of Emetine in the Rat." ASPET, 1983. Gehlert, D.R., H.I. Yamamura and J.K. Wamsley. "Autoradiographic Localization of Neurotransmitter Receptors in Brain and Periphery." Symposium on Nuerotransmitter Receptor Regulation, Interactions and Coupling to Effectors, Hiroshima, Japan, 1983. Wamsley, J.K. and D.R. Gehlert. "Autoradiographic Localization of Subtypes of Muscarinic Agonist and Antagonist Binding Sites: Alterations Following CNS Lesions." International Conference on Dynamics of Cholinergic Function, Oglebay Park, West Virginia, 1983. Yamamura, H.I., J.K. Wamsley, D.R. Gehlert, T.W. Vickroy, M. Watson and W.R. Roeske. "Differential Light Microscopic Autoradiographic Localization of Muscarinic Cholinergic Receptors in the Brainstem and Spinal Cord of the Rat Using Radiolabeled Pirenzepine." International Conference on the Dynamics of Cholinergic Function, Oglebay Park, West Virginia, 1983. Gehlert, D.R., J.K. Wamsley, W.R. Roeske and H.I. Yamamura. "Muscarinic Antagonist Binding Site Heteroge~eity as Evidenced by A~toradiography After Direct Labeling with [ H]-QNB and [ H]-Pirenzepine." Society for Neuroscience, 1983. 82 Speth, R.C., C.L. Chernicky, D.R. Gehlert and J.K. Wamsley. "Autoradiographic Localization of Angiotensin II Recognition Sites in the Dog CNS." Society for Neuroscience, 1983. G~hlert, D.R. and J.K. Wamsley. "Autoradiographic Localization of [ H]-Sulpiride Binding Sites in the Rat CNS." FASEB, 1984. Yamamura, H.I., K. Gee, DS Gehlert, J.K. Wamsley, and W. Roeske. "Specific High Affinity [ H]R05-4864 Benzodiazepine Binding Sites in the Brain and Periphery." International Symposia on Endocoids, Dallas, TX, 1984. Wamsley, J.K., R.C. Speth and D.R. Gehlert. "Microscopic Analysis of the Density and Distribution of A-II Receptors in the Brain, Adrenal and Kidney." International Symposia on Endocoids, Dal~as, TX, 1984. Gehlert, D.R., a~d J.K. Wamsley. 3"Autoradiographic ~ocalization of Alterations in [ H]-Imipramine, [ H]-Serotonin and [ H]-Ketanserin Binding Sites After Chronic Imipramine Administration." Society For Neuroscience, 1984. Gibb, J.W., C.J. Schmidt, D.R. Gehlert, M.A. Peat, P.K. Sonsalla, J.K. Wamsley and G.R. Hanson. "Studies on the Mechanism of Tolerance to Methamphetamine." Society for Neuroscience, 1984. Wamsley, J.K., D.R. Gehlert and R.W. Olsen. "Autoradiographic Localization of Brain Regions where the Distribution of Benzodiazepine and Low Affinity GABA Receptors Overlap." Society For Neuroscience, 1984. Gehlert, D.R., R.C. Speth and J.K. Wamsley. "Localization of Angiotensin II Receptors in Rat Brain and Kidney by Quantitative Autoradiography." ASPET, 1984. Gibb, J.W., C.J. Schmidt, D.R. Gehlert, M.A. Peat, P.K. Sonsalla, J.K. Wamsley and G.R. Hanson. "Studies on the Mechanism of Tolerance to Methamphetamine." Mountain-West Chapter of the Society of Toxicology Meetings, 1984. Wamsley, J.K., D.R. Gehlert and A. Barnett. "Selectivity for Benzodiazepine Receptor Subtypes; A Comparison Study for Sedative-Hypnotics in the Human Brain." American Psychiatric Association Meetings, Dallas, TX, 1985. Dawson, T.M., D.R. Gehlert and J.K. Wamsley. "Quantitative Autoradiographic Demonstration of High and Low Affinity Agonist Binding of D-2 Dopamine Receptors." Western Section of the AFCR Meetings, Carmel, CA, 1985. 83 Unis, A.S., T.M. Dawson, D.R. Gehlert, E.W. Mitchell and J.K. Wamsley. 