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Reduced number of adrenal angiotensin II receptors in the spontaneously hypertensive rat
Life Sciences, Vol. 29, pp. 2331-2335 Printed in the U.S.A.
Pergamon Press
REDUCED NLIMBER OF ADRENAL ANGIOTENSIN I I RECEPTORS IN THE SPONTANEOUSLY HYPERTENSIVE RAT Kazuyuki Shimada and Barry L. Fanburg Department of Medicine, New England Medical Center Hospital and Tufts University School of Medicine, 171 Harrison Avenue, Boston, Massachusetts 02111, U.S.A. (Received in final form October 6, 1981)
Summary [1251]angiotensin I I binding to adrenal subcellular p a r t i c l e s was compared between spontaneously hypertensive and Wistar-Kyoto normotensive control rats. The number of angiotensin I I receptors was reduced in adrenals of spontaneously hypertensive rats (P < 0.05) without a change in the a f f i n i t y of angiotensin I I for the binding sites at animal ages of 5, 10 and 15 weeks. This decline of receptor content antedated the abrupt r i s e in blood pressure noted between 5 and i0 weeks. The data suggest the presence of an a l t e r a t i o n of the receptor number in the renin-angiotensinaldosterone system in the spontaneously hypertensive r a t . The spontaneously hypertensive r a t (SHR) has been found to be a useful animal model f o r the study of hypertension and several parameters of the renin-angiotensin-aldosterone system h a v e been assessed in t h i s animal. There is some controversy in the l i t e r a t u r e on the levels of plasma and kidney renin a c t i v i t i e s and of plasma aldosterone in the SHR (1-5). The adrenal output of aldosterone a f t e r the i n j e c t i o n of angiotensin i i has been reported to be reduced in SHR (6). Recently, the adrenal receptor sites f o r angiotensin I I were shown to be dependent on the level of endogenous plasma renin and angiotensin, and i t was suggested that changes of the binding c h a r a c t e r i s t i c s of these receptors may be responsible f o r the a l t e r a t i o n s of the adrenal response to angiotensin I I (7,8). The present study was undertaken to evaluate the specific adrenal receptors for angiotensin I I in the SHR as compared with normotensive control rats. Materials and methods Male SHR of the Aoki-Okamoto s t r a i n and age-matched Wistar-Kyoto normotensive controls w e r e obtained from the Charles River Breeding Laboratories, Inc., Wilmington, Mass. A l l animals were bred in a common environment and fed the same diet containing 0.4% sodium and 0.95% potassium and were given tap water ad l i b i t u m . Systolic blood pressure was measured by a t a i l c u f f plethysmographic technique (9). Angiotensin I I binding studies were performed e s s e n t i a l l y as described by Glossmann et a l . (i0), Adrenals were collected from rats a f t e r decapitation and placed into ice-cold Krebs-Ringer-phosphate buffer (pH 7.4). Adrenals from groups of 4, 5 and 10 rats at 15, i0 and 5 weeks r e s p e c t i v e l y were pooled. Three separate experiments were performed f o r each age group and experiments on paired normotensive controls were carried out in p a r a l l e l with those on SHR. Each age group was studied separately with a
0024-3205/81/292331-05502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2332
Adrenal Angiotensin
II Receptors
in SHR
Vol. 29, No. 22, 1981
maximum of four weeks between studies. Pooled whole adrenals were cut up and homogenized with 10 strokes of a t i g h t f i t t i n g t e f l o n pestle. The homogenate was centrifuged at 1,500 x g for I0 min. The supernate was then centrifuged at 20,000 x g f o r 30 min. The 20,000 x g p e l l e t s were washed once and resuspended in assay buffer containing 120 mM NaCI, 20 mM Tris-HCl (pH 7.4), 4 mM ethylenediaminetetraacetic acid disodium s a l t , 5 mM d i t h i o t h r e i t o l and 10-4 M phenylmethylsulfonyl f l u o r i d e to give a protein concentration of 2.5-4.5 mg/ml. The constituents of the assay buffer prevent breakdown of angiotensin I I by angiotensinase a c t i v i t y ( i 0 , I i ) . For binding assays, 50 ~I aliquots of f r e s h l y prepared subcellular p a r t i c u l a t e f r a c t i o n s were incubated in the above buffer containing 0.2% bovine serum albumin and 53 p M / l i t e r of [1251]angiotensin I I (New England Nuclear; s p e c i f i c a c t i v i t y = 1472 ~Ci/~g) and unlabeled angiotensin I I at various concentrations in a f i n a l volume of 0.2 ml at 22°C f o r 45 min. At the end of the incubation, each assay tube was f i l l e d r a p i d l y with 4 ml of cold buffer and f i l t e r e d through a M i l l i p o r e n i t r o c e l l u l o s e HAWP (0.45 ~m) filter. Each f i l t e r was washed once and r a d i o a c t i v i t y on the f i l t e r was measured in an automatic v-spectrometer. Specific binding of [1251]angiotension I I is defined as the portion of t o t a l binding that is i n h i b i t e d by the presence of an excess ( 1 0 - 5 M ) of unlabeled angiotensin II. Total binding to s u b c e l l u l a r p a r t i c l e s at a [125I]angiotensin I I concentration of 53 pM accounted f o r 36-62% of t o t a l r a d i o a c t i v i t y in the system and s p e c i f i c binding accounted f o r 96-98% of t o t a l binding. Protein was determined by the method of Lowry et K+-ATPase was determined as described by Nakao et al. (13).
