- Email: [email protected]
Comparison of aldosterone binding in aortic cells from Dahl salt-susceptible and salt-resistant rats
Life Sciences, Vol. 36, pp. 1653-1660 Printed in the U.S.A.
Pergamon Press
COMPARISON OF ALDOSTERONE BINDING IN AORTIC CELLS FROM DAHL SALTSUSCEPTIBLE AND SALT-RESISTANT RATS Nancy R. Nichols, Denise F. Obert, and Walter J. Meyer,III Department of Pediatrics, The University of Texas Medical Branch, Galveston, Texas 77550-2776 (Received in final form Feburary 18, 1985) Sun,~ary The Dahl salt-resistant substrain of Sprague-Dawley rats represents a uniform population of animals that are resistant to salt and mineralocorticoid induced hypertension. Aldosterone binding in the aortae of these rats is contrasted to that of age- and sex-matched rats of the Dahl salt-susceptible strain in an effort to identify a mechanism for resistance to salt induced hypertension. Cultured smooth muscle cells of both substrains contain two classes of aldosterone binding sites: corticoid receptor I with high affinity and low capacity, and corticoid receptor II with low affinity and high capacity. No differences were found between the two substrains in the affinities or binding capacities of these receptors. Both groups of Sprague-Dawley rats had a significantly higher corticoid receptor I affinity than the salt resistant Fischer 344 rats, a strain with a twofold lower affinity than salt sensitive strains. These results indicate that an intrinsic defect in mineralocorticoid binding in aortic smooth muscle cells could not account for the resistance to salt and mineralocorticoid induced hypertension seen in Sprague-Dawley rats and that the biochemical abnormality underlying salt resistance is likely to be different from that of Fischer 344 rats. In 1962, Dahl (i) selectively bred two strains of Sprague-Dawley rats of susceptibility (S) or resistance (R) to the hypertensive effects of salt. The regulation of blood pressure in S and R rats is polygenic with the adrenal cortex kidney, nervous system, and humoral factors serving important functions (2). Physiological and biochemical data point to a role for steroid receptors in blood pressure regulation and the development of hypertensive disease through their effects on the ionic properties of vascular smooth muscle. Alterations in ionic composition and ion transport have been found in aortae of rats made hypertensive by steroid and salt treatment (3-6). We have previously described two receptors, corticoid receptor I and corticoid receptor II (glucocorticoid-specific), which bind aldosterone in aortic smooth muscle cells from Sprague-Dawley rats (7). Recently, Moura and Worcel (8) have demonstrated in arterial smooth muscle, mineralocorticoid effects on Na transport that are consistent with a receptor-initiated, mRNAmediated mechanism of steroid action. Altered mineralocorticoid binding has been associated with resistance to salt induced hypertension in two rat strains: Long-Evans and Fischer 344.
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
1654
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
The mineralocorticoid salt resistance of the Long-Evans rat can only be demonstrated when low amounts of deoxycorticosterone are used (9, 10, ii). The Long-Evans rat, which also has a decreased salt appetite, has decreased deoxycorticosterone binding in the hypothalamus, but not in other parts of the brain (12). Recently, we have identified an altered corticoid receptor I with a lower affinity for minera]ocorticoids in Fischer 344 rats, a strain that is immune to salt induced hypertension (13). Although no differences were found in renal mineralocorticoid binding between S and R rats (14), it is not known if vascular and renal type I (mineralocorticoid) receptors with the same intrinsic specificity (15) are identical in structure and function. In the present report, we have examined aldosterone binding in aortic smooth muscle cells from R rats and compared it to that of S rats and previously studied salt-resistant and salt-sensitive strains. Materials and Methods Chemicals ~H-Aldosterone (57-82 Ci/mmole) and H-dexamethasone (36-42 Ci/mmole) were purchased from New England Nuclear and routinely checked for purity. Nonradioactive a]dosterone and dexamethasone were obtained from Sigma. Steroids were stored in absolute ethanol at 4oc. Tissue culture medium components, Hank's balanced salt solution and trypsin were purchased from Gibco; calf thymus DNA and ga~naglobulin, from Sigma; and Sephadex G-25 from Pharmacia. Rat aortic cell cultures R and S rats from Junichi lwai, M.D., at Brookhaven National Laboratory were matched for age (12-14 weeks) and sex (male), and were maintained on Ralston Purina Lab Chow and deionized water. Systolic blood pressures were measured in conscious rats by the tail-cuff method with a programmed electrosphygmomanometer and physiograph (Narco Biosystems, Houston, Texas). Animals were exsanguinated by cardiac puncture under ether anesthesia and sections of the thoracic aorta were removed under sterile conditions. Aortic explants from seven R and seven S rats were cultured by conventional tissue culture techniques in minimal essential medium with Earle's salts and supplemented with non-essential amino acids, 27 n~M sodium bicarbonate, 