Arterial steroid receptors and their putative role in the mechanism of hypertension

0022-473 1/8 3 ss.oe -o.oo Cop yright © 1983 Pergamon Press Ltd

J . steroid Biochem. Vol. 19, No. I, pp. 333- 344, 1983 Printed in Great Britain. All rights reserved

ARTERIAL STEROID RECEPTORS AND THEIR PUTATIVE ROLE IN THE MECHANISM OF HYPERTENSION L. KORNEL, N. KANAMARLAPUDI, C. RAMSAY, 1. TRAVERS, S. KAMATH, D. 1. TAFF, N. PATEL, W. PACKER and W. 1. RAYNOR Steroid Un it, Rush-Pre sbyterian-St Luke's Medical Center, Chicago, IL 60612, U.S.A.

SUMMARY

Data from clinical and experimental studies indicate that mechanism(s) for action of mineralocorticoids, other than renal, must be involved in the overall effect of mineralocorticoids on circulation -increased peripheral resistance and hypertension. We have postulated existence of such a mechanism in the arterial wall and have looked for the evidence for its presence. We have found high affinity, specific binders for mineralocorticoids, and glucocorticoids, with characteristics of steroid receptors, in the cytosol of rabbit aorta and femoral and carotid arteries. These binders possess physico-chemical properties of steroid receptors and, moreover , they translocate to cell nuclei (as steroid-receptor complexes) and bind to relatively specific "acceptor-sites" on nuclear chromatin. This pro vides evidence for the existence in the arterial wall of a molecular mechanism for a direct in situ action of mineralocorticoids and glucocorticoids. The mineralocorticoid receptors are not present in veins. We have also found that chron ically elevated levels of II-desoxycorticosterone (DOC) result in a marked increase in permeabilit y of arterial smooth muscle cell membrane to sodium ions; this is in accord with findings of other investigators in the rat. Th is change presumably leads, through a chain of biochemical events, to increased arterial and arter iolar smooth muscle contractility, increased peripheral resistance and hyperten sion. Study is in progress to determine whether the effectof DOC on arterial smooth muscle cell-membrane permeability to eletrol ytes is elicited through the receptor-med iated mechanism for the in situ action of mineralocorticoids in the arterial wall. It is postulated that this mechanism is primarily responsible for "mineralocortico id hypertension ", but may well be instrumental also in pathogenese s of various other forms of hypertension, including "essential".

Evidence derived from many clinical and experimen- arterial (and arteriolar) wall we have conducted the tal studies indicates that adrenal steroids play an im- study which is summarized in this report. portant role in the development or maintenance of various types of arterial hypertension-essential, enMATERIALS docrine, renal, renoprival and experimentally steroid induced[1-4]. Extensive investigations have been Animals Normal male white New Zealand rabbits were used conducted during the last three decades aimed at the elucidation of the nature of the biochemical mechan- throughout this study. They were maintained on norismts), by means of which these steroids would cause, mal peletized Wayne rabbit diet (15% protein, 0.25% or contribute to, the elevation of blood pressure. Dif- Na, 0.9% K) and drinking water ad libitum, and were ferent mechanisms have been postulated for mineralo- acclimatized in vivarium for at least one week before corticoids[5, 6] and glucocorticoids [7, 8]. However, experimenta tion. to date, the exact nature of these mechanisms remains Steroids largely unknown. All radioactive steroids used were labelled with triResults of many recent studies [9-12J indicate that mineralocorticoids do not cause hypertension tium at C-I,2 or C-6,7 and had sp. act. 40- 60 Ci] through the renal retention of sodium and water. A mmo!. The following abbreviations will be used direct action of these steroids on arterial smooth throughout this paper for the tritiated and nonmuscles has been hypothesized to lead to biochemical radioactive ("cold") steroids: Aldo for aldosterone; alterations which would result in increased vaso- DOC for l l-deoxycorticosterone: Prog for progesterreactivity, increased peripheral resistance, and hyper- one; F< for cortisol; 9afF< for 9a-fluorocortisol; B tension [13-17]. In the search for such a mechanism for corticosterone; 18-DO-Aldo for 18-deoxyaldosterfor a direct in situ action of mineralocorticoids on the one(21-hydroxy-11 p,18-epoxy-4-pregnene-3,20-dione); Dex for dexamethasone (~'-91X-fluoro-161X -methyl-cor­ tiso1) ; l61X-CH 3-F K for 161X-methyl-cortisol ; FS for Send all correspondence to : Ludwig Kornel, Director, cortisol-2l-sulfate (II p, I71X-dihydroxy-3,20-diketo-4Steroid Unit, Rush-Presbyterian-St Luke's Medical Center, pregnene-21-yl-sulfate); and Spiro for spironolactone 1753 West Congress Parkway, Chicago, IL 60612, U.S.A. K

333

334

L. KORNEL

(17a-acetylthio-17a-(fJ-carboxyethyl)- testosterone-vlactone). All tritiated and cold steroids were checked for purity by t.l.c. and those containing impurities in excess of about 2% of the total steroid concentration were repurified, as previously described [15].

