Sodium increases angiotensin II receptors in neuronal cultures from brains of normotensive and hypertensive rats

Brain Research, 370(1986)265-272

265

Elsevier BRE 11596

Sodium Increases Angiotensin II Receptors in Neuronal Cultures from Brains of Normotensive and Hypertensive Rats JUDITH B. FELDSTEIN, COLIN SUMNERS and MOHAN K. RAIZADA

Department of Physiology, Collegeof Medicine, Universityof Florida, Gainesville, FL 32610 (U.S.A.) (Accepted August 13th, 1985)

Key words: neuronal cell culture - - brain - - angiotensin II - - receptor - - sodium - - cation - - spontaneously hypertensive rat

Neuronal cultures from one-day-old brains of Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats were used to determine the effect of sodium ions on angiotensin II (Ang II) receptors in order to understand the mechanism of action of sodium at the cellular level. Incubation with sodium chloride of neuronal cultures from WKY rats caused a rapid and dose-dependent increase in the specific binding of 125I-AngII to its receptors. A 260% increase in the binding was observed with 150 mM NaCI. Neuronal cultures from SH rat brains showed a similar sodium-stimulated increase in Ang II binding; however this increase was 150% greater than that observed in neuronal cultures from WKY rat brain. Kinetic studies of the effects of sodium ions on both WKY and SH neuronal cultures showed an increase in the dissociation of ~25I-AngII from its receptors in the presence of sodium ions. In addition, Scatchard analysis revealed that the increase in binding caused by sodium was due to an increase in the number of Ang II receptors. These observations indicate that sodium ions increase the number of Ang II specific receptors in intact neuronal cells and that this stimulation was more pronounced in neuronal cells from SH rat brains compared with WKY controls.

INTRODUCTION Numerous studies have suggested a relationship between sodium ions and the brain renin-angiotensin system2,to, 11. In particular, rats that are fed a low sodium diet show decreased pressor and dipsogenic responsiveness following central angiotensin II (Ang II) injections, along with a decreased n u m b e r of brain A n g II receptorslO. However, since low sodium diets also result in volume contraction and increased plasma A n g II levels, the question of whether sodium is directly influencing central A n g II receptors in this situation is unclear. Using brain m e m b r a n e s prepared from control and sodium depleted rats it is implied that sodium ions are able to stimulate A n g II binding to its specific receptor3,a.lt and this is not unusual considering the effects of this ion on catecholaminergic, opiate and other peptidergic systems7,8.t2,17. In the present study we have attempted to determine the role of sodium ions in regulating brain A n g II receptors using intact cultured neurons. The use of

neuronal cultures affords an excellent system for this investigation because changes in extracellular sodium levels can be tightly controlled. Further, non-specific hemodynamic or hormonal influences are excluded. In addition, neuronal cultures from the brains of spontaneously hypertensive (SH) rats have shown profound changes in the expression of the angiotensin system and its interaction with catecholamines 14A9-21. It has been shown that the influence of dietary sodium on central A n g II receptor binding2 and pressor responses to centrally injected A n g 1110 are altered in SH rats compared with W i s t a r - K y o t o (WKY) rats, Therefore, we also determined the effects of sodium ions on A n g II specific binding in neurons cultured from the brains of SH rats. These studies show that sodium ions cause increased A n g II receptors in neurons from both W K Y and SH rat brains and that A n g II receptors on the neurons from SH rats are hypersensitive to changes in sodium concentrations.

Correspondence: M. Raizada, Department of Physiology, Box J-274, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

266 MATERIALS AND METHODS One-day-old rats from the WKY and SH strains were obtained from our breeding stock. Dulbecco's modified Eagle's medium, fetal bovine serum, horse serum and Fungizone were purchased from GIBCO, Grand Island, NY. Deoxyribonuclease I and 1 x crystalline trypsin (150 units/mg) were from Worthington Biochemicals, Freehold, NJ. Bovine serum albumin fraction V, cytosine arabinoside, poly L-lysine (MW 150,000), angiotensin II, choline chloride, RbC1, and CsC1 were purchased from Sigma Chemical Co., St. Louis, MO. Ang 1I was iodinated with a resulting spec.act, of 1500-1800 ~Ci/~g 14'15. Iodinated Ang II migrated as a homogeneous peak on a reverse phase high-performance liquid chromatographic column 15.