3Autoradiographic Localization and Binding Characteristics of H(+)~Amphetamine to Rat Brain Sections." Western Section of the AFCR Meetings, Carmel, CA, 1985. ~1cCabe, R.T., D.R. Gehlert, T.M. Dawson, C.J. Schmidt, G.R. Hanson, J.W. Gibb and J.K. Wamsley. "Alterations of Dopamine Receptor Subtypes in the Rat CNS Following Methamphetamine Treatment."FASEB, 1985. Gehlert, D.R., R.C. Speth and J.K. Wamsley. "Quantitative Autoradiography of Angiotensin II (All) and Alpha-2 Receptors in the Spontaneously Hypertensive Rat (SHR)." FASEB, 1985. Concas, A., A. Barnett, J.K. Wamsley, D. Gehlert and H.I. Yamamura. "Temperature Regulation of Quazepam, A 1-N-Trifluoroethyl Benzodiazepine." FASEB, 1985. Gulya, K., D. Gehlert, S.P. Duckles, J.K. Wamsley and H.I. Yamamura. "Light Microscopic Autoradiographic Localization of Delta Opioid Receptors in Rat Brain Using A Highly Selective Bis-Penicillamine Cyclic Enkephalin Analog." FASEB, 1985. Ritter, J.K., D.R. Gehlert, J.W. Gibb, J.K. Wamsley and G.R. Hanson. "Neuronal Localization of Substance P Receptors in the Rat Striatum." FASEB, 1985. Yamamura, H.I., H.I. Mosberg, T. Pelton, K.W. Gee, K. Gulya, J.K. Wamsley, D.R. Gehlert and V.J. Hruby. "Conformationally Restricted Penicillamine Containing Cyclic Enkephalin and Somatostatin Analogs with High Delta and Mu Opioid Receptor Selectivity, Respectively." Fifth International Washington Spring Symposia, Neural and Endocrine Peptides and Receptors, Washington D.C., 1985. Dawson, T.M., D.R. Gehlert and J.K. Wamsley. "Quantitative Autoradiographic Demonstration of High and Low Affinity Agonist Binding of D-2 Dopamine Receptors." National Meeting of the AFCR, Washington, D.C., 1985. Papers Gehlert, D.R., H.I. Yamamura and J.K. Wamsley. nAutoradiographic Localization of 'Periphe3al' Benzodiazepine Binding Sites in the Rat Brain and Kidney Using [H]-R05-4864." Eur. J. Pharmacol., 95:329-330, 1983. Wamsley, J.K., D.R. Gehlert, W.R. Roeske and H.I. Yamamura. "Muscarinic Antagonist Binding Site Heteroge~eity as Evidenced by A~toradiography after Direct Labeling with [ H]-QNB and [H]-Pirenzepine." Life Sci., 34:1395-1402, 1984. 84 Gehlert, D.R., R.C. Speth and J.K. Wamsley. "Autoradiographic Localization of Angiotensin II Receptors in the Rat Brain and Kidney." Eur. J. Pharmacol., 98:311-312, 1984. Gehlert, D.R., H.I. Yamamura and J.K. Wamsley. "Use of Autoradiographic Techniques for the Localization of Neurotransmitter Receptors in the Brain and Periphery: Recent Applications." In: Neurotransmitter Receptors: Mechanisms of Action and Regulation, Ed. by S. Kito, T. Segawa, K. Kuriyama, H.I. Yamamura and R.W. Olsen, Adv. Expo Med. BioI., 175:255-270, Plenum Press, New York, 1984. G~hlert, D.R. and J.K. Wamsley. "Autoradiographic Localization of [ H]-Sulpiride Binding Sites in the Rat Brain." Eur. J. Pharmol. 98:311-312, 1984. Gehlert, D.R., R.C. Speth, D.P. Healy and J.K. Wamsley. "Autoradiographic Localization of Angiotensin II Receptors in the Rat Brainstem." Life Sci., 34:1565-1571, 1984. Gehlert, D.R., T.M3 Dawson, H.I. Yamamura and J.K. Wamsley. "Localization of [ H]-Forskolin Binding Sites in the Rat Brain by Quantitative Autoradiography." Eur. J. Pharmacol., 106:223-225, 1984. Gehlert, D.R., W.A. Morey and J.K. Wamsley. "Alterations in Muscarinic Cholinergic Receptor Densities Induced by Thiamine Deficiency: Autoradiographic Detection of Changes in High and Low Affinity Agonist Sites." J. Neurosci. Res., in press. Gehlert, D.R., R.C. Speth and J.K. Wamsley. "In-Vitro Autoradiographic Localization of Angiotensin II Receptors in the Rat and Dog Kidney." Peptides, in press. Speth, R.C., J.K. Wamsley, D.R. Gehlert, C.L. Chernicky, K.L. Barnes, and C.M. Ferrario. "Angiotensin II Receptor Localization in the Canine CNS." Brain Res., in press. Gehlert, D.R., H.I. Yamamura and J.K. Wamsley. "Autoradiographic Localization of Peripheral-type Benzodiazepine Binding Sites in the Rat Brain, Heart and Kidney." Naun-Schmeidebergs Arch. Pharmacol., in press. Gehlert, D.R., H.I. Yamamura, W.R. Roeske and J.K. Wamsley. "Autoradiographlc Localization of Subtypes of Agonist and Antagonist Binding Sites: Alterations Following CNS Lesions." In: Dynamics of Cholinergic Function, Ed. by I. Hanin, Plenum Press, New York, in press. Gehlert, D.R. and J.K. Wamsley. "Dopamine Recepto3s in the Rat Brain: Quantitative Autoradiographic Localization Using [ H]-Sulpiride." Neurochemistry Internat., in press. 