10% fetal calf serum, penicillin (25 units/ml), streptomycin (25 ~g/ml), and amphotericin B (2.5 ~g/ml) (7). Aortic cell lines were routinely subculture<] and used for experimentation at passages 8 through 20. The established cultures contained exclusively smooth muscle cells which had modulated phenotypically in culture from a contractile to a proliferative state (16). The endothelial cells did not last past the first passage. These cell lines were identified as vascular smooth muscle by morphologic characteristics as well as by antibody techniques (17). Whole cell bindin 9 assay Details of the receptor binding assay have been previously described (7). In brief, confluent multilayers were washed, then incubated with various concentrations of 3H-steroid in serum-free media at 37°C for 30 minutes. The medium was removed and an all,lot taken to determine the free steroid concentration. The cells were harvested by treatment with trypsin and suspended in a solution containing 0.02 M TRIS-HCI at pH 7.5, 0.32 M sucrose, and i mg/ml bovine gammaglobu]in at 4°C. After centrifugation cells were resuspended in 1 m] of 0.02 M Tris buffer (pH 7.5) containing
Vol. 36, No. 17, 1985
Aortic Aldosterone Binding in Dahl Rats
1655
0.5 M KCI, 1.5 r~M EDTA, and 20% (v/v) glycerol, and sonicated to break up the cell and nuclear membranes. The sonicate was centrifuged, and the supernatant was assayed for DNA concentration (18), and protein-bound steroid by gel filtration chromatography on Sephadex G-25. Non-specific binding was measured as detectable 3H-steroid binding in the presence of an excess of nonradioactive steroid and was subtracted from total binding to give specifically-bound steroid. Aldosterone was the ligand of choice for comparin 9 mineralocorticoid binding between rat strains because deoxycorticosterone exhibited extremely high non-specific binding in the aortic cell system, making the estimate of specific binding very unreliable. Analysis and source_____~soferro_____~r in the binding data Binding and blood pressure statistics are expressed as a mean + standard deviation (S.D.); composite Scatchard plots are graphed as a mean + standard error (S.E.). Data were tested for significance at the 5% level by the Student's ! test or analysis of variance. Results Blood pressure measurements The systolic blood pressures of the R and S strains were 123.3 + 6.7 and 150.1 + 8.3 mm Hg, respectively. All rats had blood pressures ]nder 140 and all S rats over 140 mm Hg. Two S rats had blood pressures greater than 150 mmHg. The differences between the two strains is highly significant (p<.OOl). Even on a low-salt diet, a difference in blood pressure is often observed between the two strains and S rats eventually become hypertensive when fed normal rat chow (19). Scatchard analysis o_~f 3H-aldosterone bindinq The aldosterone binding of seven vascular smooth muscle cell lines from seven rats of each substrain was subjected to Scatchard analysis (20). Composite Scatchard plots for the R and the S substrains are shown in Figure !- A and B, respectively. These plots show that the variation in °H aldosterone binding within a substrain using different rat cell lines was small and the binding data obtained for the two substrains was similar. ithough the specific binding at the lowest concentration of ligand (O.i nM H-aldosterone) was decreased in three out of seven S cell lines and in only one out of seven R cell lines, the difference between the substrains in the binding data at this point was not significant. The observed variation is probably attributable to inter-assay variation arising from receptor instability at low concentrations of ligand (7).
~
When curvilinear plots were resolved by the method detailed previously (7), aldosterone binding to corticoid receptor I (line a) and corticoid receptor II (line b) was detected in both substrains (Figure I, A and B). Individual, as well as composite Scatchards were analyzed in this manner and aldosterone binding statistics of each vascular smooth cell line from rats of both substrains are enumerated and compared in Table I. The K d and Bma x of corticoid receptors I and II were not significantly different betweem two substrains.
1656
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
140[ A
12o~-
~ Z
a
~ o
4o
2o
o
140
o 120 < loo Q)
~
B
1~
8o 6O
40 20 O(
~
1
20 40 60 80 100 120 140 160 180 BoundAldosterone[moles/#gDNA](x 1018)
PIG. 1 Composite Scatchard plots of 3H-aldosterone binding in aortic smooth muscle cells cultured from seven R rats (A) and seven S rats (B). The mean + S.E. of the X and Y coordinates are denoted by the points and two-dimensional fields, respectively. Curvilinear Scatchard plots were resolved by a graphical method previously described (7). Line a represents the high affinity, low capacity binding sites (corticoid receptor I). Line b represents the low affinity, high capacity binding sites (corticoid receptor II). For R rats (A), binding statistics were as follows: line a, F~ = 0.69 nM and B m a x = 45.1 moles × i0- l~Lg DNA; line b, K d = 4.42 nM and B m a x = 157.1 moles x lO-18/~g DNA. For S rats (B), binding statistics were as follows: line (a), K d = 0.82 nM and B m a x = 48.4 moles x lO-18/Lg DNA; line b, K d = 8.41 nM and B m a x = 300.0 moles x IO-I8/bg DNA.