Preparation of neuronal cultures from the brain of WKY and SH rats Primary cultures of neuronal cells from one-dayold normotensive (WKY) and spontaneously hypertensive (SH) strains of rats were prepared and maintained as described previously 13a4. Brains from oneday-old rats were removed and placed in an isotonic salt solution, (mM): NaC1 137, KC1 5.4, NazHPO4 0.17, KH2PO 4 0.22, glucose 5.5 and sucrose 59.0, pH 7,2, containing 100 U penicillin G, 100 big streptomycin and 0.25/~g Fungizone per milliliter. The brains were carefully cleansed free of blood vessels and pia mater under a magnifying glass, washed with isotonic salt solution and chopped into 1-2 mm 3 pieces. They were subjected to trypsin and deoxyribonuclease I treatment essentially as described previously13,14. Dissociated cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin G, 100 ~g/ml streptomycin and 10% fetal bovine serum, sedimented at 100 g for 10 min and washed once with D M E M containing 10% FBS. Cells, 2.8 x 106/mi, suspended in D M E M with 10% FBS, were plated in 35 mm diameter tissue culture dishes precoated with poly L-lysine 13. Cells were allowed to establish in culture for 3 days at 37 °C in a humidified incubator with 5% CO2/95% air. After this time, the growth medium was replaced with DMEM containing 10% horse serum, 5% FBS and 10/~M cytosine arabinosidel3.14. After two days, the medium containing cytosine arabinoside was re-

placed with DMEM containing 10% horse serum and cells were allowed to grow for a further 6-10 days before being used for Ang I1 binding experiments. Trypsin dissociation of one-day-old brains provided a comparable number of cells (24.7 ±~ 1.2 million cells per brain from both WKY and SH strains of rats). The neuronal cultures from both strains of rat brains have also been shown to contain a comparable number of neurons and amount of protein per culture dish and endogenous levels of norepinephrine and dopamine l~. These observations have suggested that neuronal cultures from WKY and SH rats are similar in composition and can be used for comparative studies.

Measurement of l:51-Angiotensin H binding Specific binding of lZq-Ang II to 15-day-old neuronal cultures which were prepared from the brains of one-day-old WKY and SH rat brains was determined as described elsewhere16. In summary, neuronal cells, maintained in culture for 15 days, were washed twice with phosphate buffered saline (PBS), pH 7.2 and triplicate cultures were incubated for 30 min at 24 °C with 0.5 ml PBS containing 0.05-0.15 nM 1251Ang II (100,000 cpm) depending upon spec.act. and 1.6% bovine serum albumin (BSA). In addition, triplicate cultures were also incubated for 30 min at 24 °C with 50-150 nM unlabelled Ang II, 0.05-0.15 nM 125I-Ang II and 1.6% BSA in 0.5 ml PBS for determination of non-specific binding. Following incubation, cultures were rapidly rinsed 4 times with 2 ml of ice-cold PBS, pH 7.2, to remove unbound 125I-Ang II. The cells were dissolved in 0.5 ml of 2 N N a O H and transferred to tubes. Each plate was rinsed with 0.5 ml water which was combined with original sampies. Radioactivity was determined with a Beckman 5500 gamma counter at 79% efficiency for 12sI. The specific binding of 125I-Ang II was calculated by subtracting the amount of radioactivity bound in the presence of 150 nM unlabelled Ang II from the total radioactivity bound to cells. The protein content of each culture dish used for lZq-Ang II binding was determined by the method of Lowry et al. with the use of bovine serum albumin as protein standard 9. All the data were presented as fmol lZSI-Ang II specifically bound per milligram protein or as a percent of maximum binding/mg protein.

267

Effects of cations on 125I-Ang H binding Choline chloride buffer (150 mM choline chloride, 5 mM Tris-HC1, pH 7.2) was used in the binding assays instead of PBS in order to study the effects of sodium ions on 125I-Ang II binding. For example, in dose-response experiments with NaCI, neuronal cultures were first washed twice with choline chloride buffer, then incubated with 0.15 nM ~25I-Ang II, 1.6% BSA and various concentrations of sodium chloride. The osmolarity of the reaction mixture was maintained by appropriate concentrations of choline chloride buffer. After the binding, neuronal cultures were washed with ice-cold choline chloride buffer and specific 125I-Ang II binding was determined as described above.