al
(12).
The s t a t i s t i c a l p r o b a b i l i t i e s were calculated by Student's comparing the experimental data of each group with the control data.
t
Na+, test,
Results A representative Scatchard plot [14] of specific [1251]angiotensin I I binding to adrenal preparations from one of three experiments on 10 week old animals is shown in Figure 1. The experimental curve obtained with unlabeled angiotensin I I concentrations ranging from 0 to I nM was l i n e a r . The a f f i n i t y constant, indicated by the slope of t h i s l i n e , and the binding capacity, indicated by the intercept at the abscissa, were calculated by least squares analysis. The values of the a f f i n i t y constants ere not s i g n i f i c a n t l y d i f f e r e n t f o r hypertensive (10.9 • 0.6 nM-1, mean w m t j and normotensive (9.2 • 1.6 nM-1) control rats at 10 weeks of age. However, the binding capacity was s i g n i f i c a n t l y lower for the adrenal preparations from hypertensive rats (80 • 4 fmol/mg vs. 138 ± 18 fmol/mg in Wistar-Kyoto controls, p < 0.05). The age dependency of t h i s change is shown in Table I . At each age studied (5, 10 and 15 weeks), the binding capacity of adrenal preparations from hypertensive rats was s i g n i f i c a n t l y lower than controls, whereas the a f f i n i t y constant was s i m i l a r for these two groups. The reduction of binding capacity was already evident at 5 weeks of age before the rapid r i s e in blood pressure occurred. Some v a r i a t i o n s of binding capacity with age were noted both in hypertensive and control rats. We considered the p o s s i b i l i t i e s that the decrease in number of binding s i t e s , expressed in terms of p a r t i c u l a t e f r a c t i o n protein, may represent an increase in protein content of t h i s f r a c t i o n in the SHR or a decrease in receptor-carrying membranes in adrenal subcellular p a r t i c l e preparations from SHR as compared to controls. However, measurements of protein of the p a r t i c u l a t e f r a c t i o n showed no s i g n i f i c a n t differences between the WKY and SHR rats (Table I I ) at any age (p > 0.05). The protein content decreased
Vol. 29, No. 22, 1981
Adrenal Angiotensin II Receptors in SHR
2333
with age in both the SHR and WKY homogenates and by 15 weeks of age was s i g n i f i c a n t l y decreased in the WKY homogenates (p < 0.05) as compared to the
1.5L
• WKY
m I.O U.l n~ ks_ Z
otoO.5
0
50 I00 150 BOUND ANGIOTENSIN TF {fmol/mg) Fig. 1
Scatchard plot of [1251]angiotensin II binding to adrenal subcellular p a r t i c l e s from i0 week old spontaneously hypertensive (o) and Wistar-Kyoto normotensive (e) rats. One of 3 separate experiments is shown. TABLE I Binding Constants of Adrenal Receptors f o r Angiotensin I I from Normotensive (WKY) and Spontaneously Hypertensive (SHR) Rats Age (weeks)
Systolic blood I pressure (mm Hg)
Binding capacity2 (fmol/mg protein)
Affinity 2 (nM- i )
5
WKY SHR
ii0 • 3 118 ± 2
165 • 16 114 • 3*
9.0 * 0.3 8.7 ± 0.5
i0
WKY SHR
146 * 3 206 • 4*
138 ± 18 80 ± 4*
9.2 ± 1.6 10.9 * 0.6
15
WKY SHR
156 * 3 229 * 4*
190 • 9 109 • 9*
i 0 . 0 • 1.3 10.6 * 1.5
iMeans • SE f o r 15, 15 and 12 rats in the 5, 10 and 15 week old animal groups respectively are shown. 2Means • SE f o r 3 separate experiments are shown. * S i g n i f i c a n t l y d i f f e r e n t from the values of WKY (p < 0.05).