Solvents and reagents These were either spectrograde quality, or were repurified as previously described [15]. The composition of the buffers used for the collection of various tissues, their homogenization, steroid binding studies, and the examination of the nuclear uptake and chromatin-binding of steroid-receptor complexes is described below under an appropriate procedure. The dextran-coated charcoal (DCC) suspension used for separation of bound from free steroids consisted of 1% (wIv) activated charcoal (Norit) and 0.1% (wIv) dextran in 20 mM Tris buffer, pH 7.4. The exact method of the separation procedure has been described previously [15]. METHODS

Preparation of tissues Following sacrifice and exsanguination of the animal, chest and abdomen were cut open, and aorta, femoral and carotid arteries, kidneys, inferior vena cava (IVC) and, in some instances, diaphragm and large pectoral muscle were quickly removed. Kidneys were perfused in situ (prior to removal) with ice-cold 25 mM potassium phosphate buffer, pH 7.6, containing 0.1 M sodium chloride (PC 1 buffer). All tissues were collected in the same buffer and immediately transported to the cold room (4-6°C) for further experimentation. The method of the cleaning of blood vessels from adhering extraneous tissues, the preparation of the renal tissue, and the homogenization of various tissues was previously described in detail [12, 15]. If not immediately processed, tissues were stored in liquid nitrogen. Cytosol was obtained by high speed centrifugation at 234,0009 for 30 min at 4°C. Examination of high affinity binding of various 3 [ H]-steroids in cytosol of aorta, femoral and carotid arteries, iriferior vena cava (IVC), kidneys and striated muscle These methods have been described in detail elsewhere [15]. They were based on classical steroid binding techniques, where aliquots of cytosol are incubated with increasing concentrations of [3H]-steroids, the free and bound steroids are then separated, and the concentration of each is determined by counting radioactivity present in the total and bound steroid fractions. The data were plotted according to Scatchard [18J, and binding parameters, dissociation constants (KD's) and maximum apparent numbers of binding sites (n values), were derived from these plots. Since mineralocorticoids and their antagonists yielded consistently curvilinear Scatchard plots, bind-

et al.

ing parameters for these steroids were obtained by statistical analysis (the least-squares method) of the binding data by means of a computer program; this way the curvilinear plots were resolved into two or three rectilinear slopes, each reflecting high affinity binding to another species of binding-sites. The details of the whole procedure have been previously reported [15].

Examination of the specificity of binding of various [3 H]-mineralo- and qlucocorticoids to the cytosolic high affinity binders in aorta, femoral and carotid arteries, IVC and kidney The methods used in this part of our study were previously published [15, 19]. They were based on the technique of classical steroid competition experiments, in which aliquots of cytosol are saturated with [3H]-steroid in the absence (control value = 100% binding) or presence of increasing concentrations of various cold steroids competing with the CH]-steroid for binding to the high affinity cytosolic binder(s). The results were calculated as concentrations of a competing steroid needed to decrease the binding of the CH]-steroid (control) by half (50% binding inhibition). The relative competitive potencies of various steroids were expressed as % competitive potency of the homologous (identical with the CH]-steroid) competing steroid. All the steroid binding experiments described above (examination of high affinity binding) and all the steroid competition experiments were carried out in the presence in cytosol of sodium molybdate (100mM) to prevent rapid loss of binding capacity [19J of cytosolic binders.

Search for evidence that the high affinity binders for mineralocorticoids and qlucocorticoids in the arterial wall are cytoplasmic receptors involved in the intraceilular-molecular mechanism of action of these steroids Examination of the ability of arterial receptor-steroid complexes to translocate to cell nuclei. A detailed description of this study is now in press [20]. Rabbit aorta cytosol was prepared in 0.25 M sucrose, 25% glucerol (v/v). The nuclei were prepared from rabbit kidneys and were purified as described previously [12]. Steroid-receptor complexes were formed by incubating aliquots of aorta cytosol, at 4°C with [3H]-steroids (Aldo, DOC, BK , F K' Dex, Prog) in concns. equal to 10 K D values for a given steroid. Additional analogous incubations were carried out in the presence of 1~100-fold concentration of various competing "cold" steroids. Aliquots of purified nuclei suspensions were then added to cytosol aliquots (containing preformed steroid-receptor complexes, and incubated at 24°C for 30 min. Nuclear pellet was then obtained by centrifugation, and was washed three times with the incubation buffer to remove free steroids and steroid-receptor complexes adsorbed on, but not taken up by the nuclei. Subsequently, nuclear uptake of CH]-steroid was measured, and compared

Arter ial rninera lo- and glucocort icoid recep to rs

to that of the identical free steroid from buffer alone (no cytosol present). The final data were expressed as fmol eH]-steroid nuclear upt ake/rug nuclear DNA. In a series of complementing experiments, disappearance of cytoplasmic [3H]-steroid-receptor complexes from the cytosol, following its incubation with the nuclei, was determined simultaneously with determination of the nuclear uptake of these complexes. Examination of specific-binding of arterial receptorsteroid complexes to nuclear chromatin. Aorta cytosol was prepared in 10mM Tris, 1.5 mM EDTA, and 12mM thioglycerol. Nuclear chromatin was prepared by a modification of the method of Spelsberg and Hnilica[21]. Details of this method have been described previously [12]. The aorta cytosol was incubated with various [3H]-steroids to form steroidreceptor complexes. Analogous incubations were performed also in the presence of 10-1OO-fold concentration of various competing "cold" steroids. Thus preformed receptor-steroid complexes were then incubated at 25°C for 30min with aliquots of purified chromatin (containing 15D-200 Jlg DNA). The complexes tightly bound to chromatin were then separated from the unbound complexes (and the free steroids) by sequential washings of the chromatin with a sodium-magnesium Tris buffer containing 5% propylene glycol (NMTP), under reduced pressure [12]. Chromatin with the tightly bound complexes was retained on a Milipore filter, washed twice with NMTP buffer, air-dried and the amount of radioactivity (e H]-steroid) present in the chromatin was determined by liquid scintillation counting. The final data derived from these experiments are expressed as fmol bound [3H]-steroid/mg chromatin DNA.