Kinetic analysis of l25I-Ang H binding It was found in previous studies14A 6 that the amount of bound 125I-Ang II was less than 3% of the total Ang II and no significant degradation of the peptide was detected during the binding assay. These observations suggested that the concentration of free 125I-Ang II in the reaction medium was assumed to be constant and the binding was analyzed as a first order reaction. Dissociation (k_l) rates and affinity constants (Ka) for Ang II binding to neuronal cultures from both strains of rat brain in the presence of ChC1 and NaC1 were calculated as described previously 14. The value for tl/2 w a s calculated as tl/2 = ln2/k_v RESULTS

Effect of sodium on 125I-Ang H binding to neuronal cultures of W K Y rat brain Binding of 125I-Ang II to neuronal cultures of WKY rat brains increased when they were incubated with increasing concentrations of NaCI during the Ang II binding assay. This stimulatory effect of NaC1 was dose-dependent (Fig. 1). In the absence of NaC1, the binding was 0.9 _+ 0.1 (S.E.M.) fmol/mg protein, and in the presence of 150 mM NaC1, it reached a maximum of 2.4 + 0.2 (S.E.M.) fmol/mg protein. A 50% increase in Ang II binding occurred at a concentration of 90 mM NaC1. The increase in Ang II binding was the result of an increase in the specific binding rather than any significant change in the non-specific binding, which represented only 5 - 1 0 % of the total binding. Thus 150 mM NaC1 caused a 2.6-fold

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Fig. 1. Effect of sodium concentration on the binding of 1251Ang II neuronal cultures from WKY rats. Neuronal cultures were rinsed with choline chloride buffer and incubated with 0.1 nM 125I-Ang II for 30 min at 24 °C. The reaction buffer contained 5 mM Tris adjusted to pH 7.2 with HCI, 1.6% BSA and the appropriate concentration of sodium ions (as indicated in the figure). Ionic strength was maintained using choline chloride. Specific binding was calculated as the difference between total and non-specific binding as described in the text and represented 90-95% of the total binding. Data are expressed as mean + S.E.M. Comparisons with control, 100 mM NaCI, 150 mM NaCI were significantly different at 1% level (NewmanKeuls test). Comparison with control 10, 20, 40 mM NaC1 were not significantly different.

increase in the specific binding of 125I-Ang II to these neuronal cultures. All of the monovalent cations used caused an increase in the specific binding of 1251Ang II to a varying degree (data not shown). A maximum stimulation of 370% was observed with LiC1 and a minimum stimulation of 180% was observed with RbCI, where ChCI binding was considered control (100%). All of the cations used were prepared from their chloride salts and their ability to stimulate Ang I! binding occurred in the following order of potency: Li > Na > K > Cs > Rb. Binding of 125I-Ang II to neuronal cultures in the presence and absence of sodium ion was time-dependent (Fig. 2). In both cases specific binding increased linearly up to 60 rain although the binding in the presence of the sodium ion was significantly greater at every time point. Analysis of this data, based on the first order kinetics as described previously l~, provided association rates similar for both ChC1 (4.9 x 10-4S -1) and NaC1 (3.7 x 10-4s-1). Dissociation of ~25I-Ang II bound to its receptor in the presence of ChC1 and NaC1 was determined as follows. Neuronal cultures were allowed to bind 125I-

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Fig. 2. Time course of ~25I-Ang II binding to neuronal cultures from brains of WKY rats. Fifteen-day-old neuronal cultures prepared from one-day-old WKY rats, were washed twice with 2 ml each of either 150 mM ChCI (0) or NaCI (&) buffer containing 5 mM Tris, pH 7.2 and incubated at 24 °C for the indicated times with the appropriate assay mixture as described in the text. The specific binding of 125I-Ang II was expressed as the difference between the total binding and the non-specific binding (mean + S.E.M.). Comparisons between control and NaCl-buffered cells at each time-point revealed significant differences at the 5% level, for 5 and 10 min (P < 0.05) and at 1% level (P < 0.01) for all other time-points (Student's t-test).