2334
Adrenal Angiotensin
II Receptors in SHR
Vol. 29, No. 22, 1981
p r o t e i n content at e a r l i e r ages. Furthermore, Na~,K+-~TPase as a marker of membrane content of the p a r t i c u l a t e f r a c t i o n s , was d i f f e r e n t between hypertensive and control r a t s (p > 0.05). The Na~K+-ATPase increased f o r both the SHR and WKY r a t s with age and a ' 15 weeks of age was s i g n i f i c a n t l y increased f o r both groups as compared ~ the other ages studied (p < 0.05). TABLE I I Protein Content and Na+,K+-m i:'ase A c t i v i t y of Adrenal S u b c e l l u l a r p a r t i c l e s from Normotensive (W,,.) and Spontaneously Hypertensive (SHR) Rats Age (weeks)
Protein (mg/g)
Na+,K+-ATPase (~mol Pi/mg/h)
5
WKY SHR
21.3 ± 0.6 2 5 . 1 " 3.6
3.5 • 0.1 2.9 • 0.4
10
WKY SHR
19.9 * 1.9 20.5 • 1.7
4 . 1 ± 0.2 3.6 • 0.6
15
WKY SHR
14.8 + 2.2 16.7 ~- 0.4
7.0 ± 0.7 5.0 ~" 0.5
Means • SE f o r 3 separate determlnations are shown. Protein content in p a r t i c u l a t e f r a c t i o n s is expressed as mg p r o t e i n / g wet weight of adrenal glands. Na+,K+-ATPase is expressed as ~moles Pi/mg p r o t e i n / h o u r . All comparisons between WKY and SHR were n o n - s i g n i f i c a n t (p > 0.05). Discussion Our studies show that the number of angiotensin I I receptors in adrenal membrane pr~:parations from SHR is decreased as compared with those from normotensive control r a t s and t h a t the occurrence of t h i s decrease antedates the development of hypertension. The data are derived from the whole adrenal r a t h e r than from the i s o l a t e d zona glomerulosa which is a t a r g e t of angioten',i~ I I . Hence, a decrease in binding s i t e s could be r e l a t e d to a decrease of these t a r g e t c e l l s instead of a decrease of the number of receptors on these c e l l s . However, t h i s p o s s i b i l i t y is u n l i k e l y since p r e v i o u s l y reported h i s t o m e t r i c data showed hypertrophy r a t h e r than atrophy of the zona glomerulosa l a y e r in SHR (15). In normal rats elevated or lowered plasma l e v e l s of components of the r e n i n - a n g i o t e n s i n system, including plasma r e n i n , have been shown to c o r r e l a t e r e s p e c t i v e l y with elevated or decreased number of s p e c i f i c receptors f o r angiotensin I I in membrane-rich p a r t i c l e s as well as in i s o l a t e d zona glomerulosa c e l l s from adrenal glands ( 7 , 8 , 1 6 ) . On the other hand, in nephrectomized r a t s which have low c i r c u l a t i n g l e v e l s of renin and angiotensin, the number of adrenal receptors was shown to be increased (17). Plasma renin in SHR has been v a r i a b l y reported to be elevated during the prehypertensive stage but suppressed during established hypertension (3), normal in young SHR but elevated in older r a t s [ 4 ] , suppressed a f t e r development of hypertension ( 1 , 2 ) , and normal and unchanged with age (5). Hence, with the v a r i a b i l i t y of reported l e v e l s of plasma renin in the l i t e r a t u r e , we are not able to r e l a t e our f i n d i n g s of decreased angiotensin I I receptors to l e v e l s of plasma renin in SHR. I t is also possible that adrenal angiotensin I I receptors in SHR are under d i f f e r e n t r e g u l a t o r y mechanisms from those of normal rats and there may not be a c o r r e l a t i o n with plasma renin. In any case, our data suggest the
Vol. 29, No. 22, 1981
Adrenal Angiotensin II Receptors in SHR
2335
presence of an a l t e r a t i o n of the receptor number in the renin-angiotensinaldosterone system in SHR. Decreased secretion of aldosterone secondary to angiotensin II administration in SHR was previously reported (6). I f this is true, our finding of decreased receptors for angiotensin II might be an explanation for this altered adrenal response to angiotensin I I . Acknowldegements This study was supported by Grants HL 06924 and HL 14456 from the National Heart, Lung, and Blood I n s t i t u t e , National I n s t i t u t e s of Health. Dr. Shimada is a r e c i p i e n t of a Parker Francis Foundation Fellowship. References I. 2. 3. 4. 5. 6.