85 Schmidt, C.J., D.R. Gehlert, M.A. Peat, P.K. Sonsalla, G.R. Hanson, J.K. Wamsley and J.W. Gibb. "Studies On the Mechanism of Tolerance to Methamphetamine." Brain Res., in press. Yamamura, H.I., D.R. Gehlert, K.W. Gee'3J.K. Wamsley, W.D. Horst and W.R. Roeske. "Specific High Affinity [ H]-R05-4864 Benzodiazepine Binding Sites in the Brain and Periphery." In: Endocoids, Ed. by H. Lal, Alan R. Liss Inc., N.Y., in press. Gehlert, D.R., R.C. Speth and J.K. Wamsley. "Quantitative Autoradiography of Angiotensin II Receptors in the Brain and Kidney: Focus on Cardiovascular Implications." In: Endocoids, Ed. by H. Lal, Alan R. Liss Inc., N.Y., in press. Yamamura, H.I., T.W. Vickroy, D.R. Gehlert, J.K. Wamsley and W.R. Roeske. "Autoradiographic Localization of Musc~rinic Agonist Binding Sites in the Rat Central Nervous System using [ H]-Cis­methyldioxalane." Brain Res., 325:340-344, 1985. Gehlert, D.R., H.I. Yamamura and J.K. Wamsley. "Quantitative A~toradiographic Localization of GAB~ Receptors in Rat Brain using [H](-)Baclofen." Neurosci. Lett., in press. Dawson, T.M., D.R. Gehlert, E.W. Snowhill and J.K. Wamsley. "Quantitative Autoradiographic Evidence for Axonal Transport of Imipramine Receptors in the CNS. II Neurosci. Lett., in press. Wamsley, J.K., T.M. Dawson and D.R. Gehlert. "Autoradiographic Localization of Receptors for Psychotropic Drugs in the CNS." Progress in Neurobiology, in press. Vickroy, T.W., D.R. Gehlert, J.K. Wamsley, W.R. Roeske and H.I. Y~mamura. "Quantitative Light Microscopic Autoradiography of [ H]-Hemicholinium 3 Binding Sites in the Rat Central Nervous System: A Novel Anatomical Marker for Cholinergic Nerve Terminals." Brain Res., in press. Wamsley, J.K., D.R. Gehlert and R.W. Olsen. "The Benzodiazepine­Barbiturate/ Convulsant-GABA Receptor-Chloride Ionophore Complex: Autoradiographic Localization of Individual Components." In: Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, Vol. 6, Receptor Biochemistry and Methodology, Ed. by R.W. Olsen and J.C. Venter, Alan R. Liss Inc., N.Y., in press. Gehlert, D.R., T.M. Dawson, H.I. Y~mamura and J.K. Wamsley. "Quantitative Autoradiography of [ H]-Forskolin Binding Sites in the Rat Brain." Brain Res., submitted. Gi2!ert, D.R., R.C. Speth and J.K. Wamsley. "Distribution of [ IJ-Angiotensin II Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study." J. Neurosci., submitted. Brinton, R.E., J.K. Wamsley, D.R. Gehlert, Y-P. Wan, and H.I. Yamamura. "Vasopressin Metabolite AVP 4 _ 9 Binding Sites in the Rat Kidney: Distribution Distinct from Vasopressin Binding Sites." Eur. J. Pharmacol., in press. Dawson, T.M., D.R. Gehlert, H.I. Yamamura, A. Barnett and J.K. Wamsley. "D-l Dopam~ne Receptors in the Rat Brain: Autoradiographic Localization using [ H]-SCH 23390." Eur. J. Pharmacol., in press. G~hlert, D.R. and J.K. Wamsley. "Evaluation and Recalibration of [ H]-Microscales for Quantitative Autoradiography." J. Neurosci. Meth., submitted. Gehlert, D.R., R.C. Speth and J.K. Wamsley. "Quantitative Autoradiography of Angiotensin II Receptors in the Spontaneously Hypertensive Rat Brain." Hypertension, submitted. 86 Gulya, K., J.K. Wamsley, D.R. Gehlert, T.J. Pelton, S.P. Duckles and H.I. Yamamura. "Light Microscopic Autoradiographic Localization of Somatostatin Receptors in the Rat Brain." J. Pharmacol. Exp. Therap., submitted. Ritter, J.K., D.R. Gehlert, J.W. Gibb, J.K. Wamsley and G.R. Hanson. "Neuronal Localization of Substance P Receptors in the Rat Striatum." Eur. J. Pharmacol., submitted. Gulya, K., D.R. Gehlert, J.K. Wamsley, H.I. Mosberg, V.J. Hruby, S.P. Duckles and H.I. Yamamura. "Autoradiographic Localization of Delta Opioid Receptors in the Rat Brain using a Highly Selective Bis-Penicillamine Cyclic Enkephalin Analog." Eur. J. Pharmacol., submitted. Gulya, K., D.R. Gehlert, J.K. Wamsley, H. Mosberg, V. Hruby, S.P. Duckles and H.I. Yamamura. "Light Microscopic Autoradiographic Localization of Delta Opioid Receptors Using a Highly Selective Bis-Penicillamine Cyclic Enkephalin Analog." J. Pharmacol. Exp. Therap., submitted.