摘要:

Cultured bovine adrenal fasciculata cells were used to characterize angiotensin II (A-II) and corticotropin (ACTH) receptors and to study their homologous and heterologous regulation. These cells contain one type of high affinity binding sites for A-II (KD∼2.4±0.3 10−9M) and about 100000 sites/cell. Photoaffinity labeling followed by SDS-PAGE under reducing conditions revealed a single macromolecule of apparent MR 65000. Treatment of cells with increasing concentrations of A-II produced down-regulation of its own receptors and marked homologous and heterologous (ACTH) steroidogenic desensitization. However, the desensitization was not correlated with receptor loss and was mainly due to alterations of the steroidogenic pathway. Pretreatment of cells with ACTH also reduced A-II receptors, but this was not associated with steroidogenic desensitization. Bovine fasciculata cells contain two binding sites for ACTH: one of high affinity (KD∼2.6±0.4 10−10M) and low capacity (2030±390 sites/cell) and the other of low affinity and high capacity. Affinity cross-linking of ACTH to plasma membranes prepared from adrenal cells revealed a labeled macromolecule of apparent MR 43000. However, cross-linking experiments to intact cells revealed, both under reducing and non-reducing conditions, two labeled macromolecules of apparent MR of 123000 and 43000. Pretreatment of cells with ACTH enhanced its receptor and the cAMP and cortisol responses to further ACTH stimulation. These effects were time -and dose-dependent. The maximal effects were observed at 10−10to 10−9M. A-II alone had no effect but it blocked partially the stimulatory action of ACTH.

ISSN:0743-5800    

DOI:10.1080/07435809109027186

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Y1 adrenocortical tumor cells (Y1DS) and Y1 mutants resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were transfected with a gene encoding the mouseß-adrenergic receptor (ß-AR). Transfectants expressedß-ARs that were able to stimulate adenylyl cyclase activity and steroid biosynthesis. These transfectants were used to explore the basis for the DR mutation in Y1 cells. We demonstrate thatß-adrenergic agonists desensitize the adenylyl cyclase system in transfected Y1DScells whereas transfected Y1DRcells are resistant to desensitization byß-adrenergic agonists. The fate of theß-ARs during desensitization was evaluated by photoaffinity labelling with [125I] iodocyanopindolol diazerine. Desensitization of Y1DStransfectants was accompanied by a modest loss in receptor density that was insufficient to account for the complete loss of responsiveness toß-adrenergic agonists. The extent of receptor loss induced byß-adrenergic agonists in Y1DRtransfectants exceeded that in the Y1DStransfectants indicating that the mutation which protects Y1DRcells from agonist-induced desensitization is prior to receptor down-regulation in the desensitization pathway. From these results we infer that ACTH and isoproterenol desensitize adenylyl cyclase by a common pathway and that receptor loss is not a major component of the desensitization process in these cells.

ISSN:0743-5800    

DOI:10.1080/07435809109027187

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Heterogeneity exists among humans in the responses of blood pressure to alterations in sodium and extracellular fluid volume status. A variety of approaches have been utilized to characterize sodium responsivity of blood pressure including rapid volume expansion and contraction and dietary sodium manipulation. Studies conducted in our laboratories over the past 15 years have enabled us to examine salt sensitivity of blood pressure in different ways and to characterize this phenomenon as well as to define neurohumoral, demographic and genetic factors associated with it in normotensive and hypertensive humans.

