Study on the mechanisms of glucocorticoid-induced hypertension: glucocorticoids increase transmembrane Ca2+ influx in vascular smooth muscle in vivo.

Blood pressure (BP) and ex vivo influx rate of Ca2+ in excised aortae were measured in rabbits implanted with silastic rubber strips impregnated with glucocorticoids (GC) [dexamethasone (DEX) or cortisol (FK)], or carbenoxolone (CX) [inhibitor of 11 beta-hydroxysteroid dehydrogenase (11-HSD), in a large (lg) or a small (sm) (10 times smaller) concentration], or FK plus CX (sm), or DEX plus RU 38486 (a specific GC-receptor blocker). After 4-6 weeks rabbits implanted with DEX, CX (lg), and FK+CK (sm) developed hypertension. Those implanted with FK alone (yielding physiological serum concentration of FK), CX (sm), and DEX+RU 38486 did not develop hypertension. Rates of unidirectional influx of Ca2+ measured in rings of excised aortae were in all hypertensive rabbits more than twice those in the control rabbits (implanted with silastic strips not containing any steroids). In all normotensive rabbits, Ca2+ influx rates remained normal. We conclude that, in analogy with the in vitro findings in cultured vascular smooth muscle (VSM) cells treated with GC, also in vivo, the elevation of tissue levels of GC causes an increase in the influx rate of Ca2+ in VSM. We propose that this may be the main pathogenic mechanism of GC-induced hypertension.

Arterial receptors for adrenal steroids and transport of electrolytes in vascular smooth muscle.

The role of arterial receptors to mineralocorticoids (MC) and glucocorticoids (GC) in the induction by MC and GC of changes in transmembrane transport of sodium (Na+) and water was investigated. Implantation of Silastic rubber strips impregnated with 11-desoxycorticosterone acetate (DOCA) in rabbits was followed by a marked increase in vascular smooth muscle cell membrane permeability to Na+ and hypertension. Both of these effects were preventable with progesterone, an anti-MC at the steroid-receptor level, implanted in relative excess simultaneously with DOCA, in approximately 50% of the implanted animals. The other 50% were hydroxylating in vivo progesterone to 11-desoxycorticosterone (DOC) efficiently enough not to yield the necessary ratio of progesterone to DOC for the sufficient MC receptor blockage. In vascular smooth muscle cell culture, grown in the presence of steroids, GC but not MC increased intracellular water space. This increase was preventable by a potent synthetic anti-GC,RU 38486, a steroid with high affinity for GC receptors, added to culture medium along with GC. These results provide evidence that both the in vivo effect of MC on Na+ permeability and the induction of hypertension, and the in vitro effect of GC on water transport in cultured vascular smooth muscle cells are elicited through the receptor-mediated molecular mechanism(s) for action of these steroids in the arterial wall.

The role of arterial mineralocorticoid receptors in the mechanism of hypertension: findings and hypothesis.

Alterations in electrolyte transport across cell membrane of vascular smooth muscle (VSM) and changes in hemodynamics [increased extracellular fluid volume (ECFV) and cardiac output (C.O.)] have been implicated in the pathogenetic mechanisms of both mineralocorticoid-induced hypertension (MH) and essential hypertension (EH). We have previously found that mineralocorticoids (MC) can act directly on arterial wall by means of a receptor-mediated mechanism, and have postulated that this mechanism is of critical importance in the increased reactivity of VSM to vasoconstrictive stimuli in MH. We now present evidence that a MC-antagonist at the MC-receptor level, progesterone, prevents induction of changes in VSM cell-membrane permeability to Na+ by MC, and development of hypertension. This study has been carried out on rabbits made hypertensive by s.c. implantation of silastic rubber strips impregnated with 11-desoxycorticosterone (the inducer) and/or 50 times that amount of progesterone (the anti-inducer). We hypothesize that the VSM cell-membrane defect (MC-induced in MH and congenital in EH) initiates two separate sequences of biochemical events. One leads, in early stages of hypertension, to expansion of ECFV, increase in C.O., myogenic vasoconstriction and hypertension. When kidney function matures, hypertension recedes. The second sequence of events leads to hypertension via an increase in [Na]i of VSM, leading to an increase in [Ca]i, and an increased contractility of VSM. This hypertension persists. The two sequences are concomitant but independent of each other.

Studies on arterial mineralocorticoid and glucocorticoid receptors: evidence for the translocation of steroid-cytoplasmic receptor complexes to cell nuclei.

