Ludwig Kornel

Mechanism of the effects of glucocorticoids and mineralocorticoids on vascular smooth muscle contractility

We have previously demonstrated that receptors to both mineralocorticoids (MC) and glucocorticoids (GC) exist in the arterial wall and that treatment with GC markedly increases Na+ and Ca2+ influx in cultured aortic vascular smooth muscle (VSM) cells, whereas treatment with MC increases only Na+ influx. We now report the results of the study aimed at the elucidation of the mechanism(s) of these effects. Unidirectional influx of Na+ and Ca2+ was measured in cultured cells of rabbit aortic media, using 22Na and 45Ca as tracers, in the presence of ouabain. The cells were treated for different periods with dexamethasone (DEX) or aldosterone (ALDO) in physiologic or supraphysiologic concentrations, in the presence or absence of competitive inhibitors of GC-receptor binding, RU 486, or MC-receptor binding, K-prorenoate. DEX in 50 nM concentration increased Na+ influx by 98 +/- 18% and Ca2+ influx by 100 +/- 20%, and the maximum effect was seen after 48 hour cell-treatment. ALDO in 5 nM concentration increased Na+ influx by 90 +/- 12% and had no effect on Ca2+ influx, and the maximum effect was seen after 7-10 days of cell-treatment. The enhancing effect of both DEX and ALDO on the influx rate of Na+ was prevented by actinomycin D and by cycloheximide. RU 486 completely inhibited DEX from exercising its enhancing effect on Na+ influx, but diminished influx rate of Na+ increased by ALDO only by 25%. Prorenoate (PRN) did not have any effect on DEX-increased Na+ influx, but completely inhibited ALDO from exercising its effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of vascular steroid receptors in the control of vascular contractility and peripheral vascular resistance

Evidence is presented that glucocorticoids (GC) and mineralocorticoids (MC) control contractility of vascular smooth muscle (VSM). This control is effected through the in situ mechanism for the action of these steroids in the arterial and arteriolar wall. This action is mediated through GC and MC receptors in the VSM cell. Acting through these receptors, MC and GC increase transport capacities of different transmembrane transport systems for Na+ and/or Ca2+ through induction of synthesis of proteins constituting the transport systems. Colocalization of enzymes deactivating cortisol in VSM, with VSM receptors for GC and MC further strengthens the concept that the arterial network houses an in situ molecular mechanism for the control of VSM contractility, thus peripheral vascular resistance.

Aldosterone (ALDO) increases transmembrane influx of Na+ in vascular smooth muscle (VSM) cells through increased synthesis of Na+ channels

We have previously reported our studies on glucocorticoid (GC) effects on Na+ influx in vascular smooth muscle (VSM) cells. We now report a parallel study on the effect of mineralocorticoid (MC) on Na+ influx in VSM cells. Unidirectional influx of Na+ was measured in cultured cells of rabbit aortic media with 22Na as tracer. Cells were treated with near physiologic (5 nM) or supraphysiologic (50 nM) aldosterone (ALDO) for 24 or 48 hours, or for 7 to 10 days, in the presence of competitive inhibitors of MC-receptor binding, K-prorenoate (PRN), or GC-receptor binding, RU 486. ALDO at 5 nM increased Na+ influx by 98% +/- 12%, but only after 7-10 days of treatment. This effect was inhibited by PRN, but not by RU 486, and blocked by amiloride but not by ethylisopropyl-amiloride or by dichlorobenzamil (DCB). In VSM cell membranes from aortae of rabbits treated in vivo with ALDO (2 mg/day) for 4 weeks. Na+ channels were quantified by determination of specific [3H]amiloride binding in the presence of excess of DCB and EIPA to exclude tracer binding from the Na+/Ca2+ exchanger and the Na+/H+ antiporter. ALDO doubled the number of of Na+ channels in such isolated cell membranes, as determined by Bmax per mg membrane protein. We propose that this vascular effect of ALDO may constitute an important pathogenetic mechanism of hypertension induced by chronic excess of MC, in addition to the well known renal mechanism.