Vol. 36, No. 17, 1985
Aortic Aldosterone Binding in Dahl Rats
1657
TABLE I Scatchard Analysis of Specific 3H-aldosterone Binding in Aortic Smooth Muscle Cells from R and S Rats
Strain
Culture Number
i 2 3 4 5 6 7 Mean SD + 1 2 3 4 5 6 7 Mean SD +
Corticoid Receptor I K t Bmaxt d (nM) (m xlO-18/~g DNA)
.19 .30 .29 .57 .43 .38 .90 .44 MS .24 .47 .19 .40 .ii .25 .60 1.20 .46 .37
Corticoid Receptor If Kd+ Bmaxt (nM)
(m xlO-18/ug DNA)
48.1 14.7 11.7 54.3 37.9 40.5 28.5 33.7 MS 16.2
4.70 3.70 13.10 5.16 18.80 2.10 5.00 7.5NS 6.1
160.9 154.6 198.8 198.5 934.4 96.1 172.O 273.6NS 293.4
99.0 15.2 26.1 14.5 42.0 44.5 10.8 36. O 30.8
6.45 19.49 10.76 7.30 20.92 7.63 9.20 ii. 68 6.00
432.5 479.2 412.7 89.9 662.7 337.0 303.2 388.2 175.6
t Kd and Bma x ate derived from curvilinear Scatchard plots. NS = Not significantly different from S strain. Scatchard analysis of 3H-dexamethasone bindin@ Since aldosterone does not saturate the corticoid receptor II (glucocorticoid) sites under the experimental conditions employed (7), it was necessary to further compare the binding statistics of the type II sites using the potent glucocorticoid, dexamethasone. Unlike the Scatchard plot of aldosterone binding in the aorta, the plot of dexamethasone binding is linear and saturation is achieved at 10-15 nM (not shown). The results of Scatchard analysis of 3H-dexamethasone binding in vascular smooth muscle cells from three R and three S rats ate shown in Table II. The differences in the binding statistics were not significant. Con~oarison of vascular aldosterone bindin@ in R and S rats with other saltresistant and salt-sensitive strains A second objective of the study was to compare vascular aldosterone binding in Dahl's salt-resistant and salt-susceptible substrains with that of other salt-resistant and salt-sensitive strains. Since altered aldosterone binding to corticoid receptor I but not to cortlcoid receptor II has been observed for a salt-resistant strain in comparison to a salt-sensitive strain (13), only corticoid receptor I binding characteristics ate compared in Table III. The corticoid receptor I of salt-resistant Fischer 344 rats has a significantly (p
1658
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
the salt-resistant Sprague-Dawley R strain. The binding capacity of corticoid receptor I was not different among strains, nor was the binding affinitybetween strains other than Fischer 344. TABLE II Scatchard Analysis of Specific 3H-Dexamethasone Binding Aortic Smooth Muscle Cells in R and S Rats Corticoid Receptor If Kd+ Bmax # Strain
Culture Number
(riM)
1 2 3 Mean SD +
16.89 5.79 7.09 NS 9.92 6.07
1
4.65 6.02 9.42 6.70 2.46
2 3 Mean SD +
(m xlO-18/ug DNA) 3293 3312 1890 NS 2832 816 3653 3644 3848 3715 115
%Kd~and Bmax are derived from linear Scatchard plots. NS = not significantly different from S strain. TABLE III Comparison of Corticoid Receptor I Aldosterone Binding in Aortic Smooth Muscle Cells of Salt-Resistant and Salt-Sensitive Rat Strains Kd (riM) Mean + S.E.
~ax (m xlO- ~/ug DNA) Mean + S.E.
SALT-RESISTANT STRAINS Fischer 344 % Sprague-Dawley R
6 7
1.12 + .08 * .44 % .09
105 + 7 NS 34 % 6
7 7
.46 + .14 .49 T .08
36 + 12 77 ¥ 15
8
.47 + .08
115 + 47
SALT-SENSITIVE STRAINS Sprague-Dawley S Wistar-Kyoto %% Spontaneous] y Hypertensive +%
%Ref. (13). +%Ref. (21). •By one-way analysis of variance the K d of corticoid receptor I was significantly different among strains (p<.OOl). Fischer 344 rats have by Scheffe analysis of variance a significantly different corticoid receptor I K d from that of all other strains (p<.Ol). NS = Not significantly different among ~trains by one-way analysis of variance.
Vol. 36, No. 17, 1985
Aortic Aldosterone Binding in Dahl Rats
1659
Discussion The etiology of increased vascular resistance in mineralocorticoid and salt hypertension is not completely understood. Two mechanisms have been proposed. Tobian and Binion (3) postulated that vessel wall swellin9 due to increased water and ion content would decrease the size of the lumen and increase peripheral resistance. More recently the increased sensitivity of the vasculature to catecholamines has been implicated (22,23). Since direct effects of steroids on the blood vessels are presumably mediated by specific intracellular receptors, we have examined these binding sites in aortic smooth muscle cells cultured from Sprague-Dawley S and R rats. The cultured aortic smooth muscle cells offer a opportunity to look for genetic differences between strains without interference from the extracellular environment. The existence and characteristics of these receptors has been documented by others usin 9 homogenized whole arteries of rabbits (24,25). S rats have higher plasma mineralocorticoid activity than R rats, which explains part but not all of the difference in blood pressure on a high-salt diet (26). No intrinsic differences in aortic cell aldosterone bindin 9 were found between the two substrains, but the data do not exclude in vivo differences due to extrinsic factors present in S and R rats or differences distal to receptor bindin 9 in the mechanism of steroid action. However, these findings are in agreement with those of Funder et al. (14) in the kidney, where no differences were found in mineralocorticoid bindin 9 between groups of S and R rats on low- and high-salt diets. Additional data on kallikrein induction, distal tubl]lar hypertrophy and skeletal muscle ion changes indicate that mineralocorticoid responses are intact and similar in the two substrains (27). Bindin 9 to beth classes of corticoid receptors was examined since both mineralocorticoid and glucocorticoid hormones can cause hypertension albeit by different mechanisms (28). In addition, there is considerable overlap in specificity between the two receptors and it is not known through which class of receptors adrenal steroids may he acting in the pathogenesis of hypertension. In our experiments, aldosterone always estimates fewer corticoid receptor II sites than dexamethasone, and saturation is not achieved unless much higher concentrations of aldosterone are used. Therefore, dexamethasone is a better indicator of differences in cgrticoid receptor II sites than aldosterone. ~o differences were found in ~Hdexamethasone binding between the two substrains. The relationship between mineralocorticoid- and salt-induced hypertension and vascular steroid receptors has been investigated by comparin 9 aldosterone bindin 9 in aortic smooth muscle cells of salt-resistant and salt-sensitive rat strains. There is good evidence that the resistance of Fischer 344 rats to mineralocorticoid and salt hypertension is due to a generalized defect in response to mineralocorticoids since these animals have increased plasma renin activity, decreased salt appetite, and a vascular steroid receptor with a two-fold lower affinity for mineralocorticoids (13,29). In contrast, the Long-Evans salt-resistant strain, appears to have a localized defect in the hypothalamus characterized by deficient mineralocorticoid bindin 9 and decreased salt appetite (12). The Sprague-Dawley salt-resistant substrain has similar vascular (Table 3) and renal (14) aldosterone bindin 9 characteristics, and mineralocorticoid responses (27) compared to the salt-sensitive substrain. Collectively, these data indicate that the three rat models have a different molecular basis for their resistance to salt-induced hypertension and the role of mineralocorticoid receptors varies considerably between strains.