7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17.
R.H. FREEMAN, J.O. DAVIS, N. VERSANO-AHARON, S. ULICK and M.H. WEINBERGER, Circ. Res. 37 66-71 (1975). K. SHIONO and H. SOKABE,---Am. J. Physiol. 231 1295-1299 (1976). S. SEN, R.R. SEMBY and F.M. BUMPUS, Circ. Res. 31 876-880 (1972). W. DEJONG, W. LOVENBERG and A. SJOERDSMA, Pro-C. Soc. Exp. Biol. Med. 139 1213-1216 (1972). B.H. FORMAN and P.J.V. MULROW, Circ. Res. 35 215-221 (1974). K. NAKAMURA, K. NAKAMURA and T. SUZUKI, Tpontaneous Hypertension, Its Pathogenesis and Complications in Proceedings of the Second International Symposium on the Spontaneously Hypertensive Rat. 149-158 (1976). G. AGUILERA, R.L. HAUGER and K.J. CATT, Proc. Natl. Acad. Sci. U.S.A. 75 975-979 (1978). R.---L. HAUGER, G. AGUIERA and K.J. CATT, Nature 271 176-178 (1978). J.M. PFEFFER, M.A. PFEFFER and E.D. FROHLICH, J. Lab. Clin. Med. 78 957-962 (1971). H. GLOSSMANN, A.J. BAUKAL and K.J. CATT, J. Biol. Chem. 249 825-834 (1974). K. SHIMADA, Y. KATO and Y. YAZAKI, Jap. Circ. J. 43 843-852 (1979). O . H . LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). T. NAKAO, K. NAGANO, K. ADACHI and M. NAKAO, Biochem. Biophys. Res. Commun. 13 444-448 (1963). G. SCATC~RD, N.Y. Acad. Sci. 51 660-672 (1949). K. AOKI, H. TANKAWA, T. FUJIN-A-BII, A. MIYAZAKI and Y. HASHIMOTO, Jap. Heart J. 4 426-442 (1963). J. DOUGLATand K.J. CATT, J. Clin. Invest. 58 834-843 (1976). M.G. PERNOLLET, M.A. DEVYNCK, P.G. MATTHEWS and P. MEYER, P. Eur. J. Pharmacol 43 361-372 (1977).