Studies on the mechanism of mineralocorticoid-induced hypertension: evidence for the presence of an In-Situ mechanism in the arterial wall for a direct 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 of its presence. We have found high affinity, specific binders for mineralocorticoids, and glucocorticoids. with characteristics of steroid receptors, in rabbit and lamb arterial homogenate cytosol. Moreover, these binders were found to possess also other fundamental properties of cytoplasmic steroid receptors: they translocate to cell nuclei (as steroid-receptor complexes) and bind to relatively specific “acceptor-sites” on nuclear chromatin. The mineralocorticoid with the highest affinity for the arterial mineralocorticoid receptor is 11-desoxycorticosterone (DOC); however, the possibility of a parallel existence of a specific aldosterone receptor is not ruled out. Cytoplasmic mineralocorticoid receptors are not present in veins. The steroid with the highest affinity for the arterial glucocorticoid receptor is dexamethasone. similarly as in other target tissues to glucocorticoids. A highly specific transcortinlike cortisol binder was also found in aorta cytosol. In parallel with this binder a true cortisol-receptor (translocating to cell nuclei and binding to nuclear chromatin) was also found. This points to heterogeneity of cytoplasmic glucocorticoid receptors. Furthermore, we have studied the effect of chronic administration of DOC acetate (DOCA) on intracellular sodium concentration [Na i .] and on the rate of passive Na + -influx into the cells of aorta. Both were found to be significantly higher in DOCA-induced hypertensive rabbits than in normotensive control animals. This indicates that cell-membrane permeability to electrolytes is increased as a result of chronic elevation of circulating levels of DOC, and is in accord with the findings of other investigators in other animal species. We postulate that this effect of DOC is elicited through the receptor-mediated mechanism for action of mineralocorticoids in the arterial wall; and that increased [Na i ] in arterial and arteriolar smooth muscles leads to their increased contractility, hyperresponsiveness to vasoconstrictive stimuli, hypertrophy, increased peripheral resistance, and hypertension.

Studies on steroid conjugates—VIII: Isolation and characterization of glucuronide-conjugated metabolites of cortisol in human urine

Abstract A total spectrum of glucuronide-conjugated metabolites of cortisol has been isolated from human urine and characterized: 22 steroid monoglucuronides and 7 steroid diglucuronides. Following i.v. administration of a tracer dose of [4-14C]-cortisol to 10 normal subjects, urine was collected for 2 consecutive 24-h periods. Free steroids were removed with ethyl acetate. All conjugated metabolites were extracted by means of an Amberlite XAD-2 column, and were further purified by chromatography on a polyethyleneimine-impregnated cellulose column. Groups of mono- and di-glucuronide conjugated steroids were then separated from each other, and from other groups of steroid conjugates by means of high voltage paper electrophoresis. Individual monoglucuronide-conjugated metabolites were then separated from each other by means of 15 consecutive paper chromatographies, and their homogeneity was ascertained. Each steroid conjugate was then subjected to the following characterization: (1) identification by RID of the steroid moiety released by β-glucuronidase hydrolysis; (2) identification of the glucuronide moiety; (3) determination of the steroid/glucuronide molar ratio; (4) determination of the site of conjugation. The following monoglucuronide-conjugated (-G) metabolites of cortisol were isolated: cortisol-21-G, cortisone-21-G, 20β-dihydrocortisol-21-G, tetrahydrocortisol-3-G, tetrahydrocortisol-21-G, 5α-tetrahydrocortisol-3-G,tetrahydrocortisone-3-G,5α-tetrahydrocortisone-3-G,cortol-20α-3-G, 5α-cortol-20α-3-G, cortol-20β-3-G, 5α-cortol-20β-3-G, cortolone-20α-3-G, 5α-cortolone-20α-3-G, cortolone-20β-3-G, 5α-cortolone-20β-3-G, ll-hydroxyaetiocholanolone-3-G,11-hydroxyandrosterone-3-G, 11-oxoaetiocholanolone-3-G, 11-oxoandrosterone-3-G. In addition, 6β-hydroxy-20β-dihydrocortisol and 6β-hydroxy-20β-cortol were also isolated as monoglucuronides, but their site of conjugation was not determined. The steroids identified as diglucuronide conjugates were: cortol-20α and −20β, cortolone-20α and −20β, tetrahydrocortisol, 5α-tetrahydrocortisol and tetrahydrocortisone. Most of these steroid metabolites have not been heretofore isolated and characterized as conjugates. Three of the isolated metabolites were not known to exist as glucuronide conjugates. The quantitation of individual steroid conjugates revealed interesting relationships between various metabolites, which are discussed.

Isolation of cytoplasmic glucocorticoid-binding protein(s) from rat liver by means of affinity chromatography, and its partial characterization.