1660
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
Acknowled@ements We are grateful to Mrs. Maria Barrett for her secretarial
assistance and to Dr. Eric Hall for the use of the electrosphygmomanometer and physiograph. HL 20201.
This work was supported by USPHS Grant
References i.
L.K. DAHL, M. HOLENE and L. TASSENARI, J. Exp. Med. 115 1173-1190 (1962). 2. J.P. RAPP, Hypertension 4 753-763 (1982). 3. L. TOBIAN and J.T. BINION, Circulation 5 754-758 (1952). 4. L. TOBIAN and P.D. REDLEAF, Am. J. PhysTol. 189 451-454 (1957). 5. A.W. JONES and R.G. HART, Circ. Res. 37 333-341 (1975). 6. E.T. GARWITZ and A.W. JONES, Hypertension 4 374-381 (1982). 7. W.J. MEYER and N.R. NICHOLS, J. Steroid Bi~chem. 14 1157-1168 (1981). 8. A.M. MOURA and M. WORCEL, The Adrenal Gland and Hypertension, in press Raven Press, New York (1985). 9. C.E. HALL, S. AYACHI, and O. HALL, Texas Rep. Biol. Meal. 30 143-153 (1972). 10. A.C. BROWNIE, S. GALLANT, P.A. NICKERSON and L.M. JOSEPH, Endocr. Res. Commun 5 71-80 (1978). Ii. O.B. HOLLAND, C. GOMEZ-SANCHEZ and T. ZIEGLER, Clin. Sci. 56 109-113 (1979. 12. M.N. LASSMAN and P.J. MULROW, Endocrinology 94 1541-1546 (1974). 13. N.R. NICHOLS, C.E. Hall and W.J. MEYER, J. Hypertension 1 393-397 (1983). 14. J.W. FUNDER, D. DUVAL, P. MEYER and L.K. DAHL, Endocrinology 94 1739-1743 (1974). 15. Z.S. KROZOWSKI and J.W. FUNDER, Proc. Natl. Acad. Sci. 80 60256O60 (1983). 16. J. CHAMLEY-CAMPBELL, G.R. CAMPBELL and R. ROSS, Physiol. Rev. 59 1-61 (1979). 17. N.R. NICHOLS, C.A. OLESON and J.W. FUNDER, Endocrinology 113 1096-1101 (1983). 18. K. BURTON, Biochem. J. 62 315-323 (1956). 19. D.L. SUSTARSIC, R.P. MCPARTLAND and J.P. RAPP, J. Lab. Clin. Med. 98 599-606 (1981). 20. G. SCATCHARD, Ann. N.Y. Acad. Sci. 51 660-672 (1949). 21. N.R. NICHOLS, C.E. HALL and W.J. MEYER Ill, Hypertension 4 646652 (1982). 22. J. DECHAMPLAIN, L. KRAKOFF and J. AXELROD, Circ. Res. 24 I75-92 (1969). 23. F.M. ABBOUD, Fed, Proc. 33 143-149 (1974). 24. L. KORNEL, N. KANAMARLAPUDI, T. TRAVERS and D.J. TAFF, J. Steroid Biochem. 16 245-264, (1982). 25. L. KORNEL, N. KANAMARLAPUDI, C. RAMSAY, T. TRAVERS and S. KAMATH, J. Steroid Biochem. 19 333-344 (1983). 26. J.P. RAPP and L.K. DAHL, Endocrinology 88 52-65 (1971). 27. J.P. RAPP, R.P. MCPARTLAND and D.L. SUSTARSIC, Hypertension 4 2026 (1982). 28. D. HAACK, J. MOHRING, B. MOHRING, M. PETRI and E. HACHKENTHAL, Am. J. Physiol. 233 F403-411 (1977). 29. C.E. HALL and O. HALL, Life Sci. 20 1239-1248 (1977).
