摘要:

The relative effectiveness of succinate and isocitrate in supplying NADPH for cholesterol and deoxycorticosterone (DOC) metabolism by rat adrenal mitochondria has been investigated. As previously seen for cholesterol metabolism, isocitrate supported DOC metabolism at a higher rate than succinate. Maximal rates of DOC metabolism, however, required 10 times more precursor (10mM) than cholesterol metabolism. Addition of DOC to mitochondria inhibited cholesterol metabolism, indicating competition for NADPH between these pathways. Coaddition of these reducing precursors resulted in a substantially greater than additive rate of DOC hydroxylation, but not cholesterol metabolism. The synergistic effect was seen with both 11β- and 18 hydroxylation. For both precursors, the synergism was maximal upon addition of only 1mM of the second precursor. The synergistic effect was far more resistant to added KCN and malonate than succinate supported DOC metabolism, and neither inhibitor affected isocitrate supported DOC metabolism. These results suggest that while cytochromes P450sccβand P45011βuse a common supply of NADPH generated by each precursor, there is a pool of NADPH that is only effectively synthesised upon coaddition of precursors and only utilised by cytochrome P45011β. This second NADPH pool may be produced in response to potentiated isocitrate dehydrogenase activity or activation of a different NADPH generating system.

ISSN:0743-5800    

DOI:10.1080/07435809109027196

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

In the guinea pig adrenal the cells of the outer zone secrete high levels of steroid and respond to ACTH with increased synthesis of cholesterol and steroid. The outer zone consists of two cell types: zona fasciculata (ZF) and zona glomerulosa (ZG). To determine the relative contribution of ZF and ZG to the outer zone's ACTH response, purified populations of each cell type were prepared from ACTH-treated and control animals. Levels of proteins potentially involved in the ACTH response were measured by ELISA. HMG CoA reductase, the rate limiting enzyme of cholesterol synthesis, and two cytochrome P450s, P45017αand P45021, were studied. P45017αis required for production of cortisol, but not for corticosterone or aldosterone. P45021is required for production of all of these corticosteroids. ZF cells had 4-5 fold greater concentration of all three proteins, but the proteins in ZG cells showed a greater response to ACTH (3 fold). The response of ZG cells to ACTH and their possession of P45017αis consistent with observations made in vitro that ZG cell populations synthesize cortisol and respond to ACTH with increased output of cortisol as well as of corticosterone and aldosterone. In ZG cells to a greater extent than ZF cells, the response to ACTH involved increases in levels of enzymes of both cholesterol and steroid synthesis, suggesting that new protein synthesis is an important component of their ACTH response. On the other hand, the fact that ZF cells can increase steroid output in response to ACTH with a lesser increase in these proteins suggests a different mechanism of regulation. Mobilization of stored cholesterol may be more important in the ACTH-responsiveness of the lipid-filled ZF cells than in the lipid-poor ZG cells.

ISSN:0743-5800    

DOI:10.1080/07435809109027197

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

The present study offers evidence indicating that acrylamide a polar molecule inhibits substrate-binding to P450c-21in a competitive manner and quenches tryptophanyl fluorescence in bovine adrenocortical microsomes, similar to that in the purified lipid-free enzyme. Resolution of tryptophanyl fluorescence of the microsomes revealed an acrylamide quenching constant (K2= 9.9M, is the association constant for the quencher-fluorophore complex) which was similar to the reciprocal of its inhibition constant (1/K1= Ka = 8.3±0.9M) for substrate-binding. The substrate inhibited the fluorescence quenching by acrylamide which was in accordance with partial competition. In addition the substrate dissociation, acrylamide inhibiton and fluorescence quenching constants and tryptophanyl fluorescence maximum (340-342nm) were essentially the same in the microsomes and the purified enzyme. These results suggest that, similar to that in the purified enzyme, a tryptophan in a polar environment in the membrane-bound P450, may serve as a reporter group for the substrate binding site and the site in the membrane-bound enzyme, is accessible to the substrate in aqueous phase.