Pergamon Press
REDUCED NLIMBER OF ADRENAL ANGIOTENSIN I I RECEPTORS IN THE SPONTANEOUSLY HYPERTENSIVE RAT Kazuyuki Shimada and Barry L. Fanburg Department of Medicine, New England Medical Center Hospital and Tufts University School of Medicine, 171 Harrison Avenue, Boston, Massachusetts 02111, U.S.A. (Received in final form October 6, 1981)
Summary [1251]angiotensin I I binding to adrenal subcellular p a r t i c l e s was compared between spontaneously hypertensive and Wistar-Kyoto normotensive control rats. The number of angiotensin I I receptors was reduced in adrenals of spontaneously hypertensive rats (P < 0.05) without a change in the a f f i n i t y of angiotensin I I for the binding sites at animal ages of 5, 10 and 15 weeks. This decline of receptor content antedated the abrupt r i s e in blood pressure noted between 5 and i0 weeks. The data suggest the presence of an a l t e r a t i o n of the receptor number in the renin-angiotensinaldosterone system in the spontaneously hypertensive r a t . The spontaneously hypertensive r a t (SHR) has been found to be a useful animal model f o r the study of hypertension and several parameters of the renin-angiotensin-aldosterone system h a v e been assessed in t h i s animal. There is some controversy in the l i t e r a t u r e on the levels of plasma and kidney renin a c t i v i t i e s and of plasma aldosterone in the SHR (1-5). The adrenal output of aldosterone a f t e r the i n j e c t i o n of angiotensin i i has been reported to be reduced in SHR (6). Recently, the adrenal receptor sites f o r angiotensin I I were shown to be dependent on the level of endogenous plasma renin and angiotensin, and i t was suggested that changes of the binding c h a r a c t e r i s t i c s of these receptors may be responsible f o r the a l t e r a t i o n s of the adrenal response to angiotensin I I (7,8). The present study was undertaken to evaluate the specific adrenal receptors for angiotensin I I in the SHR as compared with normotensive control rats. Materials and methods Male SHR of the Aoki-Okamoto s t r a i n and age-matched Wistar-Kyoto normotensive controls w e r e obtained from the Charles River Breeding Laboratories, Inc., Wilmington, Mass. A l l animals were bred in a common environment and fed the same diet containing 0.4% sodium and 0.95% potassium and were given tap water ad l i b i t u m . Systolic blood pressure was measured by a t a i l c u f f plethysmographic technique (9). Angiotensin I I binding studies were performed e s s e n t i a l l y as described by Glossmann et a l . (i0), Adrenals were collected from rats a f t e r decapitation and placed into ice-cold Krebs-Ringer-phosphate buffer (pH 7.4). Adrenals from groups of 4, 5 and 10 rats at 15, i0 and 5 weeks r e s p e c t i v e l y were pooled. Three separate experiments were performed f o r each age group and experiments on paired normotensive controls were carried out in p a r a l l e l with those on SHR. Each age group was studied separately with a
0024-3205/81/292331-05502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2332
Adrenal Angiotensin
II Receptors
in SHR
Vol. 29, No. 22, 1981
maximum of four weeks between studies. Pooled whole adrenals were cut up and homogenized with 10 strokes of a t i g h t f i t t i n g t e f l o n pestle. The homogenate was centrifuged at 1,500 x g for I0 min. The supernate was then centrifuged at 20,000 x g f o r 30 min. The 20,000 x g p e l l e t s were washed once and resuspended in assay buffer containing 120 mM NaCI, 20 mM Tris-HCl (pH 7.4), 4 mM ethylenediaminetetraacetic acid disodium s a l t , 5 mM d i t h i o t h r e i t o l and 10-4 M phenylmethylsulfonyl f l u o r i d e to give a protein concentration of 2.5-4.5 mg/ml. The constituents of the assay buffer prevent breakdown of angiotensin I I by angiotensinase a c t i v i t y ( i 0 , I i ) . For binding assays, 50 ~I aliquots of f r e s h l y prepared subcellular p a r t i c u l a t e f r a c t i o n s were incubated in the above buffer containing 0.2% bovine serum albumin and 53 p M / l i t e r of [1251]angiotensin I I (New England Nuclear; s p e c i f i c a c t i v i t y = 1472 ~Ci/~g) and unlabeled angiotensin I I at various concentrations in a f i n a l volume of 0.2 ml at 22°C f o r 45 min. At the end of the incubation, each assay tube was f i l l e d r a p i d l y with 4 ml of cold buffer and f i l t e r e d through a M i l l i p o r e n i t r o c e l l u l o s e HAWP (0.45 ~m) filter. Each f i l t e r was washed once and r a d i o a c t i v i t y on the f i l t e r was measured in an automatic v-spectrometer. Specific binding of [1251]angiotension I I is defined as the portion of t o t a l binding that is i n h i b i t e d by the presence of an excess ( 1 0 - 5 M ) of unlabeled angiotensin II. Total binding to s u b c e l l u l a r p a r t i c l e s at a [125I]angiotensin I I concentration of 53 pM accounted f o r 36-62% of t o t a l r a d i o a c t i v i t y in the system and s p e c i f i c binding accounted f o r 96-98% of t o t a l binding. Protein was determined by the method of Lowry et K+-ATPase was determined as described by Nakao et al. (13).
al
(12).