Pergamon Press
COMPARISON OF ALDOSTERONE BINDING IN AORTIC CELLS FROM DAHL SALTSUSCEPTIBLE AND SALT-RESISTANT RATS Nancy R. Nichols, Denise F. Obert, and Walter J. Meyer,III Department of Pediatrics, The University of Texas Medical Branch, Galveston, Texas 77550-2776 (Received in final form Feburary 18, 1985) Sun,~ary The Dahl salt-resistant substrain of Sprague-Dawley rats represents a uniform population of animals that are resistant to salt and mineralocorticoid induced hypertension. Aldosterone binding in the aortae of these rats is contrasted to that of age- and sex-matched rats of the Dahl salt-susceptible strain in an effort to identify a mechanism for resistance to salt induced hypertension. Cultured smooth muscle cells of both substrains contain two classes of aldosterone binding sites: corticoid receptor I with high affinity and low capacity, and corticoid receptor II with low affinity and high capacity. No differences were found between the two substrains in the affinities or binding capacities of these receptors. Both groups of Sprague-Dawley rats had a significantly higher corticoid receptor I affinity than the salt resistant Fischer 344 rats, a strain with a twofold lower affinity than salt sensitive strains. These results indicate that an intrinsic defect in mineralocorticoid binding in aortic smooth muscle cells could not account for the resistance to salt and mineralocorticoid induced hypertension seen in Sprague-Dawley rats and that the biochemical abnormality underlying salt resistance is likely to be different from that of Fischer 344 rats. In 1962, Dahl (i) selectively bred two strains of Sprague-Dawley rats of susceptibility (S) or resistance (R) to the hypertensive effects of salt. The regulation of blood pressure in S and R rats is polygenic with the adrenal cortex kidney, nervous system, and humoral factors serving important functions (2). Physiological and biochemical data point to a role for steroid receptors in blood pressure regulation and the development of hypertensive disease through their effects on the ionic properties of vascular smooth muscle. Alterations in ionic composition and ion transport have been found in aortae of rats made hypertensive by steroid and salt treatment (3-6). We have previously described two receptors, corticoid receptor I and corticoid receptor II (glucocorticoid-specific), which bind aldosterone in aortic smooth muscle cells from Sprague-Dawley rats (7). Recently, Moura and Worcel (8) have demonstrated in arterial smooth muscle, mineralocorticoid effects on Na transport that are consistent with a receptor-initiated, mRNAmediated mechanism of steroid action. Altered mineralocorticoid binding has been associated with resistance to salt induced hypertension in two rat strains: Long-Evans and Fischer 344.
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
1654
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
The mineralocorticoid salt resistance of the Long-Evans rat can only be demonstrated when low amounts of deoxycorticosterone are used (9, 10, ii). The Long-Evans rat, which also has a decreased salt appetite, has decreased deoxycorticosterone binding in the hypothalamus, but not in other parts of the brain (12). Recently, we have identified an altered corticoid receptor I with a lower affinity for minera]ocorticoids in Fischer 344 rats, a strain that is immune to salt induced hypertension (13). Although no differences were found in renal mineralocorticoid binding between S and R rats (14), it is not known if vascular and renal type I (mineralocorticoid) receptors with the same intrinsic specificity (15) are identical in structure and function. In the present report, we have examined aldosterone binding in aortic smooth muscle cells from R rats and compared it to that of S rats and previously studied salt-resistant and salt-sensitive strains. Materials and Methods Chemicals ~H-Aldosterone (57-82 Ci/mmole) and H-dexamethasone (36-42 Ci/mmole) were purchased from New England Nuclear and routinely checked for purity. Nonradioactive a]dosterone and dexamethasone were obtained from Sigma. Steroids were stored in absolute ethanol at 4oc. Tissue culture medium components, Hank's balanced salt solution and trypsin were purchased from Gibco; calf thymus DNA and ga~naglobulin, from Sigma; and Sephadex G-25 from Pharmacia. Rat aortic cell cultures R and S rats from Junichi lwai, M.D., at Brookhaven National Laboratory were matched for age (12-14 weeks) and sex (male), and were maintained on Ralston Purina Lab Chow and deionized water. Systolic blood pressures were measured in conscious rats by the tail-cuff method with a programmed electrosphygmomanometer and physiograph (Narco Biosystems, Houston, Texas). Animals were exsanguinated by cardiac puncture under ether anesthesia and sections of the thoracic aorta were removed under sterile conditions. Aortic explants from seven R and seven S rats were cultured by conventional tissue culture techniques in minimal essential medium with Earle's salts and supplemented with non-essential amino acids, 27 n~M sodium bicarbonate, 10% fetal calf serum, penicillin (25 units/ml), streptomycin (25 ~g/ml), and amphotericin B (2.5 ~g/ml) (7). Aortic cell lines were routinely subculture<] and used for experimentation at passages 8 through 20. The established cultures contained exclusively smooth muscle cells which had modulated phenotypically in culture from a contractile to a proliferative state (16). The endothelial cells did not last past the first passage. These cell lines were identified as vascular smooth muscle by morphologic characteristics as well as by antibody techniques (17). Whole cell bindin 9 assay Details of the receptor binding assay have been previously described (7). In brief, confluent multilayers were washed, then incubated with various concentrations of 3H-steroid in serum-free media at 37°C for 30 minutes. The medium was removed and an all,lot taken to determine the free steroid concentration. The cells were harvested by treatment with trypsin and suspended in a solution containing 0.02 M TRIS-HCI at pH 7.5, 0.32 M sucrose, and i mg/ml bovine gammaglobu]in at 4°C. After centrifugation cells were resuspended in 1 m] of 0.02 M Tris buffer (pH 7.5) containing
Vol. 36, No. 17, 1985
Aortic Aldosterone Binding in Dahl Rats
1655
0.5 M KCI, 1.5 r~M EDTA, and 20% (v/v) glycerol, and sonicated to break up the cell and nuclear membranes. The sonicate was centrifuged, and the supernatant was assayed for DNA concentration (18), and protein-bound steroid by gel filtration chromatography on Sephadex G-25. Non-specific binding was measured as detectable 3H-steroid binding in the presence of an excess of nonradioactive steroid and was subtracted from total binding to give specifically-bound steroid. Aldosterone was the ligand of choice for comparin 9 mineralocorticoid binding between rat strains because deoxycorticosterone exhibited extremely high non-specific binding in the aortic cell system, making the estimate of specific binding very unreliable. Analysis and source_____~soferro_____~r in the binding data Binding and blood pressure statistics are expressed as a mean + standard deviation (S.D.); composite Scatchard plots are graphed as a mean + standard error (S.E.). Data were tested for significance at the 5% level by the Student's ! test or analysis of variance. Results Blood pressure measurements The systolic blood pressures of the R and S strains were 123.3 + 6.7 and 150.1 + 8.3 mm Hg, respectively. All rats had blood pressures ]nder 140 and all S rats over 140 mm Hg. Two S rats had blood pressures greater than 150 mmHg. The differences between the two strains is highly significant (p<.OOl). Even on a low-salt diet, a difference in blood pressure is often observed between the two strains and S rats eventually become hypertensive when fed normal rat chow (19). Scatchard analysis o_~f 3H-aldosterone bindinq The aldosterone binding of seven vascular smooth muscle cell lines from seven rats of each substrain was subjected to Scatchard analysis (20). Composite Scatchard plots for the R and the S substrains are shown in Figure !- A and B, respectively. These plots show that the variation in °H aldosterone binding within a substrain using different rat cell lines was small and the binding data obtained for the two substrains was similar. ithough the specific binding at the lowest concentration of ligand (O.i nM H-aldosterone) was decreased in three out of seven S cell lines and in only one out of seven R cell lines, the difference between the substrains in the binding data at this point was not significant. The observed variation is probably attributable to inter-assay variation arising from receptor instability at low concentrations of ligand (7).
~
When curvilinear plots were resolved by the method detailed previously (7), aldosterone binding to corticoid receptor I (line a) and corticoid receptor II (line b) was detected in both substrains (Figure I, A and B). Individual, as well as composite Scatchards were analyzed in this manner and aldosterone binding statistics of each vascular smooth cell line from rats of both substrains are enumerated and compared in Table I. The K d and Bma x of corticoid receptors I and II were not significantly different betweem two substrains.
1656
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
140[ A
12o~-
~ Z
a
~ o
4o
2o
o
140
o 120 < loo Q)
~
B
1~
8o 6O
40 20 O(
~
1
20 40 60 80 100 120 140 160 180 BoundAldosterone[moles/#gDNA](x 1018)
PIG. 1 Composite Scatchard plots of 3H-aldosterone binding in aortic smooth muscle cells cultured from seven R rats (A) and seven S rats (B). The mean + S.E. of the X and Y coordinates are denoted by the points and two-dimensional fields, respectively. Curvilinear Scatchard plots were resolved by a graphical method previously described (7). Line a represents the high affinity, low capacity binding sites (corticoid receptor I). Line b represents the low affinity, high capacity binding sites (corticoid receptor II). For R rats (A), binding statistics were as follows: line a, F~ = 0.69 nM and B m a x = 45.1 moles × i0- l~Lg DNA; line b, K d = 4.42 nM and B m a x = 157.1 moles x lO-18/~g DNA. For S rats (B), binding statistics were as follows: line (a), K d = 0.82 nM and B m a x = 48.4 moles x lO-18/Lg DNA; line b, K d = 8.41 nM and B m a x = 300.0 moles x IO-I8/bg DNA.