Fig. 3. Dissociation of bound ~5I-Ang II from neuronal cultures of WKY rats. Neuronal cultures were grown for 15 days and incubated with 125I-Ang II in NaC1 buffer (&) or in ChCI buffer (0) at 24 °C for 30 min under the standard binding assay conditions as described in Materials and Methods. The cultures were washed with either ChCI buffer (0) or NaCI buffer (A) to remove unbound radioactivity and were then incubated with 2 ml of the appropriate buffer containing 150 nM unlabelled Ang II at 24 °C. At the indicated times triplicate cultures were washed 4 times with ice-cold buffer. Specific binding was determined and the data were expressed as the mean of percentage of the maximum ~25I-Ang II specifically bound at each time _+ S.E.M. One hundred percent was 5.6 _+ 0.8 fmol of 12-SI-AngII bound/mg protein for NaCI and 2.7 ___0.4 fmol/mg protein for ChCI.

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belled A n g II to d e t e r m i n e the rate of dissociation of ~251-Ang II b o u n d to m e m b r a n e r e c e p t o r s . A l t h o u g h dissociation of b o u n d ~25I-Ang II f r o m the cells was

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time d e p e n d e n t in b o t h buffers, in 90 min 9 0 % of the radioactivity was dissociated f r o m cells i n c u b a t e d with NaC! in c o n t r a s t with only 6 0 % f r o m cells incubated with ChC1 (Fig. 3). This r e s u l t e d in a tv~ of dissociation of 29 and 84 min in the p r e s e n c e of s o d i u m ion and choline c h l o r i d e r e s p e c t i v e l y , indicating that s o d i u m caused an a c c e l e r a t e d dissociation of 1251A n g II f r o m the r e c e p t o r s . In o r d e r to f u r t h e r support this c o n t e n t i o n , the d a t a w e r e s u b j e c t e d to firsto r d e r kinetic analysis 14by p l o t t i n g ln[B]/[Bo] against time (Fig. 4). Such analysis p r o v i d e d straight lines

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Fig. 4. Kinetic analysis of dissociation of 1251-Ang II to neuronal cultures from WKY rats. Dissociation data from Fig. 3 were plotted according to a first-order rate equation and dissociation rate constants were calculated from the slopes as described previously t4. A linear regression analysis was performed to obtain the value for the slope, y = 0.98 and 0.99 for ChCI (0) and NaCI (A), respectively. Values for the slope of ChCI vs 150 mM NaCI wash revealed a significant difference according to Student's t-test, P << 0.01.

269 (correlation coefficients of 0.98 and 0.99) with significantly different slopes for NaCI and ChCI. The dissociation rate constants k_ t obtained in the presence of ChC1 were 1.4 × 10 -4 s -1 and in the presence of NaCI, 4.0 × 10 -4 s -t. T h u s , the data obtained from these experiments suggest that the dissociation of ]25I-Ang II from its receptors was significantly increased in the presence of sodium ions. This finding is distinct from the observation that sodium ions cause a concentration and time-dependent increase in the specific binding (Figs. 1 and 2). Specific binding of ]25I-Ang II was studied as a function of Ang II concentrations in the presence and absence of sodium ions to clarify these observations. Neuronal cultures were incubated with increasing concentrations of ]25I-Ang II for 90 min at 24 °C. Cultures were also incubated with similar concentrations of 125I-Ang II containing 150 nM unlabelled Ang II (for non-specific binding). Specific binding of 1251Ang II increased with increasing concentrations of labelled Ang II in cultures incubated with either ChC1 or NaCI and the binding was 3 - 4 fold greater in the presence of sodium ions (Fig. 5). Scatchard analysis 18 provided straight lines for both treatments suggesting the presence of homogenous sites (Fig. 6). The affinity constants (Ka) calculated from these data gave

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Fig. 5. Effect of Ang II concentration of ~25I-Ang II binding to neuronal cultures from WKY rats. Neuronal cultures were grown for 15 days and the binding of 125I-Ang II was determined in the presence of either 150 mM ChCI (Q) or 150 mM NaCI (&) buffer. Assay mixtures were prepared in these different concentrations of labelled Ang II (0.05-1 nM). Data are presented as specific binding (mean + S.E.M.). Comparisons between control and 150 mM NaCI groups were significantly different for each point according to a Student's t-test, P < 0.01.

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Fig. 6. Scatchard analysis of 1251-AngII binding to neuronal cultures in the presence of ChCI (O) and NaCI (A). Data from Fig. 6 were plotted as bound hormone [B], abscissa against bound/free ([B]/[F]) ordinate. Slopes and intercepts were used to calculate affinity constants and number of binding sites.

values of 4 x 109M -1 in the presence of ChCI and 1.8 × 109 M -t in the presence of NaC1. The total number of binding sites per cell were 40 fmol/mg protein in the absence and 280 fmol/mg protein in the presence of sodium ion. Thus an increase in the binding of Ang II stimulated by sodium is due to its profound effects on the binding capacity.

Effect of sodium on ~esI-Ang H binding to neuronal cultures of SH rat brain Previous studies from our laboratory have demonstrated that neuronal cultures prepared from 1day-old rat brain from SH rats possess a 2-3-fold greater number of Ang II-specific receptors 14 and significantly lower levels of endogenous Ang IllL It has been concluded from these observations that changes in Ang II systems demonstrated under in vivo conditions are preserved in these neuronal cultures. The effect of sodium ions on the binding of 125IAng II in neuronal cultures of SH rat brain was studied to determine if these cells respond in a manner similar to cultures made from WKY rat brain. Sodium ion caused a concentration-dependent increase in the binding of 125I-Ang II (Fig. 7) with 50% stimulation occurring at a concentration of 90 mM NaCI and a maximum stimulation with 150 mM NaC1. Comparison of the data from both strains of neuronal cultures revealed that the binding levels in SH neuronal cultures were greater (+ 4.5 fmol/mg protein) compared with WKY cultures (+ 2.4

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Fig. 7. Effect of sodium ion concentration on the binding of t25IAng II in neuronal cultures from SH rats. Fifteen-day-old neuronal cultures from both WKY and SH rat brains were washed with 150 mM ChCl/5 mM Tris buffer, pH 7.2. Binding was performed essentially as described in the legend to Fig. 1. Specific binding was determined and the data were expressed as the mean percentage of maximum 125I-Ang II bound at each concentration + S.E.M. One hundred percent for WKY cultures ( 0 ) at zero NaCl concentration was 0.94 + 0.1 fmol 125I-Ang II specifically bound/rag protein and for SH cultures (&) it was 1.2 + 0.05 fmol 125I-Ang II specifically bound/mg protein bound. Comparisons of data in the absence of NaCl (control) with 100 mM NaCl and 150 mM NaCl showed a significant difference for both WKY and SH rat brain cultures at 1% level (Newman-Keuls test). i

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their WKY counterpart. Binding in the presence and absence of sodium ions was time-dependent and analysis of the data provided straight lines for the slope in a manner similar to that of WKY (data not shown). A time-dependent dissociation of bound 1251-Ang II was observed in which 85% and 42% of the ligand was dissociated in 90 min, in the presence and absence of sodium ions, respectively (Fig. 8). Analysis of the dissociation data according to first-order kinetics was performed. The dissociation rate constants (k_t) were 4 x 10-5 s-~ in the absence of sodium ions and 4 x 10-a s-1 in the presence of sodium (Fig. 9). DISCUSSION

This study represents the first demonstration of the stimulation of Ang II binding by sodium ions in intact neuronal cells derived from the brains of normotensive and spontaneously hypertensive rats. The increased binding of Ang II to its receptor in the presence of sodium ions appears to be a unique characteristic of this receptor that this laboratory as well as others have s h o w n 2'4"22. In contrast to this finding, sodium has been shown to decrease the binding of many other agonists to their hormone receptors 5.6,s.t2-17. Three distinct regulatory effects of sodi-

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Fig. 8. Dissociation of boundX25I-Ang II from neuronal cultures of SH rats which were grown for 15 days and then incubated with 125I-Ang II in 150 mM NaCI buffer, pH 7.2 at 24 °C for 30 min essentially as described in the legend to Fig. 3. Cultures were washed 4 times, 2 ml each with ice-cold ChCI buffer and incubated with 150 mM NaCI buffer ( A ) or with 150 mM ChCI buffer (O) containing 150 nM unlabelled Ang II at 24 °C. At the indicated time-periods, the buffer was removed from the dishes, cultures were washed 4 times with ice-cold ChCI or NaCI buffer and binding of lz5I-Ang II was determined. Data were presented as mean of percentage of the maximum 125IAng II bound at each time + S.E.M. One hundred percent was 75 + 5 fmol 125I-Ang II bound/mg protein.

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Fig. 9. Kinetic analysis of dissociation of 125I-Ang lI from neuronal cultures of SH rats. Dissociation data from Fig. 8 were plotted according to a first-order rate equation and dissociation rate constants were calculated from the slope as described previously 14. A linear regression analysis was performed to obtain values for the slope which were significantly different, P < < 0.01.

271 um ions upon the binding of A n g II to its receptors on neuronal cultures from the rat brain have been observed. These are: an increase in the rate of dissociation of A n g II from its receptors, an increase in the number of Ang II receptors and a consistently greater magnitude of stimulation of A n g II receptors in SH rat brain neurons compared with those observed in neuronal cultures. Thus, neurons from SH rats do not only possess an increased number of Ang II receptors 14 but are also more sensitive to the presence or absence of sodium than are neurons from brains of W K Y rats. Kinetic analysis of 125I-Ang II binding revealed no significant change in the association characteristics of the binding in the presence of sodium ions. In contrast, dissociation characteristics showed a 3-fold and 10-fold decrease in k_ 1 observed in W K Y and SH neuronal cultures, respectively, in the presence of 150 mM NaCI. Binding of 125I-Ang II to neuronal cultures was dependent on the peptide concentration and Scatchard analysis of these data revealed that 150 mM NaCI caused a 7-fold increase in the binding capacity in W K Y neuronal cultures. An increase in the Bmax of Ang II binding to calf cerebellar membranes and rat mesenteric artery have also been reported TM. The increase in the binding capacity of Ang II receptors observed in the presence of sodium ions may be the result of unmasking A n g II receptors which were already present on the surface of the cell, since the stimulation occurred within 30 min. This contention is further supported by the observation that the binding of 125I-Ang II was maximally influenced by incubation with sodium for 30 min during the binding assay. No further change in the binding

REFERENCES 1 Aguilera, G. and Catt, K., Regulation of vascular angiotensin II receptors in the rat during altered sodium intake, Circ. Res., 49 (1981) 751-758. 2 Ashida, T., Ohuchi, Y., Saito, T. and Yazaki, Y., Effects of dietary sodium on brain angiotensin II receptors in spontaneously hypertensive rats, Jpn. Circ. J., 46 (1982) 1328-1336. 3 Bennett, J.P., Jr. and Snyder, S.H., Angiotensin II binding to mammalian brain membranes, J. Biol. Chem., 251 (1976) 7423-7430. 4 Bennett, J.P., Jr. and Snyder, S.H., Regulation of receptor binding interactions of lzsI-Ang II and 125I-AngI-[sarcosine l, leucine8] angiotensin II, an angiotensin antagonist, by sodium ion, Eur. J. Pharmacol., 67 (1980) 1-10.

was observed when cultures were preincubated for 1 h or more in the presence of sodium. The differences in the effects of sodium upon SH and W K Y neuronal cultures were compared. There was a 150% increase in the levels of Ang II receptor binding in SH neuronal cultures compared with those of W K Y at normal physiological (150 mM) concentration of sodium. This difference reflects a greater sensitivity of the effect of sodium ions upon Ang II receptors in SH neurons. The physiological significance of these findings from neuronal cells in culture is highly speculative at the present time. It may be possible that previously reported central effects of sodium on blood pressure and water intake may be mediated by alterations in Ang II receptors. One important conclusion which could be drawn from this study is that neuronal cultures from SH rat brains express hypersensitivity to sodium. This supports our previous observations demonstrating that the expression of Ang II and its receptors is significantly altered in neuronal cells and may be genetically linked with the expression of hypertensive state in the animals 14.15.

ACKNOWLEDGEMENTS The authors wish to thank Diane Childs for typing the manuscript. This work was supported by grants from American Heart Association and N I H HL33610 and NS 19441. M.K.R. is an Established Investigator of the American Heart Association. J.B.F. is a postdoctoral fellow, supported by A H A - Florida Affiliate.