The s t a t i s t i c a l p r o b a b i l i t i e s were calculated by Student's comparing the experimental data of each group with the control data.
t
Na+, test,
Results A representative Scatchard plot [14] of specific [1251]angiotensin I I binding to adrenal preparations from one of three experiments on 10 week old animals is shown in Figure 1. The experimental curve obtained with unlabeled angiotensin I I concentrations ranging from 0 to I nM was l i n e a r . The a f f i n i t y constant, indicated by the slope of t h i s l i n e , and the binding capacity, indicated by the intercept at the abscissa, were calculated by least squares analysis. The values of the a f f i n i t y constants ere not s i g n i f i c a n t l y d i f f e r e n t f o r hypertensive (10.9 • 0.6 nM-1, mean w m t j and normotensive (9.2 • 1.6 nM-1) control rats at 10 weeks of age. However, the binding capacity was s i g n i f i c a n t l y lower for the adrenal preparations from hypertensive rats (80 • 4 fmol/mg vs. 138 ± 18 fmol/mg in Wistar-Kyoto controls, p < 0.05). The age dependency of t h i s change is shown in Table I . At each age studied (5, 10 and 15 weeks), the binding capacity of adrenal preparations from hypertensive rats was s i g n i f i c a n t l y lower than controls, whereas the a f f i n i t y constant was s i m i l a r for these two groups. The reduction of binding capacity was already evident at 5 weeks of age before the rapid r i s e in blood pressure occurred. Some v a r i a t i o n s of binding capacity with age were noted both in hypertensive and control rats. We considered the p o s s i b i l i t i e s that the decrease in number of binding s i t e s , expressed in terms of p a r t i c u l a t e f r a c t i o n protein, may represent an increase in protein content of t h i s f r a c t i o n in the SHR or a decrease in receptor-carrying membranes in adrenal subcellular p a r t i c l e preparations from SHR as compared to controls. However, measurements of protein of the p a r t i c u l a t e f r a c t i o n showed no s i g n i f i c a n t differences between the WKY and SHR rats (Table I I ) at any age (p > 0.05). The protein content decreased
Vol. 29, No. 22, 1981
Adrenal Angiotensin II Receptors in SHR
2333
with age in both the SHR and WKY homogenates and by 15 weeks of age was s i g n i f i c a n t l y decreased in the WKY homogenates (p < 0.05) as compared to the
1.5L
• WKY
m I.O U.l n~ ks_ Z
otoO.5
0
50 I00 150 BOUND ANGIOTENSIN TF {fmol/mg) Fig. 1
Scatchard plot of [1251]angiotensin II binding to adrenal subcellular p a r t i c l e s from i0 week old spontaneously hypertensive (o) and Wistar-Kyoto normotensive (e) rats. One of 3 separate experiments is shown. TABLE I Binding Constants of Adrenal Receptors f o r Angiotensin I I from Normotensive (WKY) and Spontaneously Hypertensive (SHR) Rats Age (weeks)
Systolic blood I pressure (mm Hg)
Binding capacity2 (fmol/mg protein)
Affinity 2 (nM- i )
5
WKY SHR
ii0 • 3 118 ± 2
165 • 16 114 • 3*
9.0 * 0.3 8.7 ± 0.5
i0
WKY SHR
146 * 3 206 • 4*
138 ± 18 80 ± 4*
9.2 ± 1.6 10.9 * 0.6
15
WKY SHR
156 * 3 229 * 4*
190 • 9 109 • 9*
i 0 . 0 • 1.3 10.6 * 1.5
iMeans • SE f o r 15, 15 and 12 rats in the 5, 10 and 15 week old animal groups respectively are shown. 2Means • SE f o r 3 separate experiments are shown. * S i g n i f i c a n t l y d i f f e r e n t from the values of WKY (p < 0.05).