Vol. 36, No. 17, 1985
Aortic Aldosterone Binding in Dahl Rats
1657
TABLE I Scatchard Analysis of Specific 3H-aldosterone Binding in Aortic Smooth Muscle Cells from R and S Rats
Strain
Culture Number
i 2 3 4 5 6 7 Mean SD + 1 2 3 4 5 6 7 Mean SD +
Corticoid Receptor I K t Bmaxt d (nM) (m xlO-18/~g DNA)
.19 .30 .29 .57 .43 .38 .90 .44 MS .24 .47 .19 .40 .ii .25 .60 1.20 .46 .37
Corticoid Receptor If Kd+ Bmaxt (nM)
(m xlO-18/ug DNA)
48.1 14.7 11.7 54.3 37.9 40.5 28.5 33.7 MS 16.2
4.70 3.70 13.10 5.16 18.80 2.10 5.00 7.5NS 6.1
160.9 154.6 198.8 198.5 934.4 96.1 172.O 273.6NS 293.4
99.0 15.2 26.1 14.5 42.0 44.5 10.8 36. O 30.8
6.45 19.49 10.76 7.30 20.92 7.63 9.20 ii. 68 6.00
432.5 479.2 412.7 89.9 662.7 337.0 303.2 388.2 175.6
t Kd and Bma x ate derived from curvilinear Scatchard plots. NS = Not significantly different from S strain. Scatchard analysis of 3H-dexamethasone bindin@ Since aldosterone does not saturate the corticoid receptor II (glucocorticoid) sites under the experimental conditions employed (7), it was necessary to further compare the binding statistics of the type II sites using the potent glucocorticoid, dexamethasone. Unlike the Scatchard plot of aldosterone binding in the aorta, the plot of dexamethasone binding is linear and saturation is achieved at 10-15 nM (not shown). The results of Scatchard analysis of 3H-dexamethasone binding in vascular smooth muscle cells from three R and three S rats ate shown in Table II. The differences in the binding statistics were not significant. Con~oarison of vascular aldosterone bindin@ in R and S rats with other saltresistant and salt-sensitive strains A second objective of the study was to compare vascular aldosterone binding in Dahl's salt-resistant and salt-susceptible substrains with that of other salt-resistant and salt-sensitive strains. Since altered aldosterone binding to corticoid receptor I but not to cortlcoid receptor II has been observed for a salt-resistant strain in comparison to a salt-sensitive strain (13), only corticoid receptor I binding characteristics ate compared in Table III. The corticoid receptor I of salt-resistant Fischer 344 rats has a significantly (p
1658
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
the salt-resistant Sprague-Dawley R strain. The binding capacity of corticoid receptor I was not different among strains, nor was the binding affinitybetween strains other than Fischer 344. TABLE II Scatchard Analysis of Specific 3H-Dexamethasone Binding Aortic Smooth Muscle Cells in R and S Rats Corticoid Receptor If Kd+ Bmax # Strain
Culture Number
(riM)
1 2 3 Mean SD +
16.89 5.79 7.09 NS 9.92 6.07
1
4.65 6.02 9.42 6.70 2.46
2 3 Mean SD +
(m xlO-18/ug DNA) 3293 3312 1890 NS 2832 816 3653 3644 3848 3715 115
%Kd~and Bmax are derived from linear Scatchard plots. NS = not significantly different from S strain. TABLE III Comparison of Corticoid Receptor I Aldosterone Binding in Aortic Smooth Muscle Cells of Salt-Resistant and Salt-Sensitive Rat Strains Kd (riM) Mean + S.E.
~ax (m xlO- ~/ug DNA) Mean + S.E.
SALT-RESISTANT STRAINS Fischer 344 % Sprague-Dawley R
6 7
1.12 + .08 * .44 % .09
105 + 7 NS 34 % 6
7 7
.46 + .14 .49 T .08
36 + 12 77 ¥ 15
8
.47 + .08
115 + 47
SALT-SENSITIVE STRAINS Sprague-Dawley S Wistar-Kyoto %% Spontaneous] y Hypertensive +%
%Ref. (13). +%Ref. (21). •By one-way analysis of variance the K d of corticoid receptor I was significantly different among strains (p<.OOl). Fischer 344 rats have by Scheffe analysis of variance a significantly different corticoid receptor I K d from that of all other strains (p<.Ol). NS = Not significantly different among ~trains by one-way analysis of variance.
Vol. 36, No. 17, 1985
Aortic Aldosterone Binding in Dahl Rats
1659
Discussion The etiology of increased vascular resistance in mineralocorticoid and salt hypertension is not completely understood. Two mechanisms have been proposed. Tobian and Binion (3) postulated that vessel wall swellin9 due to increased water and ion content would decrease the size of the lumen and increase peripheral resistance. More recently the increased sensitivity of the vasculature to catecholamines has been implicated (22,23). Since direct effects of steroids on the blood vessels are presumably mediated by specific intracellular receptors, we have examined these binding sites in aortic smooth muscle cells cultured from Sprague-Dawley S and R rats. The cultured aortic smooth muscle cells offer a opportunity to look for genetic differences between strains without interference from the extracellular environment. The existence and characteristics of these receptors has been documented by others usin 9 homogenized whole arteries of rabbits (24,25). S rats have higher plasma mineralocorticoid activity than R rats, which explains part but not all of the difference in blood pressure on a high-salt diet (26). No intrinsic differences in aortic cell aldosterone bindin 9 were found between the two substrains, but the data do not exclude in vivo differences due to extrinsic factors present in S and R rats or differences distal to receptor bindin 9 in the mechanism of steroid action. However, these findings are in agreement with those of Funder et al. (14) in the kidney, where no differences were found in mineralocorticoid bindin 9 between groups of S and R rats on low- and high-salt diets. Additional data on kallikrein induction, distal tubl]lar hypertrophy and skeletal muscle ion changes indicate that mineralocorticoid responses are intact and similar in the two substrains (27). Bindin 9 to beth classes of corticoid receptors was examined since both mineralocorticoid and glucocorticoid hormones can cause hypertension albeit by different mechanisms (28). In addition, there is considerable overlap in specificity between the two receptors and it is not known through which class of receptors adrenal steroids may he acting in the pathogenesis of hypertension. In our experiments, aldosterone always estimates fewer corticoid receptor II sites than dexamethasone, and saturation is not achieved unless much higher concentrations of aldosterone are used. Therefore, dexamethasone is a better indicator of differences in cgrticoid receptor II sites than aldosterone. ~o differences were found in ~Hdexamethasone binding between the two substrains. The relationship between mineralocorticoid- and salt-induced hypertension and vascular steroid receptors has been investigated by comparin 9 aldosterone bindin 9 in aortic smooth muscle cells of salt-resistant and salt-sensitive rat strains. There is good evidence that the resistance of Fischer 344 rats to mineralocorticoid and salt hypertension is due to a generalized defect in response to mineralocorticoids since these animals have increased plasma renin activity, decreased salt appetite, and a vascular steroid receptor with a two-fold lower affinity for mineralocorticoids (13,29). In contrast, the Long-Evans salt-resistant strain, appears to have a localized defect in the hypothalamus characterized by deficient mineralocorticoid bindin 9 and decreased salt appetite (12). The Sprague-Dawley salt-resistant substrain has similar vascular (Table 3) and renal (14) aldosterone bindin 9 characteristics, and mineralocorticoid responses (27) compared to the salt-sensitive substrain. Collectively, these data indicate that the three rat models have a different molecular basis for their resistance to salt-induced hypertension and the role of mineralocorticoid receptors varies considerably between strains.
