Monographs on endocrinology最新文献

The regulation of tyrosine aminotransferase activity by glucocorticoids and cyclic AMP was investigated in isolated liver parenchymal cell suspensions. The induction and maintenance of elevated levels of tyrosine aminotransferase activity in liver cells were completely dependent upon the presence of both the synthetic glucocorticoid, dexamethasone, and glucagon of dibutyryl cyclic AMP. No induction was observed when any of these compounds were tested alone. Immunotitration experiments revealed that the 6- to 7-fold increase in tyrosine aminotransferase activity following the addition of dexamethasone and glucagon was accompanied by a parallel increase in the amount of immunologically reactive enzyme protein. Pulse-labeling experiments established that this increase in enzyme protein could be fully accounted for by a corresponding increase in rate of synthesis of tyrosine aminotransferase. Neither hormone had any effect on the rate of degradation of the enzyme. The increase in tyrosine aminotransferase synthesis evoked by the presence of both hormones was blocked by the addition of actinomycin D or cycloheximide to the medium, demonstrating that RNA and protein synthesis were required for the induction of enzyme activity. By varying the time and order of addition of the inducers and inhibitions, evidence was obtained that the hormones act sequentially. The steroid hormone acts first, presumably to increase the level of functional tyrosine aminotransferase mRNA or its precursor. The processing of this precursor to a translatable form or the specific translation of tyrosine aminotransferase mRNA is apparently dependent upon a specific cyclic AMP-controlled process. In vivo experiments demonstrated that both glucocorticoids and cyclic AMP increase the level of functional tyrosine aminotransferase mRNA in the liver. The actions of the steroid hormone and cyclic nucleotide were blocked by alpha amanitin, establishing the requirement for ongoing gene transcription. The protein synthesis inhibitors, cycloheximide, emetine, and puromycin, were as effective as cyclic AMP in increasing tyrosine aminotransferase mRNA activity. The action of these inhibitors is probably related to their ability to elevate hepatic intracellular cyclic AMP levels, thus mimicking cyclic AMP administration. Extension of these in vivo studies to isolated liver cells will provide a valuable system for investigating the regulation of gene expression by glucocorticoids and cyclic AMP.

Steroids in general and glucocorticoids in particular affect lysosomes in various ways. The explanation of these effects remains in dispute, however. Theories include the view that steroids interact directly with lysosomal membranes, that steroids provoke induced changes in lysosomes, and that classical steroid receptors originate in lysosomes. Experiments pertaining to these views are discussed, particularly with respect to steroid specificity and tissue specificity of effects and to dose-response considerations.

Relying heavily on studies of TAT regulation in cultured rat hepatoma cell lines, we have attempted in this brief review to discuss possible mechanisms for posttranscriptional regulation of glucocorticoid-sensitive enzymes and to chronicle the evidence for and against posttranscriptional mechanisms for specific enzyme induction by glucocorticoids. Initially, mechanisms were considered that would reconcile results showing sensitivity of both induction and deinduction of TAT to inhibitors of RNA synthesis with studies demonstrating first that glucocorticoids regulate the rates of specific enzyme synthesis and, then, that glucocorticoids regulate levels of enzyme-specific mRNA. Such reconciliation proved unnecessary when it was demonstrated that inhibitors of RNA synthesis such as actinomycin D were not specific for RNA synthesis, but also had effects on mRNA turnover and protein metabolism. The bulk of evidence to date establishes that glucocorticoids promote the production of enzyme-specific mRNA for the proteins whose synthesis is regulated by thses steroids. Nevertheless, there is still very little direct evidence that steroids can modulate rates of specific gene transcription. The glucocorticoid stimulation of mouse mammary tumor virus RNA production in cultured cell lines is the only example to date where such a mechanism is supported by RNA-DNA hybridization studies. Posttranscriptional actions of steroids on the turnover, processing, or extranuclear transport of specific mRNA precursors remain potential steps at which glucocorticoids might function. The rapid turnover of some glucocorticoid-regulated enzymes and their mRNAs not only ensures a rapid response to steroid addition or withdrawal, but also subjects these proteins to relatively large fluctuations upon alterations in overall protein or mRNA metabolism. Thus many of the inductions and repressions of hepatic TAT and TO by mediators other than the glucocorticoids may be attributable entirely to nonspecific mechanisms.

For over a decade, tyrosine aminotransferase induction in tissue culture cells has been a useful model system in which to study glucocorticosteroid action. In the 1960s, the establishment in culture of rat hepatomas expressing the inducible enzyme, already known to be induced in liver in vivo, provoked a wide-ranging series of experiments. The data from these experiments have provided considerable information regarding the mechanism of action of steroids. These include the fundamental facts that the steroids act directly on the induced cell in unmetablized form, that removal of steroid results in deinduction, that induction does not require DNA synthesis or massive changes in RNA synthesis, and that cytoplasmic receptor occupancy by active steroids correlates closely with the steroids' ability to affect inductions. Studies in tissue culture cells have led to the analysis of transcriptional and posttranscriptional models attempting to explain enzyme induction. The effects on enzyme induction of nonsteroid hormones and other factors have been studied through the use of tissue culture cells. Finally, cells and clones of cell variants are being used to study enzyme induction, through biochemical analysis and cell genetic approaches, including somatic cell hybridization.