335

The gels were formed in pyrex tubes (6 mm i.d.), in two lengths, 11 0 mM and 55 mm. The electrode buffer consisted of 10 mM Tris, 77 mM glycine, 1 mM dithiothreitol (OTT) and 10% glycerol. Separat ion gels contained 6--7% of total monomer and 15% bisacrylamide. Except for the addition of 1 mM DDT and 10% glycerol to both separation and stacking gels they were prepared as in the method of Miller et al.[22]. Electrophoresis was carried out for 210min or until the bromophenol blue dye had reached 0.5 ern from the bottom of the gel tubes. The gels were gently flushed out of the tubes, sliced into 0.5 ern pieces, left overnight each in 10 ml toluene-butyl PBD and then counted for radioactivity. Study of the effec t of mineralocorticoids on the rate of net sodiumflux into the cells of aortae of DOCA-hypertensive and normotensive control rabbits

The method used in this part of our investigation is a modification [12] of the technique of Friedman et 01. [23]. Silastic rubber strips impregnated with DOCA were implanted subcutaneously in nine male New Zealand rabbits, all 13 weeks of age, and silastic rubber strips without DOCA were implanted in eight control rabbits. Blood pressure (BP) was measured in all animals first twice-weekly, then weekly, using a Doppler ultrasound probe and a modified pediatric blood pressure cuff. Plasma levels of DOC were determined at weekly intervals by radioimmunoassay [24]. Serum levels of sodium and potassium were also determined periodically. The pre-implantation BP in both groups of rabbits was 120-135/65-75 mmHg. Rabbits implanted with DOCA-containing silastic rubber strips develFurther characterization of arterial mineralocorticoid oped hypertension, which stabilized at a level of and glucocorticoid receptors: their separation on poly- 160-1 90/85-95 mmHg during the 6-8th week after imacrylamide gel electrophoresis (PA GE) plantation. BP of the control rabbits remained Rabbit aorta cytosol was prepared as for the unchanged throughout the study. After 6 months, steroid binding experiments [ 15]. but without sodium both hypertensive and control normotensive rabbits molybdate. [3H]-steroid-receptor complexes were were sacrificed. in groups of 2 or 3, and aortae were formed by incubating cytosol with 2 x 10- 8 M removed. Total tissue sodium concentrations (in strips of aoreH]-DOC or [3H]-Dex and 10 x 10- 8 M cold heterologous steroid, with or without 1.0 x 10- 0 M tae), intracellular sodium (Nail, and net sodium ion homologous cold steroid, to check the specificity of fluxes into the cells of aortae were measured as debinding of the respective [3HJ-steroids. The labelled scribed [12]. The results are presented as mEq cytosol was then "activated" by heating at 25"C for sodium/kg dry weight of tissue (concentrations) and 20 min in a gyrotory water bath. The activated cyto- as mfiq/kg dry tissue wt/min. (flux rates). sol was then cooled on ice, and ice-cold Na2Mo0 4 RESULTS was added so as to make its concentration approx. 20 mM. The cytosol was then stored in liquid N 2 until High affinity binding of [3H]-mineralocorticoids and [ j H)-glucocorticoids in cytosols of aorta, femoral and the electrophoresis run. carotid arteries, I VC, and kidney The PAGE procedure was a modification of the Scatchard plots-binding affinity and capacity. technique of Miller et al.[22]. Electrophoresis was performed in a Bio-Rad electrophoresis cell kept at Typical Scatchard plots for DOC, Aldo and Prog 4°C. Ice-cold aqueous ethylene glycol (501:" v/v) was obtained with the aorta and carotid artery cytosols circulated through the jacket of the lower chamber by are shown in Fig. I. All three steroids yielded curvimeans of a peristaltic pump. The entire apparatus was linear plots, which were resolved each into two rectisurrounded by frozen cold packs. linear slopes by computer analysis. B" F" and Dex

336

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Fig. 1. High affi nity binding of tritiated DOC, Aldo and Prog in the cytosol of rabbit aorta (panel A) and carotid artery (panel C). Comparison of typical Scatchard plots ; experiments performed in the presence of 100 mM sodium molybdate [ 19]. Each point in the graph represents mean of duplicate determinations. All three steroids yielded consistently in repeated experiments curvilinear plots. The resolution of these plots was obtained by means of statistical analysis with the aid of a computer program (see text). The computer-generated slopes are shown as straight lines. K D and n values, computer-generated dissociation constants and numbers of binding sites. which is represented in Fig. I by the steeper slopes, the binding of these steroids to this binder could not be detected by Scatchard analysis ; presumably, because of the much lower BfF ratio with respect to mineralocorticoid binders, which was "oblitera ted" by many-fold higher ratio exhib ited b y these steroids in the ir binding to the glucocorticoid binders. The binding parameters obtained for all the

yielded rectilinear Scatchard plots (these are shown in Fig. 2). Both mineralo- and glucocorticoids gave very similar plots for the b inding to aorta cytosol and for b inding to sma ller arter ies, femoral a nd carotid [cf. panels (A) a nd (C) of Figs I a nd 2]. Although each of the glu co corticoids tested exhibited also a significant affinit y for the "mineralocorticoid b inde r" (Ty pe A; cf. below the results of ster oid competitio n experiments),

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Fig. 2. High affinity binding of tritiated F. , B. and Dex in the cytosol of rabb it aorta (panel A) and caro tid artery (panel C). Comparison of typical Scatchard plots; experiments performed in the presence of 100mM sodium molybdate [19]. Each point in the graph represents mean of duplicate determinations. Note two different scales, one for B. and Dex, the other for F•. F. and Dex yielded consistently in repeated experiments rectilinear plots. B. yielded usually also only one rectilinear slope, but occasionally, it gave a curvilinear plot which was resolved into two slopes by means of computer analysis (cf. legend to Fig. 1).