1660
Aortic Aldosterone Binding in Dahl Rats
Vol. 36, No. 17, 1985
Acknowled@ements We are grateful to Mrs. Maria Barrett for her secretarial
assistance and to Dr. Eric Hall for the use of the electrosphygmomanometer and physiograph. HL 20201.
This work was supported by USPHS Grant
References i.
L.K. DAHL, M. HOLENE and L. TASSENARI, J. Exp. Med. 115 1173-1190 (1962). 2. J.P. RAPP, Hypertension 4 753-763 (1982). 3. L. TOBIAN and J.T. BINION, Circulation 5 754-758 (1952). 4. L. TOBIAN and P.D. REDLEAF, Am. J. PhysTol. 189 451-454 (1957). 5. A.W. JONES and R.G. HART, Circ. Res. 37 333-341 (1975). 6. E.T. GARWITZ and A.W. JONES, Hypertension 4 374-381 (1982). 7. W.J. MEYER and N.R. NICHOLS, J. Steroid Bi~chem. 14 1157-1168 (1981). 8. A.M. MOURA and M. WORCEL, The Adrenal Gland and Hypertension, in press Raven Press, New York (1985). 9. C.E. HALL, S. AYACHI, and O. HALL, Texas Rep. Biol. Meal. 30 143-153 (1972). 10. A.C. BROWNIE, S. GALLANT, P.A. NICKERSON and L.M. JOSEPH, Endocr. Res. Commun 5 71-80 (1978). Ii. O.B. HOLLAND, C. GOMEZ-SANCHEZ and T. ZIEGLER, Clin. Sci. 56 109-113 (1979. 12. M.N. LASSMAN and P.J. MULROW, Endocrinology 94 1541-1546 (1974). 13. N.R. NICHOLS, C.E. Hall and W.J. MEYER, J. Hypertension 1 393-397 (1983). 14. J.W. FUNDER, D. DUVAL, P. MEYER and L.K. DAHL, Endocrinology 94 1739-1743 (1974). 15. Z.S. KROZOWSKI and J.W. FUNDER, Proc. Natl. Acad. Sci. 80 60256O60 (1983). 16. J. CHAMLEY-CAMPBELL, G.R. CAMPBELL and R. ROSS, Physiol. Rev. 59 1-61 (1979). 17. N.R. NICHOLS, C.A. OLESON and J.W. FUNDER, Endocrinology 113 1096-1101 (1983). 18. K. BURTON, Biochem. J. 62 315-323 (1956). 19. D.L. SUSTARSIC, R.P. MCPARTLAND and J.P. RAPP, J. Lab. Clin. Med. 98 599-606 (1981). 20. G. SCATCHARD, Ann. N.Y. Acad. Sci. 51 660-672 (1949). 21. N.R. NICHOLS, C.E. HALL and W.J. MEYER Ill, Hypertension 4 646652 (1982). 22. J. DECHAMPLAIN, L. KRAKOFF and J. AXELROD, Circ. Res. 24 I75-92 (1969). 23. F.M. ABBOUD, Fed, Proc. 33 143-149 (1974). 24. L. KORNEL, N. KANAMARLAPUDI, T. TRAVERS and D.J. TAFF, J. Steroid Biochem. 16 245-264, (1982). 25. L. KORNEL, N. KANAMARLAPUDI, C. RAMSAY, T. TRAVERS and S. KAMATH, J. Steroid Biochem. 19 333-344 (1983). 26. J.P. RAPP and L.K. DAHL, Endocrinology 88 52-65 (1971). 27. J.P. RAPP, R.P. MCPARTLAND and D.L. SUSTARSIC, Hypertension 4 2026 (1982). 28. D. HAACK, J. MOHRING, B. MOHRING, M. PETRI and E. HACHKENTHAL, Am. J. Physiol. 233 F403-411 (1977). 29. C.E. HALL and O. HALL, Life Sci. 20 1239-1248 (1977).