Meaningful answers to the question of the relationship between glucocorticoid structure and activity have emerged. Structural change has predictable effects on susceptibility to the action of metabolizing enzymes, on receptor affinity, and on intrinsic activity. These effects are, in principle, amenable to mathematical modeling techniques. The fascinating possibility of being able to calculate receptor affinity directly from chemical structure has already been realized through the development of an equation [19] that allows the calculation of receptor binding of any glucocorticoid from structural parameters. Utilizing knowledge of the free energy contributions of the substituents and the hydrophobicity and A-ring conformation of the steroids, receptor affinity for a large number of compounds could be described in terms of four parameters. A general relationship was derived relating the equilibrium dissociation constant to a surface area term, a polar interaction term, and A-ring tilt term, and a size limitation function for the 9 alpha-substituent. The excellent correlation obtained suggests that these four factors are the major determinants of glucocorticoid receptor interactions. It is clear that the use of a mathematical relationship that defines the strength of steroid-receptor interaction is a valuable tool for investigating structure-activity relationships. This would be especially true in the design of steroid drugs. The use of a linear free-energy equation is superior to the assumption of substituent additivity in predicting binding affinities. This type of relationship will be useful in the preparation of steroids for use in affinity labeling studies and should be adaptable to other binding systems in which it is desirable to obtain synthetic analogs for more potent activity or specificity.

In this article, we have provided two examples of pleiotropic regulation by specific effector molecules as assayed by two-dimensional gel electrophoresis. In one case, catabolite repression in the bacterium Escherichia coli was examined by measuring the response to cyclic cAMP. In the other, the effect of dexamethasone on the rate of synthesis of over a thousand cell proteins was analyzed in HTC cells. It was found that in E. coli, cAMP regulates the synthesis of about 10 percent of the cell's proteins; both inductions and repressions are observed, but inductions clearly predominate. In HTC cells, dexamethasone induces the synthesis of seven proteins, or about 0.7 percent of the total cellular proteins; repression was not consistently observed. In another rat hepatoma line (FAZA) a similar number but essentially different set of proteins was induced. These data are discussed in terms of the notion of domains of response originally proposed by TOMKINS [1].

1. Regulation of gluconeogenic substrate supply and modulation of the gluconeogenic pathway in the liver are both important in the control of gluconeogenesis by glucocorticoids. 2. Adrenal deficiency decreases the release of gluconeogenic and other amino acids from skeletal muscle during starvation. The effect is reversed by glucocorticoid replacement. The changes in amino acid release are accompanied by similar alterations in tissue amino acid levels and are not explained by alterations in net protein breakdown. Glucocorticoids do not alter protein catabolism and cause a small inhibition of protein synthesis. The biochemical alterations underlying the changes in amino acid metabolism induced by these steroids remain to be elucidated. Glucocorticoids may also regulate the supply of gluconeogenic substrates through permissive effects on the lipolytic action of catecholamines and other hormones in adipose tissue and on the glycogenolytic action of catecholamines on skeletal muscle. 3. Glucocorticoids are required for the increases in gluconeogenesis in starvation and diabetes. Part of their action is exerted directly on the liver and appears to involve modulation of P-enlopyruvate carboxykinase levels. Glucocorticoids increase the synthesis of this enzyme apparently through effects at the level of transcription. 4. Glucocorticoids exert permissive effects on the stimulation of gluconeogenesis in the liver by glucagon and epinephrine. The steroids are not required for cAMP generation or protein kinase activation by these hormones, but appear to act by maintaining the responsiveness of certain enzymes to the effects of the cAMP and alpha-adrenergic systems. It is proposed that this involves the maintenance of a normal intracellular ionic environment.

Attempts to reconstruct, in a test tube, the steroid-hormone system of a responsive cell are fraught with enumerable difficulties. In this chapter I have attempted to point out some of the factors that affect receptor-steroid complexes and their interactions with acceptors. In most cases there is a quantitative influence of these factors on the level of steroid complex binding to acceptors. In some cases, selected experimental designs that neglect these factors and methods of presenting the observed data may lead to artifactual conclusions. Several of these problems should disappear when the prospect of pure receptor-steroid complexes [127, 147, 150, 181, 247, 248] becomes a common occurrence. Nevertheless much has already been learned about the interactions of complexes with acceptors, which in turn have been used to help formulate models of steroid-hormone action.