ISSN:0743-5800    

DOI:10.1080/07435809109027198

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Fast and slow K+efflux components, independently regulated by angiotensin II (AII), have been identified in bovine adrenocortical cells. We have further investigated the role of potassium in the control of aldosterone synthesis in two ways. Firstly, isotopic tracers, in conjunction with channel modulators, have been used to study the interrelationship of K+and Ca2+in the control of AII-stimulated aldosterone synthesis. Secondly, electron probe X-ray microanalysis (EPXMA) was used to quantify potassium, sodium, chlorine and phosphorous in control and AII-stimulated cells.The effects of verapamil on43K efflux were measured at two stages during AII stimulation. During the first ten minutes of treatment, when efflux via the fast component predominates, AII and verapamil both slowed efflux and their effects were additive. If verapamil was added later, at the time when efflux by the fast component appeared exhausted and the stimulatory effect of AII on the slow efflux component was apparent, it again slowed efflux. These data suggest that verapamil prevents calcium-gated K+channels from opening by blocking Ca2+channels. However, verapamil had no effect on AII-stimulated calcium efflux. In addition to blocking Ca2+channels, verapamil may directly inhibit potassium efflux.EPXMA showed a bimodal distribution of potassium concentrations in control cells. However, in cells stimulated with All for five minutes, the mean potassium content was less than in controls and was not bimodally distributed. Sodium content was increased by AII-treatment, chlorine was lowered and phosphorus remained unchanged. The data confirm previous observations that AII inhibits Na+/K+ATPase activity.

ISSN:0743-5800    

DOI:10.1080/07435809109027199

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Inner zone cells, isolated from bovine adrenal cortex, secrete cortisol in response to both adrenergic and cholinergic agonists.The response to adrenaline (and other catecholamines) appears during culture, is evident by 24 h and reaches a maximum by 48–72 h, but is absent in freshly isolated cells. Pre-incubation of cultured cells with adrenaline leads to homologous desensitisation; the possibility that this may explain the absent response in freshly isolated cells is discussed. Cells show a dose-dependent cyclic AMP response but no increased membrane phosphoinositide turnover. In agreement, cortisol secretion is blocked byβ-receptor, but notα-receptor, antagonists. Schild analysis established that the response occurs through binding to aβ1,-receptor subtype, consistent with adrenergic innervation as opposed to an effect of circulating catecholamines.In contrast, cortisol secretion to AcCh was present in both freshly isolated cells and those in culture, reaching a maximum by 48–72 h in culture. The response was specifically blocked by muscarinic, but not nicotinic, antagonists. No effect on cyclic AMP formation was observed, but dose-dependent stimulation of phosphoinositide turnover occurred. HPLC analysis of the time-course of appearance of3H-inositol labelled head

ISSN:0743-5800    

DOI:10.1080/07435809109027200

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Transforming growth factorß1 (TGFß1) is a member of a large family of structurally related regulatory polypeptides which comprises both functionally similar (TGFß1, TGFß2, TGFß3, TGFß4 and TGFß5) and functionally distinct proteins. In the past few years, TGFß1 has emerged as a multifunctional protein. One of its remarkable properties is its capacity to negatively modulate the differentiated, steroïdogenic adrenocortical functions. We present here a review of the results from our recent work related to the effects of TGFß1 on bovine adrenocortical cell (zona fasciculata-reticularis) functions. We identified the steroid 17α-hydroxylase (P-45017α) biosynthetic enzyme and the angiotensin II receptor as major targets whose expression are negatively regulated by TGFß1 in these cells. We characterized TGFß1 receptors at the surface of adrenocortical cells (mainly type I and type III receptors) and observed that their number is increased under ACTH treatment. Furthermore, we could detect the presence of immunoreactive TGFß1 in the bovine adrenal cortex whereas it was undetectable in the adrenal medulla and in the capsule. We also observed that adrenocortical cells secrete TGFß1 under a latent form together with large amounts ofα2-macroglobulin, a protease inhibitor known to be implied in the latency of TGFß1 in serum. Taken together, these observations led us to a working hypothesis, proposing TGFß1 as an autocrine and/or paracrine regulator of adrenocortical steroidogenic functions. This concept points out the physiological activation of the latent TGFß1 complex as the important limiting step controlling its action in the adrenal cortex.