2334
Adrenal Angiotensin
II Receptors in SHR
Vol. 29, No. 22, 1981
p r o t e i n content at e a r l i e r ages. Furthermore, Na~,K+-~TPase as a marker of membrane content of the p a r t i c u l a t e f r a c t i o n s , was d i f f e r e n t between hypertensive and control r a t s (p > 0.05). The Na~K+-ATPase increased f o r both the SHR and WKY r a t s with age and a ' 15 weeks of age was s i g n i f i c a n t l y increased f o r both groups as compared ~ the other ages studied (p < 0.05). TABLE I I Protein Content and Na+,K+-m i:'ase A c t i v i t y of Adrenal S u b c e l l u l a r p a r t i c l e s from Normotensive (W,,.) and Spontaneously Hypertensive (SHR) Rats Age (weeks)
Protein (mg/g)
Na+,K+-ATPase (~mol Pi/mg/h)
5
WKY SHR
21.3 ± 0.6 2 5 . 1 " 3.6
3.5 • 0.1 2.9 • 0.4
10
WKY SHR
19.9 * 1.9 20.5 • 1.7
4 . 1 ± 0.2 3.6 • 0.6
15
WKY SHR
14.8 + 2.2 16.7 ~- 0.4
7.0 ± 0.7 5.0 ~" 0.5
Means • SE f o r 3 separate determlnations are shown. Protein content in p a r t i c u l a t e f r a c t i o n s is expressed as mg p r o t e i n / g wet weight of adrenal glands. Na+,K+-ATPase is expressed as ~moles Pi/mg p r o t e i n / h o u r . All comparisons between WKY and SHR were n o n - s i g n i f i c a n t (p > 0.05). Discussion Our studies show that the number of angiotensin I I receptors in adrenal membrane pr~:parations from SHR is decreased as compared with those from normotensive control r a t s and t h a t the occurrence of t h i s decrease antedates the development of hypertension. The data are derived from the whole adrenal r a t h e r than from the i s o l a t e d zona glomerulosa which is a t a r g e t of angioten',i~ I I . Hence, a decrease in binding s i t e s could be r e l a t e d to a decrease of these t a r g e t c e l l s instead of a decrease of the number of receptors on these c e l l s . However, t h i s p o s s i b i l i t y is u n l i k e l y since p r e v i o u s l y reported h i s t o m e t r i c data showed hypertrophy r a t h e r than atrophy of the zona glomerulosa l a y e r in SHR (15). In normal rats elevated or lowered plasma l e v e l s of components of the r e n i n - a n g i o t e n s i n system, including plasma r e n i n , have been shown to c o r r e l a t e r e s p e c t i v e l y with elevated or decreased number of s p e c i f i c receptors f o r angiotensin I I in membrane-rich p a r t i c l e s as well as in i s o l a t e d zona glomerulosa c e l l s from adrenal glands ( 7 , 8 , 1 6 ) . On the other hand, in nephrectomized r a t s which have low c i r c u l a t i n g l e v e l s of renin and angiotensin, the number of adrenal receptors was shown to be increased (17). Plasma renin in SHR has been v a r i a b l y reported to be elevated during the prehypertensive stage but suppressed during established hypertension (3), normal in young SHR but elevated in older r a t s [ 4 ] , suppressed a f t e r development of hypertension ( 1 , 2 ) , and normal and unchanged with age (5). Hence, with the v a r i a b i l i t y of reported l e v e l s of plasma renin in the l i t e r a t u r e , we are not able to r e l a t e our f i n d i n g s of decreased angiotensin I I receptors to l e v e l s of plasma renin in SHR. I t is also possible that adrenal angiotensin I I receptors in SHR are under d i f f e r e n t r e g u l a t o r y mechanisms from those of normal rats and there may not be a c o r r e l a t i o n with plasma renin. In any case, our data suggest the
Vol. 29, No. 22, 1981
Adrenal Angiotensin II Receptors in SHR
2335
presence of an a l t e r a t i o n of the receptor number in the renin-angiotensinaldosterone system in SHR. Decreased secretion of aldosterone secondary to angiotensin II administration in SHR was previously reported (6). I f this is true, our finding of decreased receptors for angiotensin II might be an explanation for this altered adrenal response to angiotensin I I . Acknowldegements This study was supported by Grants HL 06924 and HL 14456 from the National Heart, Lung, and Blood I n s t i t u t e , National I n s t i t u t e s of Health. Dr. Shimada is a r e c i p i e n t of a Parker Francis Foundation Fellowship. References I. 2. 3. 4. 5. 6.
7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17.
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