Abstract Intracellular corticosteroid-binding protein(s) was isolated from rat liver cytosol by means of affinity chromatography, using amino-sepharose coupled to cortisol hemisuccinate. This protein material displayed apparent homogeneity on Sephadex G-100 and G-200 gel filtrations. Its molecular weight is 93 000 (average of two estimations), as calculated from sedimentation and diffusion constants. The amino acid composition of this protein closely resembles that of transcortin, although some quantitative differences between the two exist. The carbohydrate content of this protein was found to be appreciably smaller than that of transcortin. Calculation of the amount of possible contamination of the isolated protein(s) with residual blood present within the vascular bed of the liver tissue prior to homogenization indicates that at most one-fourth of the cytoplasmic corticosterone-binding protein(s) could be due to contamination with transcortin from plasma. On starch gel and polyacrylamide gel electrophoresis, the isolated protein material resolved into two major and one minor components, migrating in the α 1 - and α 2 - globulin region . Competitive binding studies with different steroids revealed that the isolated protein(s) has high affinity for progesterone, corticosterone, and cortisol. For all the three steroids, at least two binding components were present on the Scatchard plot: one possessing high affinity but low capacity ( K A = 10 −8 M, n = 15 · 10 −9 M ), the other, lower affinity but higher capacity ( K A = 10 −6 M , n = 160 · 10 −9 M .

Corticosteroids in human blood--VII. Isolation, characterization and quantitation of glucuronide-conjugated metabolites of cortisol in human plasma.

A total spectrum of glucuronide-conjugated metabolites of cortisol has been isolated from human plasma, and their structure and quantitative relationship have been determined: 22 steroid monoglucuronides (MG), 7 steroid-diglucuronides (DG), and 6 steroid-sulfoglucuronides (GS). Large heparinized blood specimens (450–550 ml) were obtained from 15 normal subjects 214–212 h after i.v. administration of a tracer dose of [4-14C]-cortisol. Plasma was deproteinized and defatted and the “free” steroids were extracted with ethyl acetate. All the conjugated steroid metabolites were then extracted by means of an Amberlite XAD-2 column and purified on a polyethyleneimine-impregnated cellulose column. The three groups of conjugates (MG, DG, GS) were then separated from each other, and from sulfate conjugates by means of high voltage paper electrophoresis. Individual MG conjugated steroid metabolites were then separated as conjugates by means of 15 consecutive paper chromatographies. Each steroid conjugate was subjected to the following characterization studies: (1) identification by reverse isotope dilution (RID) of the steroid moiety released by β-glucuronidase hydrolysis; (2) identification of the glucuronide moiety; (3) determination of the steroid-glucuronide molar ratio; (4) determination of the site of conjugation. Steroid DG and GS were also characterized by similar studies. The following steroids have been isolated and identified as MG: cortisol, cortisone, 20β-dihydrocortisol, 6β-hydroxy-20β-dihydrocortisol, tetrahydrocortisol, 5α-tetrahydrocortisol, tetrahydrocortisone, 5α-tetrahydrocortisone, cortol-20α, 5α-cortol-20α, cortol-20β, 5α-cortol-20β 60β-hydroxy-cortol-20β cortolone-20α, 5β-cortolone-20α, cortolone-20β, 5α-cortolone-20β, 11-hydroxyetiocholanolone, 11-hydroxyandrosterone, 11-oxoetiocholanolone, 11-oxoandrosterone. The following steroids have been isolated and identified as DG: cortol-20α, and -20β, cortolone-20α, and -20β tetrahydrocortisol, 5α-tetrahydrocortisol and tetrahydrocortisone. The following steroids have been isolated as GS: cortols, cortolones (individual isomers not separated), tetrahydrocortisol and tetrahydrocortisone. All ring-A reduced steroid MG were found to be conjugated at C-3, with the exception of tetrahydrocortisol which was also found as a C-21 conjugate. All 4-en-3-oxosteroids were conjugated at C-21. Steroid DG were conjugated at C-3 and C-21. Steroid GS were found to have glucuronide moiety at C-3 and sulfate moiety at C-21. All individual MG, DG and GS conjugates identified were identical with those previously isolated by us from urine. Most of these conjugated metabolites of cortisol have not been heretofore isolated from plasma as conjugates. Of all plasma metabolites of cortisol, total “free” metabolites constituted 47.5 ± 8.6%, and total conjugated metabolites 52.5 ± 9.8%. Of the latter, MG constituted 86.0 ± 2.7%, DG 1.8 ± 1.2%, and GS 1.3 ± 1.1%. The relative plasma concentrations of individual MG, DG and GS conjugates were found to be very similar to those of the urinary steroids. Only concentrations of C-21 MG of 4-en-3-oxosteroids were 2–3 times higher than those in urine, presumably due to the binding of these conjugates to transcortin. The quantitative relationships between concentrations of various individual conjugates suggest certain substrate predilections of steroid glucuronyl-transferases and sulfotransferases, which are discussed.

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.