337

Arter ial mineralo- and glucocorticoid recept or s Table I. Binding characteristics of arterial steroid receptors (AI Co mputer-resolved curvili near Scatchard plots"

x; [3H)- steroid DOC Aldo Prog

F and

At

nit

cr

K D2

A

F and C

A

F and C

A

8.0 ± 4.6 10- 1 1 M

42

± 15

38

± 18

4.2 ± 1.3 X 10- 9 M

4.0 + 1.5 X 10-= 9 M

338

± 80

320

± 100

2.1 ± 0.7 10- 10 M

34

± 13

36

± 16

6.5 ± 2.3 x 10- 9 M

5.6 + 2.0 X 10-= 9 M

330

± 100

307

± 76

2.0 ± 0.9 x lO- lo M

36

± 14

38

± 10

5.3 ± 1.6 x 10- 9 M

5.4 ± 1.8 X 10- 9 M

314

± 90

298

± 88

1.9 ± 0.9 x 10- 10 M

X

3.8 ± 1.0 x 10- 10 M

X

3.2 + 1.1 x 10=-1 0 M

(B) Rectiline ar Scatch ard plot s

Kv

nt A

F and C

A

F and C

B.

1.8 ± 0.34 x 10- 9 M

2.0 ± 0.8 x 10- 9 M

660

± 108

648

± 120

F.

4.3 ± 1.9 x 10- 10 M

4.4 ± 1.6 x 10- 10 M

820

± 125

816

±

Dex

4.8 ± 1.3 x 10- 9 M

6.0 + 2.3 x 10-=9 M

660

± 92

626 ± 140

110

• All tissues were homogenized in 20 mM sodium molybdate, then the concentration of molybdate in the cytosol was increased to 100 mM, before the incub at ion with trit iated stero ids (cr. Ref. [19] ). All data in the table were der ived by co mputer anal ysis. t fmol/mg cytosol protein. t A-aorta ; Fs--femoral artery; C-ea rot id artery.

eH}steroids tested are summarized in Table 1. It will be seen that the affinity of mineralocorticoids (and their physiological antagonist, progesterone) for the mineralocorticoid binders (K Dt ) was about 20 times higher than that for the glucocorticoid binder s (K D2 ). At the same time the app arent maximal number of binding-sites for glucocort icoids (n for B and Dex) was about 20 times larger than that for mineralocorticoids (nIl. The lower apparent number of glucocorticoid binding-sites associated with mineralocorticoids (n2) most likely stems from the markedly lower stability of these binding-sites when they are occupied by mineralocorticoids [19]. Furthermore, it appears that the affi nity of mineralocorticoid binders for the same steroids is higher in the smaller arteries (femoral and carot id) than in the aorta. However, because of the inherent large methodological error in this kind of study, many more binding experiments have to be conducted , or purified receptors have to be used, to ascertain the existence of this difference. In preliminary studies, the corresponding affinities of mineralocorticoids to their receptors in still smaller arteries (arterioles) were found to be still higher. If confirmed, this phenomenon may have an important biological implication : the smaller the art ery, the greater the sensitivity of its receptors to the action of mineralocorticoids.

with the highest affinity for DOC; aldosterone exhibited abou t 45% of the potenc y of DOC in compet ing for this binder ; Type B, a very specific cort isol binder, with high affinity for cortisol-sulfate and a very low one for dexamethasone ; this is a tran scortin-like binder [15J ; Type C, closely resembling the classical glucocort icoid receptor in other target organ s to glucocorticoids, with highest affinity for dexamethasone. Furthermore, the results of our studies on the competitive inhibition of the translocation of [3H} steroid-receptor complexes to cell nuclei by various "cold" steroid s (see below) revealed the existence in the aorta cytosol of still another glucocort icoid binder with the highest affinity for cortisol , and a somewhat lesser one for dexamethasone, which exhibits the abilit y to translocate to cell nuclei (FK-receptor). The n value for this binder is about 20 times lower than that for the abundant, non-translocating transcortin-like F K-binder exhibiting a very low affinity for Dex (Type B). We are designating this binder as Type D. Heterogeneity of glucocorticoid binders in various known target organs to glucocortico ids has been reported also by other investigators [25-27]. In the cytosol of IYC we could not detect any high affinity mineralocorticoid binders, whereas high affinity binding of glucocorticoids was similar to that in the aort a cytosol [15]. An identical situat ion was seen Steroid competition experiments- binding specificity. to exist in the cytosol of the large pectoral and diaTable 2 lists the results of these experiment s. These phragm muscles [15, 20]. results indicate that three different types of high affinIn the rabbit kidney cytosol, we found analogous ity binding sites for adrenal steroids are present in the four types of high affinity steroid binders [15, 28J, but cytosol of rabbit aorta and femoral and carotid the mineralocorticoid binder differed from that in the arteries: Type A, most specific for mineralocorticoids, arterial cytosol in that aldosterone had the highest K

L.

338

KORNEL

et a/.

Table 2. Comparison of relative competitive potencies of various steroids for binding to cytosolic rnineralo- and glucocorticoid binders in rabbit aorta (A), femoral (F) and carotid (C) arteries Type A* [3H)-Aldo

['H)-DOC Competing Steroid DOC Aldo

9rxjFK Prog 18-DO-Aldo BK FK Dex FS

A 100 48 46 22 29 88 26 6.2 l.l

F and Ct 100 44 82 18 20 93 27 6.2 1.0

A 196 100 185 44 38 132 38

IS 1.2

Type B* Displaceable [3H)-steroid [3H)-F

F and C 191 100 183 40 29 136 40 17 1.0

A

F and C

14 1.0 1.7 4.0 2.1 35 100 0.03 41

16 1.6 2.3 7.1 2.0 45 lao 0.05 55

A 48 3.6 6.6 12.1 5.2 100 219 0.1 128

Type C* [3H)-B F and C 45 4.2 3.7 9.1 lOa 182 0.1

[3H)-Dex A 3.8 1.4 90 2.0 0.2 12 32 100 0.5

F and C 4.4 1.9 91 1.8 0.3 15 34 100 0.4

* The distinction between the three types of high affinity cytoplasmic binders, A, B, and C, is based on the order of relative potencies of various mineralo- and glucocorticoids in competing with various tritiated steroids for binding; thus Type A is most specific for DOC, Type B for F K' and Type C for Dex. It should be noted that the hierarchy of binding potencies of various steroids to the three types of binders is virtually the same for aorta and for femoral and carotid arteries. The only conspicuous difference is in the potency of 9~fF K in displacing [3H)-DOC from aortic vs femoral and carotid artery binders. t Average values obtained for F and C arteries. For individual values see Ref. [19].

affinity, and DOC exhibited only 50% competitive potency for that binder (classical mineralocorticoid receptor). Mineralocorticoid antagonists, progesterone and 18-desoxyaldosterone exhibited high competitiveness for the mineralocorticoid binder in the arteries, as well as in the kidney. These antagonists revealed very low competitiveness for the glucocorticoid binders (Table 2).

Translocation of arterial cytoplasmic receptor-steroid complexes to cell nuclei The ability of the steroid-receptor complexes to migrate to cell nuclei, and the binding of these complexes to relatively specific "acceptor-sites" on nuclear chromatin are the fundamental properties of steroid receptors. Thus, the demonstration that the newly discovered arterial cytoplasmic high affinity binders to mineralo- and glucocorticoids possess such properties would shed an important additional light on the role of these binders. A detailed description of this study is now in press [20]. The results obtained are presented in Figs 3 and 4. It will be seen that: (1) The nuclear uptake of [3H]-steroids was 3-10 times greater in the presence of cytosol than from the buffer only; in analogous experiments utilizing cytosol of rabbit diaphragm muscles, instead of aorta cytosol (not shown in the figures), the nuclear uptake of [3H]-mineralocorticoids was not enhanced by the presence of cytosol, but that of [3H]-glucocorticoids was. (2) The uptake of [3H]-mineralocorticoids was decreased much more by the presence in the cytosol of an excess of "cold" mineralocorticoids than of "cold" glucocorticoids; the order of the potency of various cold steroids to decrease the nuclear uptake of an CH]-steroid corresponds well with the order of the relative competitive

potencies of these steroids in competition for binding to the cytosolic receptors (cf. above, steroid competition studies). The apparent decrease in the nuclear uptake of an [3H]-steroid is thus the result of the displacement of that steroid from the cytoplasmic receptor-site by the competing steroid. Consequently, the majority of the receptor-steroid complexes translocated to the nucleus contain the "cold" competing steroids. However, the apparent decrease in the nuclear uptake of an [3H]-steroid may also be a sequence of the binding to the cytosolic receptor of a steroid not capable of inducing an "active conformation" of the receptor, which then stays in the cytoplasm and does not migrate to the cell nucleus. Such was the case when "cold" progesterone was competing for binding with CH]-DOC or [3H]-Aldo. In a series of separate experiments (not shown in the figures) it was established that the nuclear uptake of [3H]-progesterone was not increased in the presence of aorta cytosol. Thus, progesterone acts as an "antiinducer" with respect to the mineralocorticoid cytoplasmic receptors. A similar situation was noticed when synthetic compounds with anti-mineralocorticoid activity were used, spironolactone and 18desoxyaldosterone. In a series of experiments in which receptor disappearance from the cytosol during the incubation with nuclei was measured, the amount of radioactivity ([3H]-steroid) remaining in the cytosol after the incubation was significantly lower than that present in the cytosol before the incubation with nuclei. Moreover, the sum of the cytosolic "post-incubation radioactivity" plus that translocated to the nuclei, plus the amount of radioactivity found in the nuclear washes (see "Methods") was in each experiment almost identical with the amount of radioactivity present in the cytosol before its incubation with nuclei (Table 3).