The high affinity binders for mineralocorticoids and glucocorticoids, previously reported by us as present in arterial cytosol have been further characterized. The results of this study demonstrate that these binders translocate under appropriate conditions to cell nuclei as complexes with mineralocorticoids and glucocorticoids, respectively. Thus, they exhibit a fundamental property of steroid receptors. This provides evidence for the presence in the arterial wall of a molecular mechanism(s) for the in-situ action of both mineralocorticoids and glucocorticoids.

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

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 provides 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 chronically elevated levels of 11-desoxycorticosterone (DOC) result in a marked increase in permeability of arterial smooth muscle cell membrane to sodium ions; this is in accord with findings of other investigators in the rat. This change presumably leads, through a chain of biochemical events, to increased arterial and arteriolar smooth muscle contractility, increased peripheral resistance and hypertension. Study is in progress to determine whether the effect of DOC on arterial smooth muscle cell-membrane permeability to electrolytes is elicited through the receptor-mediated mechanism for the in situ action of mineralocorticoids in the arterial wall. It is postulated that this mechanism is primarily responsible for "mineralocorticoid hypertension", but may well be instrumental also in pathogeneses of various other forms of hypertension, including "essential".

Studies on high affinity binding of mineralo- and glucocorticoids in rabbit aorta cytosol.

High affinity, specific binding-sites to mineralocorticoids and glucocorticoids, with characteristics of steroid receptors, have been found in rabbit aorta cytosol. Binding parameters (dissociation constants and number of binding sites per mg of cytosol protein) were determined from Scatchard plots, after statistical treatment of the data with the aid of a computer program, for the following tritiated steroids: 11-desoxycorticosterone (DOC), aldosterone (Aldo), progesterone (Prog), corticosterone (BK), cortisol (FK) and dexamethasone (Dex). The specificity of binding was then examined by means of steroid competition studies. The results of these experiments indicate that three different types of high-affinity binding sites to adrenal steroids are present in aorta cytosol: Type A, with the highest affinity for DOC; Type B, with the highest affinity for FK; Type C, with the highest affinity for Dex. In accordance with the relative competitive potencies of various steroids for these binding sites, Type A is designated as the "arterial mineralocorticoid binder", clearly differing in its binding characteristics from the cytoplasmic mineralocorticoid binders in known target tissues to these steroids (e.g. the renal receptor), while Type C is designated as the "arterial glucocorticoid binder", closely resembling the classical glucocorticoid receptor in known target tissues to glucocorticoids. Type B exhibited some of the binding characteristics of transcortin and may represent a modified, intracellular transcortin. While Types B and C are present also in the cytosol of inferior vena cava. Type A was only in the aorta. The role of these binders is not known at present. Arguments are presented in favor of a hypothesis that the Type A (mineralocorticoid) binder represents an arterial wall; and that, under certain conditions, this action leads to an increased contractility of arterial and arteriolar smooth muscles, increased peripheral resistance and hypertension.

Evidence for the presence in arterial walls of intracellular-molecular mechanism for action of mineralocorticoids.

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 physicochemical 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 provides evidence for the existence of an intracellular-molecular mechanism for a direct in situ action of mineralocorticoids, and also glucocorticoids, in the arterial wall. We postulate that the demonstrated previously effect of mineralocorticoids on arterial smooth muscle cell-membrane permeability to electrolytes, leading presumably to increased peripheral resistance and hypertension, is elicited through the receptor-mediated mechanism for action of mineralocorticoids in the arterial wall.

Synthesis and study of the estrogenic activity of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether.

Synthesis of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether (BBE2M) was accomplished by reducing a methanolic solution of 2,4-bis(bromomethyl)estrone methyl ether with sodium borohydride. In 0.5 M phosphate buffer, pH 7.0, 25 degrees, BBE2M readily reacts with Ellman's anion and alkylates cysteine to form a steroid-amino acid conjugate. Stoichiometry of the reaction indicates that the bromosteroid is divalent with cysteine. Tryptophan and histidine react more slowly with the bromosteroid. Estrogenic activity of BBE2M was evaluated in ovariectomized rats by uterine intraluminal administration and quantitation of glucose-6-phosphate dehydrogenase (D-glucose-6-P:NADP+ oxidoreductase, EC 1.1.1.49) activity in the uterus. BBE2M induced glucose-6-phosphate dehydrogenase activity as did estradiol-17 beta or estradiol-17 beta 3-methyl ether (E2M). BBE2M was more persistent in activity than E2M. Histological examination of uterus following BBE2M treatment shows classic estrogenic morphology. BBE2M covalently binds to the cytoplasmic estrogen receptor of calf uterus. Such binding is prevented by pretreatment of the receptor protein with estradiol-17 beta. The covalently bound steroid-receptor complex appears to stimulate RNA synthesis in isolated nuclei from calf endometrium.