5 Chang, R.S.L. and Snyder, S.H., Histamine Hi-receptor binding sites in guinea pig brain membranes: regulation of agonist interactions by guanine nucleotides and cations, J. Neurochem. 34 (1980) 916-922. 6 Goodman, R.R., Cooper, M.J., Gavish, M. and Snyder, SH., Guanine nucleotide and cation regulation of the binding of [3H]cyclohexyladenosine and [3H]diethylphenylxanthine to adenosine A l receptors in brain membranes, Mol. Pharmacol., 21 (1982) 329-335. 7 Hinkle, P.M. and Kinsella, P.A., Regulation of thyrotropin-releasing hormone binding by monovalent cations and guanyl nucleotides, J. Biol. Chem., 259 (1984) 3445-3449. 8 Innis, R.B., Manning, D.C., Stewart, J.M. and Snyder, S.H., [3H]Bradykinin receptor binding in mammalian tissue membranes, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 2630-2634.

272 9 kowry. O.H., Rosebrough, N.J.. Fa,r, A.L. and Randall, R.J.. Protein measurement with the folin-phenol reagent, J. Biol. ('hem.. 193 (1951) 265-275. 10 Mann, J.F.E., Schiffrin, E i . , Schiller, P.W., Rascher, W., Boucher. R. and Genest, J., Central actions and brain receptor binding of angiotensin II: influence of sodmm intake, Hypertension, 2 (1980) 437-443. 11 Mann, J.F.E., Schiffrin, E.L., Schiller, P.W., Boucher, R. and Genest, J.. The physiological significance of brain angiotensin receptors. In J.P. Buckley and C.M. Ferrario (Eds.), Central Nervous System Mechanisms in lqvpertension, Raven, New York, 1981, pp. 337-349. 12 Pert, C.B. and Snyder, S.H., Opiate receptor binding of agonists and antagonists affected differentially by sodium, Mol. Pharmacol., 10(1974) 868-879. 13 Raizada, M.K., Localization of insulin-like immunoreactivity in the neurons from primary cultures of rat brain, Exp. (Tell Res., 143 (1983) 351-357. 14 Raizada, M.K., Muther, T.F. and Sumners, C., Increased angiotensin lI receptors in neuronal cultures from hypertensive rat brain, Am. J. Physiol., 247 (1984) C364-372. 15 Raizada, M.K., Stenstrom. B., Phillips, M.I. and Sumners, C., Angiotensin lI in neuronal cultures from the brains of normotensive and hypertensive rats, Am. J. Physiol., 247 (1984) Cll5-119.

16 Raizada, M.K., Yang, j.\x,,., Phillips, M.I. and Fellows, R.E., Rat brain cells in primary cuhure: characterization ol angiotensin II binding sites. Brain Research, !(]7 (1981) 343-355. 17 Rouot, B.M., U'Prichard, I).C. and Snyder, S.I [., Multiple (x:-noradrenergic receptor sites in rat brain: selective regulation of high affinity [~H]clonidine binding by' guanine nucleotides and divalenl cations. J. Neurochem., 34 (1980) 374-384. 18 Scatchard, G., The attraction of protein for small molecules and ions, Ann. N. Y. A cad. Sci. , 5I (1949)66(I-672. 19 Sumners, C. and Raizada, M.K., Catecholamine-angiotensin lI receptor interaction in primary cultures of rat brain, Am. J. Physiol., 246 (1984) C502-509. 20 Sumners, C., Phillips, M.I. and Raizada, M.K., Angiotensin II stimulates changes in the norepinephrine content of primary cultures of rat brain, Neurosci, Lett., 36 (1983) 305-309. 21 Sumners, C., Phillips, M.h and Raizada, M.K., Rat brain cells in primary culture: visualization and measurement of catecholamines, Brain Research. 264 (1983) 267-275. 22 Wright, G.B., Alexander, R.W., Ekstein, L.S. and Gimbrone, M.A., Jr., Sodium, divalent cations and guanine nucleotides regulate the affinity of the rat mesenteric artery angiotensin If receptor, Circ. Res., 50 (1982) 462-469,