Arterial mineralo- and glucocorticoid receptors 20,-19

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Fig. 3. Nuclear uptake of CH]-aldosterone (panel A) and [3H]-11-desoxycorticosterone (panel B) from rabbit aorta cytosol, in the presence or absence of various "cold" steroids competing for binding to the cytoplasmic receptors (hatched or dotted bars). Each of the competing steroids was used in three different concentrations, as indicated. The uptake shown is in fmol [3H]-steroid/mg of nuclear DNA. The first single bar on the left (-) indicates the nuclear uptake on an [3H]-steroid from aorta cytosol in the absence of a competing "cold" steroid; the last single bar on the right (BUFF*) indicates the uptake of the same CH]-steroid from buffer alone, in the absence of cytosol, also with no competing steroid present. Each bar in the graph represents a mean of 3--6 separate experiments. (Reprinted with permission of Endocrinologv.)

Specific binding of arterial receptor-steroid complexes to nuclear chromatin

complexes. (2) Some degree of chromatin binding of free steroids was also noted with all the steroids examined. This binding of the free steroid to chromaThe results obtained are presented in Figs 5 and 6. tin was seen to be surprisingly extensive in the case of These results closely resemble those of the experi- DOC. This phenomenon is under investigation at ments on the translocation of the steroid-receptor present. From the above experiments it is concluded that complexes to cell nuclei. However, it will be noticed that: (1) "cold" mineralocorticoids affected the bind- both mineralocorticoid- and glucocorticoid-receptor ing of glucocorticoid-receptor complexes to chroma- complexes from aorta cytosol bind tightly to relatin to much greater extent than did "cold" glucocorti- tively specific "acceptor-sites" on nuclear chromatin. coids with respect to the binding of mineralocorti- Furthermore, the results of these experiments, as well coid-receptor complexes. This suggests that the bind- as those of the experiments on the translocation of ing specificity of "acceptor-sites" on chromatin for steroid-receptor complexes to cell nuclei seem to be mineralocorticoid-receptor complexes is greater than at some variance with the results of the steroid comthat of acceptor-sites for glucocorticoid-receptor petition experiments described in the appropriate

340

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Endocrinology.) section of this paper: (1) the strongest "suppressor" of the translocation and of the chromatin-binding of the [3H}Aldo-cytoplasmic-receptor complex is "cold" Aldo, whereas that of the [3H}DOC-receptor complexes is "cold" DOC. This suggests that separate receptors (or receptor-sites) exist for DOC and for Aldo, even though there appears to be a considerable degree of cross-reactivity between the two. There is, however, also another possible explanation of this finding. Since we have used in our translocation experiments kidney nuclei, and the steroid with the highest affinity for the renal mineralocorticoid receptor is Aldo, it is likely that the renal nuclear chromatin binding-sites exhibit a higher affinity for the conformation of Aldo--receptor complexes than for the DOC-receptor ones. Thus, as a result of a continuous dissociation-reassociation between the steroids and the receptors which have translocated to the nuclei, cold Aldo would be displacing [3H]-Aldo from the intranuclear receptors more efficiently than does cold DOC. A study has been designed to resolve this issue.

Further characterization of arterial mineralocorticoidand glucocorticoid-receptors: their separation on polyacrylamide gel electrophoresis This study is in progress. The results obtained are shown in Fig. 7. To achieve the separation of the mineralocorticoid from the glucocorticoid cytosolic binders it was necessary to extend the time of electrophoretic run to 3t h, so that the receptors travelled close to the end of the gel-column . The specificity of the separated binders is indicated by an almost complete disappearance of the respective radioactive bands by suppression with an excess of homologous cold steroid .

Study of the effea ofchronically administered mineralocorticoids On arterial smooth muscle cell membrane permeability to sodium ions Total tissue sodium concentration in aortae of DOCA-hypertensive rabbits was 332 ± 16 mEqjkg dry wt ; the initial intracellular sodium concentration

341

Arterial mineralo- and glucocorticoid receptors Table 3. Correlation of nuclear uptake of [3H]-steroids with the amount of specifically bound [3H]-steroids present in the cytosol before and after incubation with nuclei (fmol/sample) [3H]-Steroid fraction

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(without any treatment) was 152 ± 14 mEqjkg dry wt. In the control rabbits the corresponding sodium concentrations were 198 ± 18 and 81 ± 7 mEqjkg dry wt. The results of the experiments on passive sodium influx are shown in Fig. 8. The rate of sodium flux 10 ,..--

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chromatin. Thus, the described findings provide evidence for the presence in the arterial wall of a molecular mechanism for the in situ action of rnineralocorticoids and glucocorticoids. What is the role of this mechanism in the overall effect of mineralocorticoids on circulation-increased peripheral resistance and hypertension? The results of our studies on the rate of passive sodium ion flux into the cellsof aortae of DOCA-hypertensive rabbits corroborate findings of Jones et al.[ll , 32-34J and Friedman et al. [23, 35-37J in hypertensive rat tail artery: chronically elevated levels of mineralocorticoids increase arterial, presumably smooth muscle, cell membrane permeability to electrolytes. As a result of this, intracellular concentrations of certain ions, such as sodium and magnesium, are increased, whereas those of other electrolytes are maintained within normal range, presumably by an adequate compensatory trans-membrane active transport mechanism [34]. Increased intracellular sodium concentrat ions are thought to enhance contractility of actonmyosin fibres. The mechanism by means of which this may occur has been discussed by us recently [19]. The increased contractility of actomyosins results in hyperresponsiveness of arterial and most importantly, arteriolar muscles to various humoral vasoconstrictive agents, as well as neurogenic vasoconstrictive stimuli. It is tempting to hypothesize that mineralocorticoids alter the permeability of the smooth muscles membrane to electrolytes through induction of synthesis of structural membrane proteins which would increase the "porosity" of the membrane. We have 240 2 20 ~