ISSN:0743-5800    

DOI:10.1080/07435809109027201

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid metabolizing enzymes. In the steroidogenic pathway, the ratio of 3β-hydroxysteroid dehydrogenase (3β-HSD) to 17α-hydroxylase cytochrome P450 (P-45017α) plays a key role in determining the final steroid products released by adrenal cells. The differences in these enzymes are particularly important when one considers the adrenal zones and the secretion of the zone-specific steroids. In the present study we have investigated the regulation of 3βHSD with regard to its enzyme activity, levels of protein and changes in specific mRNA encoding for this enzyme. Following eight days in primary culture, bovine adrenocortical (BAC) cells were found to respond to both ACTH and Bu2cAMP by increased cortisol production. In addition, 3βHSD activity, enzyme protein and mRNA levels were increased in response to both factors. The increases varied from 2-fold for activity to 5-7 fold for mRNA. ACTH and Bu2cAMP also greatly increased P-45017αfrom the near undetectable levels in control cells. In order to examine the possibility of differential regulation of these adrenal steroidogenic enzymes we determined the effects of angiotensin II (A-II) and transforming growth factor beta (TGFβ) on the levels of these enzymes. Both of these factors decreased the ACTH-stimulated levels of P-45017αenzyme and mRNA to near nondetectable levels observed within control cells. In addition, these compounds inhibited the ACTH induction of 3βHSD. While the mechanism of TGFβaction is not clear, A-II probably is acting through protein kinase C. Indeed the protein kinase C activating phorbol ester, TPA, mimicked the inhibitory effects of A-II on 3βHSD and P45017α. It is important to point out, however, that the effects of A-II and TGFβon P45017αactivity appeared more pronounced than their action of 3βHSD. This observation may relate to the relative stability of 3βHSD as compared to P45017α. Taken together these data indicate that, while A-II and TGFβeach decrease the levels of steroid-metabolizing enzymes, a differential regulation is observed in that P-45017αprotein and activity levels are much more sensitive to treatment with these factors.

ISSN:0743-5800    

DOI:10.1080/07435809109027202

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Cholesterol sulfate inhibits cholesterol side-chain cleavage in adrenal mitochondria. In this study, analogs of cholesterol sulfate were evaluated for their ability to inhibit steroidogenesis. Structural requirements for inhibitory activity included a planar A-B ring junction, an intact side chain, and a 3β-ester group containing a single negative charge. This structural specificity argues against cholesterol sulfate acting solely as a membrane perturbing agent or a detergent, and also differs in some features from the specificity for binding to cytochrome P-450scc

ISSN:0743-5800    

DOI:10.1080/07435809109027203

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

A 30-residue peptide corresponding to the amino acid sequence of steroidogenesis activator peptide (SAP) from rat Leydig tumor cells has been synthesized by the solid-phase method using Boc protection. SAP is the putative cycloheximide-sensitive, cAMP-regulated mediator of ACTH-stimulated conversion of cholesterol to pregnenolone in adrenal cortex. Na-acetyl-SAP(11-30), an NH2-terminally truncated steroidogenesis activator peptide analog that is missing the most hydrophobic portion of SAP, was also prepared. In addition to these two peptides, Na-acetyl-(Cys0)SAP was synthesized in both non-radiolabeled and tritiated forms for coupling to carrier proteins for use as an immunogen to raise anti-SAP antibodies. Chain elongation during synthesis of SAP on PAM resin proceeded with an average coupling yield of 99.3% as determined by quantitative ninhydrin tests. After HF cleavage at -7°, the crude products were purified by semi-preparative HPLC. Peptides were analyzed by analytical HPLC, amino acid analysis, tryptic peptide mapping, and by UV and CD spectroscopy. As determined by CD spectra, SAP showed little evidence of preferred structure either in aqueous solution in the presence of divalent cations or in micelles of reduced Triton X-100 in the absence or presence of either cholesterol or phosphatidylcholine. SAP, in conjunction with GTP, enhanced side chain cleavage activity in isolated adrenal mitochondria.

ISSN:0743-5800    

DOI:10.1080/07435809109027204

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

ISSN:0743-5800    

DOI:10.1080/07435809109027185

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor