Isidore S. Edelman

Subcellular mechanisms in the action of adrenal steroids

Abstract The available evidence indicates that induction of de novo synthesis of proteins is the common although not necessarily the sole pathway in the action of adrenocortical steroids in a variety of tissues and physiologic processes. Plausible but as yet unproved models have been proposed to account for the major actions of these steroids (e.g., Na + retention, gluconeogenesis, cytolysis). The details of the molecular mechanics of steroidreceptor interaction, gene activation and of the physiologic roles assigned to the induced proteins under active study and the subject of lively debate. The most promising area of study presently appears to be the nature and identity of the receptor proteins. Current understanding of receptor mechanisms, albeit incomplete, provides an explanation of steroid specificity and overlapping physiologic effects—such as the glucocorticoid effects of aldosterone and mineralocorticoid effects of cortisol or corticosterone. Similarly, this understanding enables us to design new and more potent steroid antagonists and to rationalize resistance or sensitivity of tumor cells to specific steroids. Ultimately, perhaps, studies on the interactions of the steroid receptor complex within the nucleus may serve as a means of clarifying some of the mechanisms of gene regulation in animal cells.

Mechanism of action of steroid hormones.

Abstract Despite the diversity in the target sites and physiological actions of the steroids, an impressive body of evidence has been assembled in support of a unitary theory of the mechanism of action of these hormones in vertebrates. The steroids set in motion a train of events, as follows: (a) penetration into the target cell, (b) stereospecific binding to high affinity receptors, (c) temperature-sensitive activation of the steroid-receptor complex, (d) attachment of the active complex to chromatin, (e) induction of RNA and protein synthesis, and (f) physiological expression of the induced protein. Although the overall sequence is well-defined, our knowledge of the molecular processes involved in each of these steps is still quite incomplete. Two of the major efforts now underway to elucidate these molecular processes involve, (a) purification and characterization, in terms of structure-function relations, of the putative receptors, and (b) studies on the nature of the interaction between steroid-receptor complexes and the genome. It is now apparent that steroids induce de novo synthesis of both messenger RNA (mRNA) and ribosomal RNA (rRNA); the role of the former in directing the synthesis of specific proteins is reasonably clear but that of the latter remains to be elucidated. The mechanism of induction is also under scrutiny since the observed increases in mRNA synthesis could arise in a variety of ways, e.g. negative or positive regulation of chromatin template (gene) activity, changes in processing of heterogeneous RNA to mRNA, effects on RNA polymerase or ribonuclease activities. Although steroidal regulation of RNA and protein synthesis is a dominant pathway, the possibility of direct actions on membranes or regulatory enzymes in some circumstances can not be excluded at the present time.

Glucocorticoid and mineralocorticoid receptors for aldosterone

Abstract Specific binding of aldosterone and dexamethasone by rat kidney and hepatoma tissue culture (HTC) cell cytosol has been studied. In cytosol of HTC cells, aldosterone and dexamethasone bind to a single class of sites with affinities that correspond with their potencies as inducers of tyrosine aminotransferase. Kidney cytosol, however, contains two classes of specific aldosterone binding sites. The higher affinity sites bind aldosterone with an affinity (equilibrium, dissociation, constant) which is similar to the plasma concentration of aldosterone required for antinatriuresis. The lower affinity aldosterone-binding sites are present at a higher concentration than the higher affinity sites and also bind dexamethasone with a high affinity. We have tentatively identified these two classes of binding sites in renal cytosol as “mineraloeonicoid” and “glucocorticoid” receptors, respectively.

The Action of Aldosterone and Related Corticosteroids on Sodium Transport across the Toad Bladder

The importance of aldosterone and related adrenal steroids in the regulation of renal conservation of Na+ and excretion of K+ has been established by extensive studies in man and other mammals (1). The complexity of the mammalian kidney, however, makes it difficult to study the chemical pathways involved in the action of mineralocorticoids on cation transport. The prospects for achieving a solution of this problem should be improved considerably by the development of an in vitro preparation with a relatively simple histological structure that responds reproducibly and predictably to aldosterone. Previous studies on the action of mineralocorticoids on active Na+ transport by anuran skin in vivo and in vitro made it likely that such a preparation could be developed (24). Significant progress in the development of an in vitro system suitable for an analysis of the mechanism of action of aldosterone was made by Crabbe (7-9), who showed that the rate of endogenous secretion of d-aldosterone in Bufo marinus varies with the salinity of the environment and that aldosterone stimulates active transport of Na+ across the isolated urinary bladder and ventral skin of anurans. Our objectives were 1) to improve the sensitivity and reproducibility of the response of the toad bladder system to aldosterone, 2) to obtain information on the dependance of the action of

Calcium Inhibition of the Action of Vasopressin on the Urinary Bladder of the Toad

Earlier studies showed that exposure of the isolated urinary bladder of the toad to vasopressin produces striking increases in passive permeability to water and urea as well as increases in active transport of Na-(1-4). Bentley (5) observed that raising the concentration of Ca++ in the sero-sal bathing medium depressed the response to a 1 mU per ml concentration of vasopressin, as measured by the flow of water along a fixed os-motic gradient. In a later study, he reported that Ca++ failed to antagonize the action of vasopres-sin on active sodium transport (6). The nature of the antagonism was not explored further. A detailed study of Ca++ inhibition of the action of vasopressin in vitro should provide additional evidence bearing on the single site theory advanced by Koefoed-Johnson, Ussing, and Ander-sen on the basis of studies on anuran skin (7, 8). According to their hypothesis, transepithelial movement of water and small solutes is limited by a diffusion barrier and a porous barrier arranged in series. The neurohypophyseal hormones increase the pore diameter of the porous boundary, thus producing marked decreases in the resistances to the bulk flow of water and to Na+ transfer, as well as significant increases in the permeability coefficients of urea and related small nonelectrolytes. Leaf and his collaborators (3, 4, 9-11), in an extensive series of studies, also found that the pore theory can be applied to the action of vasopressin on the movement of * water and urea across the toad bladder. Bentleys findings, however, suggest that the effect of vaso-pressin on Na+ transport is not a simple consequence of increased porosity at the surface of the epithelial cells. Three possibilities are raised by these observations: a) Vasopressin may contain two active sites, one of which regulates water transport (subject to Ca++ inhibition) and the other Na+ transport (not subject to Ca++ inhibition); b) Na+ may enter the cell via a nonaqueous pathway that is also altered by vasopressin, while not subject to Ca++ antagonism; or c) vasopressin may have a separate action on the Na+ pump, and Ca++ has no effect on the pump site. Because of these possibilities, we elected to explore the modifying effects of Ca++ on the action of vasopressin in further detail and to examine these effects on all three parameters in the domain of vasopressin action, namely, resistance to osmotic flow of water , the permeability coefficient of urea, and …

Specific aldosterone binding in rat kidney and parotid

The specific intracellular binding of [3H]-aldosterone was studied in tissue slices of kidney and parotid from adrenalectomized rats. Specific aldosterone binding proteins were isolated from (1) cytosol by G-50 Sephadex chromatography, (2) nuclei by an initial osmotic shock procedure (2.2 M sucrose followed by extraction with 0.1 M tris-3 mM CaCl2 and 50% (NH4)2SO4 precipitation = “soluble nuclear”) and (3) nuclei by a subsequent 04 M KCl-3 mM CaCl2 extraction and 50% (NH4)2SO4 precipitation (“chromatin bound”). In both tissues, the time course of uptake into the three intracellular compartments was studied by incubation at 25°C for 0–4 h with 5.2 × 10−9 m [3H]-aldosterone. Both kidney and parotid show the same three-step time sequence of specific intracellular binding—first cytosol, then soluble nuclear, and then chromatin bound. The time course and extent of [3H]-aldosterone binding in kidney slices was unaffected by concentrations of cycloheximide sufficient to lower protein synthesis by 67%. Cytosol binding proteins in kidney and parotid have an identical affinity for aldosterone but their concentration per g wet weight tissue in the kidney is twice that in the parotid. Despite this difference in cytosol donor concentration, and the presumed identity of active sites, levels of intranuclear [3H]-aldosterone-protein complexes are considerably higher in parotid than in kidney (soluble nuclear × 2, chromatin bound × 15).

Effects of Ca++ and Prostaglandin E1 on Vasopressin Activation of Renal Adenyl Cyclase

Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl(2) inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca(++) inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl(2) reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E(1) (PGE(1) = 10(-7)m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10(-4)m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10(-7)m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE(1) modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed.

Molecular modifications of anti-aldosterone compounds: Effects on affinity of spirolactones for renal aldosterone receptors☆

Abstract The currently accepted hypothesis of the anti-mineralocorticoid action of spirolactones is that of competition for specific aldosterone receptors in target tissues. Binding of aldosterone to cytoplasmic receptors was studied by incubating adrenalectomized rat kidney slices with 3 H-aldosterone in the presence of 10-fold non-radioactive dexamethasone to prevent binding of tracer to glucocorticoid receptors. From the ability of a series of 24 spirolactone analogues to compete for 3 H-aldosterone binding sites under these conditions, the relative affinities for this receptor have been determined. Affinity for the receptor is decreased by B ring unsaturation at the C-6/C-7 position, by γ-lactone unsaturation, or by γ-lactone ring opening with the formation of the water-soluble K + salt. Affinity is markedly increased by esterification, or thioesterification, at the C-7 position in the B ring. Various 19-nor spirolactones show greater, equivalent or lesser affinity vis-a-vis their parent compounds. These structure-affinity relationships should define one of the determinants of pharmacologic activity.

Thyroid Thermogenesis and Active Sodium Transport

Publisher Summary This chapter presents an evidence in support of the hypothesis that thyroid hormone stimulates energy expended in active Na + transport in three of the primary thermogenic target tissues; liver, kidney, and skeletal muscle. In these tissues, this effect mediates a significant fraction and in some circumstances virtually all the thermogenic effect. This pathway does not appear to be the only mechanism involved in thermogenesis, however, in that from, one-half to two-thirds of the increase in Q O2 may be ouabain, or Na + , insensitive. It has been found that Ouabain has no effect on Q O2 of white fat from euthyroid controls or T 3 -treated rats. The chapter also explains that in this tissue, T 3 -stimulated respiration is secondary to changes in energy turnover in electron transport. The basic pathways involved in Na + transport-independent thermogenesis, however, remain undefined.

Induction of citrate synthase by aldosterone in the rat kidney.

SummaryThe possible induction of renal citrate synthase (E.C. 4.1.3.7), by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 μg/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968.Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on theKms for acetyl-CoA and oxalacetate but augmentedVmax of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation.The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 μg/100 g body wt) or the diluent, and simultaneously with3H or35S methionine (500 μCi/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 μg/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either3H-or35S-methionine at 20° for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70–80 μg/100 g body wt) or spirolactone (SC-26304) (80 μg/100 g body wt). An equimolar dose of dexamethasone (0.8 μg/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.