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The postulate of the direct action of mineralocorticoids on arterial smooth muscles implies existence of an intracellular-molecular mechanism for action of these steroids in the arterial wall. High affinity binding to stereospecific intracellular receptor proteins in the cytoplasm of target tissue cells is now considered to be the initial obligatory step in the intracellular-molecular mechanism of action of steroidal hormones [29, 31]. The results of the reported studies reveal presence of high affinity binders to mineralocorticoids (and glucocorticoids) in the arterial waIl, fitting the criteria of steroid receptors: high affinity, low, limited binding capacity, and relative stereospecificity. Recently, high affinity binders to adrenal steroids, with similar characteristics of those described by us, were demonstrated in the smooth muscle culture of explants of rat aortae [16]. Moreover, the binders described by us possess also other fundamental properties of intracellular steroid receptors: they exhibit ability to migrate to ceIl nuclei, and to bind tightly to specific "acceptor-sites" on nuclear

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Arterial mineralo- and glucocorticoid recept ors recently initiated work aimed at inves tiga ting such a po ssibility, utilizing aortic strips grown as organ culture in the presence or absence of mineralocorticoids and/or their antagonists. We are po stulating that the effect of chronicalIy increased levels of rnineralocorticoids on arterial cell membrane permeability to electrol yte s is elicited through the discovered mechanism for the in sitlt action of mineralocorticoids in the walls of arteries. The absence of suc h mechanism in the walI s of veins adds to the plausibilit y of thi s postulate. Finall y, does all this reasoning appl y only to the so -ca lled mineralocorticoid h ypertension? Recent evi dence indicates [38, 39J that a lso in patients with essential hypertension sodium and potassium fluxes across the celI membrane (of er ythrocytes and lymphocytes) are altered, with the re sulting increase in the intracellular sodium concentrations. Although this appears to be a hereditary thus presumably genetic abnormality [38J, the finding of the sa me abnormality in so m e of the normotensive offspring s of the hypertensi ve individuals, raises the possibility that this abnormality alone is not sufficient to ac count for hyperten sion . Thus an "en ha nced activit y" of mineralocorticoid receptors ma y be an additional fac to r necessary for the development of increased vasoreactivity, peripheral resi stance and hypertension. Such an enhanced activity could be either du e to increased levels o f mineralocorticoids, found in man y patients with essential hypertension [40-45J o r, in some patients, perhaps due to an increased density of m ineralocorticoid receptors in the arterial waIls . Finally, it should be mentioned that in so me patients with essential [46J and renal [47J hypertension, and in experimental renal hypertension [48, 49J, a decreased activity of vasodilating substances (Iysylbradykinin, PGE 2 ) has been demonstrated. Thus, the delicate equilibrium between the vasodilator and vasoconstrictor activity in the arterial walI may be shifted in favour of the latter. In which case, mineralocorticoids and their arterial cytoplasmic receptors may also be a sine qua non in the development of hypertension, even in the face of normal plasma levels of mineralocorticoids and normal densit y of their receptors in the arteries of such indi viduals. The elucidation of these hypothetical possibilities awaits further investigation.

343

Adrenal mineralocort icoids cau sing hypertension . Am. J. Med. S2 (1972) 623-
6. Biglieri E. G. and Forsham P. H.: Stud ies on the expanded extracellular fluid and the response to various stimuli in pr imary aldo ster onism. Am. J . Med. 30 (1961) 564-57 6. 7. Swingle W. W.. deVanzo 1. P., Glenister D., Gross field H. C and Wagle G.: Role of gluco- and mineralocorticoids in salt and water metaboli sm of adrenalectomized dogs. Am. J. Physiol. 196 (1959) 283-286. 8. Baxter 1. D. and For sham P. H.: Tissue effec ts of glucocorticoids. Am. J. Med. 53 (1972) 573-589 . 9. Berecek K. H. and Bohr D. F.: Stru ctu ral and functional cha nges in vascular resistance and reactivity in the desoxycorti costerone acetate (DOC Al-hyper tensive pig. Circulation Res. 40 Suppl [ (l 977l [-146-1 52. 10. Chobanian A. V., Volicer L., Tifft C P.. Gav ras H.. Liang C· S. and Faxon D.: Mineralocort icoid-induced hyperten sion in patients with orthostat ic hypotension. New Engl. J. Med.301 (1979) 68-73. II. Jone s A. W.: Kinetics of active sodium transport in aorta s from co ntrol and deoxycorticosterone hypertensive rat s. Hypertension 3 (1981) 631-640. 12. Kornel L. : Studies on the mechan ism of mineralocorticoid-indu ced hypertension: evidence for the presence of an in-situ mechan ism in the arterial wall for a direct action of mineralocorticoids. Clin. Biochem. 14 (1981) 282- 293. 13. Gene st 1., Nowaczynski W., Kuchel 0 ., Boucher R. and Rojo-Orterga J. M.: The role of the adrenal cor tex in hum an essential hyperten sion : keynote address. Mayo Clin. Proc. S2 (1977) 291- 307. 14. Korn el L.. wu F. T. and Saito Z.: Essential hyperten sion : a derangement in corticosteroid metabolism? R.P.S.L. Med. Ctr Bull. 14 (1975) 3- 16. 15. Kernel L., Kanamarlapudi N., Travers To. Taff D. 1., Patel N.. Chen C , Baum R. M. and Raynor W. J.: Stud ies of high affi nity bind ing of mineral o- and glucocorticoids in rabbit aorta cytosol. J. steroid Biochem. 16 (1982) 245-2 64. 16. Meyer W. J. and Nichols N. R.: Mineralocort icoid binding in cultured smooth muscle cells and fibroblast s from rat aorta. J. steroidBiochem. 14 (1981) 11571168. 17. On oyama K., Bravo E. L. and Tarazi R. D.: Sodium, extracellul ar fluid volume, and cardiac output changes in the genesis of mineralocorticoid hyperten sion in the intact dog. Hypertension 1 (1979) 331-336. 18. Scatchard G.: The attractions of prot eins for small molecules and ions. Ann. N. Y. Acad. Sci. SI (1949) 660-6 72. 19. Kornel L., Ramsay C , Kanamarlapudi N., Tra vers T. and Packer W.: Evidence for the presence in arterial walls of intracellular-molecular mechan ism for actio n of mineralocorticoids. Clin. Exp. Hypertension 4 (1982) 1561-1 582. 20. Kornel L., Kan amarlapudi N., Ramsay C and Taff D. 1.: Stud ies on arterial mineralocorticoid and glucocorticoid receptors : II. Evidence for the tran slocation of REFERENCES steroid-cytoplasmic receptor comple xes to cell nuclei. Endocrinology (1983). 1. Kernel L. : Kidne y, adrenal corte x and hyperten sion : 21. Spelsberg T. C and Hnilica L. S.: Proteins of chromatheory and facts. Archs Int. Med. 103 (1959) 820--831. tin in template restriction in RNA synthesis in vitro. 2. Kornel L., Riddle M. and Schwar tz T. B.: The manageBiochem. biophys. Acta 228 (1972) 202-21 I. ment of hypertension asso ciated with disorders of function of the endoc rine gland s ("endocrine hyper- 22. Miller L. K., Diaz S. C and Sherman M. R.: Steroidreceptor quantitation and characterizat ion by electrotension" ). Med. Clin. N. Am. 55 (1971) 23-45 . phoresis in highly cross-linked pol yacrylamide gels. 3. Genest J., Nowacz ynski W., Boucher R. and Kuchel Biochemistry 14 (1975) 4433-4443. 0 .: Role of the adrenal cortex and sodium in the pathogenesis of human hypertension. CMA J. 118 23. Friedman S. M., Nakashima M. and Friedman C L.: Cell Na and K in the rat tail artery dur ing the develop(1978) 538-549. ment of hypertension induced by desoxycorticosterone 4. Mendlowitz M.: Some theor ies of hypertension: fact acetate . Proc. Soc. expo BioI. Med. ISO (1975) 171-176. and fancy. Hypertension 1 (1979) 435-441. 5. Biglieri E. G., Stockigt J. R. and Schambelan M. D.: 24. Kornel L. and Mitka 1.: An improved method for de-

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37. Fr iedm an S. M.: Evidence for enhanced sodium transport in the tail artery of the spo nta neously hypertensive rat. Hypertension 1 (1979) 572. 38. Garay R. P. and Meyer P .: A new test showing abnormal net Na + and K + fluxes in er ythrocytes of essential hypertensive patients. Lancet 1 (1979) 349-353. 39. Ambrosioni E., Tartagni F., Montebugnoli L. and Magnani B.: Intralymphocytic sodium in hypertensive patients : A significant correlation . International Society of Hypertension 6th Scientific Meeting. Goteborg, Sweden . Abstract 1-P (1979) p. 8. 40. Genest 1., No waczynski W.. Kuchel O . and Sasak i C : New evidences of disturbances of mineralocorticoid activity in ben ign, uncomplicated essenti al hypertension . Trans. Am. din. d im. Ass. 83 (1971) 134-146. 41. Nowaczynski W ., Kuchel 0. , Genest J. et al.: Dynamic aldosterone and 18-hydroxydeoxycorticosterone studies in labile and stable benign essent ial hyperten sion. J . steroid Biochem. 6 (1975) 767-778 . 42. Woods 1. W ., Liddle G. W., Slant E. G . et al.: Effect of an adrenal inhibitor in hypertensive patients with suppressed ren in. Archs Int. Med. 123 (1969) 366-370. 43. Spark R. F. and Melby 1. C: Hypertension and low plasma renin activity : Presumptive evidence for mineralocorticoid excess. Ann. into Med. 75 (1971) 831- 836. 44. Melb y J. C, Dale S. L. and Wilson T . E .: l S-H ydroxydeox ycorticosterone in human hypertension . Circulation Res. 28 Suppl. 2 (1971) 143-150. 45. Brown 1. J., Fraser R., Lo ve D. R. et al.: Apparently isolated excess deoxycorticosterone in hypertension : a variant of the mine ralocorti coid excess syndrome. Lancet l (1972) 243-247. 46. Tan S. Y., Sweet P. and Mulrow P. 1.: Impaired renal production of prostaglandin E 2 : A newly identified lesion in human essent ial hypertension. Prostaglandins 15 (1978) 139-150. 47. Margolius H . S., Geller R., Pisano J. J. et al.: Altered urinary kallikrein excretion in human hypertension . Lancet 2 (1971) 1063-1065. 48. Croxatto H. R. and San Martin M.: Kalli krein-like act ivity in the ur ine of renal hypertensive rats. Experientia 26 (1970) 1216-1217 . 49. Terragno N . A. and Terragno A.: Role of naturally occurring vasoactive principles in hypertension : State of the art. Mayo Clin. Proc. 52 (1977) 449--458.