ISSN:0743-5800    

DOI:10.1080/07435809109027188

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

ISSN:0743-5800    

DOI:10.1080/07435809109027189

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Rats have been bred for susceptibility and resistance to the hypertensive effect of dietary salt (S/JR&R/JR). S/JR have an abnormal adrenal steroid 11β,18-hydroxylase activity resulting in increased production of 18-OH-DOC. S/JR also produce increased quantities of 19-nor-DOC, which may be related, since the 11β,18-hydroxylase also catalyzes the 19-hydroxylation of DOC, a pivotal step in 19-nor-DOC biosynthesis. The purpose of the present studies was to further characterize the mutant S/JR adrenal steroid 11β,18-hydroxylase. Preliminary studies are also presented on assessing the renal 19-desmolase, the last step in 19-nor-DOC biosynthesis. Adrenal glands were harvested from R/JR and S/JR and prepared for incubation studies, protein immunoblotting, and RNA analysis. Kidneys from Sprague-Dawley rats were also used for isolated renal perfusion studies.Both S/JR and R/JR strains had a single immunostaining band for 11β,18-hydroxylase at 51,000 molecular weight which were equal in intensity. Both strains had a single RNA transcript at 4.3 kilobases which hybridized with equal intensity to the bovine cDNA (pB11-9). The Kmfor 11β,18- and 18-hydroxylation was identical within strains but was different between strains. The Kmfor 19-hydroxylation was different between S/JR and R/JR, and was much greater than 11β,18 -and 18-hydroxylation in both strains. This suggests that the catalytic site for 19-hydroxylation is different than that for 11β- and 18-hydroxylation and that the S/JR

ISSN:0743-5800    

DOI:10.1080/07435809109027190

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Deficiency of steroid 11β-hydroxylase, which is a mitochondrial cytochrome P450 required for cortisol and aldosterone synthesis, causes hypertension as well as virilization. In addition, abnormal regulation of this enzyme or a closely related isozyme may be involved in an autosomal dominant form of inherited hypertension, dexamethasone-suppressible hyperaldosteronism. An enzyme that catalyzes the interconversion of cortisol and cortisone, 11β-hydroxysteroid dehydrogenase, may be defective in an autosomal recessive form of hypertension termed apparent mineralocorticoid excess. The molecular bases of these forms of hypertension will be elucidated by identifying mutations in the 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase genes and expressing normal and mutagenized enzymes in cultured cells.

ISSN:0743-5800    

DOI:10.1080/07435809109027191

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Over long periods of growth in culture, bovine adrenocortical cells lose the ability to express the steroid hydroxylase genes. For 17α-hydroxylase, cells show a stochastic pattern of phenotypic switching from a state in which they express this gene in response to cyclic AMP to a state in which the gene is no longer inducible. Introducing SV40 T antigen into bovine adrenocortical cells greatly increases their replicative potential; steroid hydroxylase expression in these clones resembles that of the precursor cells before transfection. The other steroid hydroxylases (21-hydroxylase and 11β-hydroxylase) appear to undergo phenotypic switching like 17ß-hydroxylase. The loss of expression of these genes appears to be more rapid, but there are differences in the requirements of 21-hydroxylase and 11β-hydroxylase versus 17ß-hydroxylase for induction by cyclic AMP; additionally, growth of cells in extracellular matrix Matrigel was required for expression of 21-hydroxylase and 11β-hydroxylase in long-term cultures of either normal or SV40 T antigen-transfected cells. Understanding the molecular basis for the phenotypic switching of steroid hydroxylases that occurs in bovine adrenocortical cells may elucidate mechanisms for cellular senescence and for maintenance of tissue-specific functions during long-term growth in culture.

ISSN:0743-5800    

DOI:10.1080/07435809109027192

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

It was found that homogenous 11β-hydroxylase from bovine and porcine adrenals catalyzes the conversion of DOC to aldosterone. Mitochondria from both glomerulosa and fasciculata also convert DOC to aldosterone but glomerulosa is much more active than fasciculata. Cholate extracts of mitochondria from the two zones were equally active in converting DOC to aldosterone. Moreover all the enzyme activities of 11β-hydroxylase (including 18-hydroxylation and aldehyde synthetase) were precipitated by a polyclonal antibody raised in rabbit against the pure 11β-hydroxylase. It is concluded that in beef and pig a single adrenocortical 11β-hydroxylase is responsible for the synthesis of aldosterone. To determine the influence of the mitochondrial membrane from glomerulosa and fasciculata on the activities of 11β-hydroxylase we examined the activities of rotenone-insensitive reductase enzymes in mitochondria from the two zones. Semidehydroxyascorbate reductase and NADH-cytochrome C reductase activities are considerably

ISSN:0743-5800    

DOI:10.1080/07435809109027193

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

The molecular nature of the aldosterone synthesizing enzymes of cattle and rat is discussed. In bovine adrenal cortex, one molecular species of cytochrome P-45011βcatalyzes aldosterone synthesis as well as 11β-hydroxylation. The intactness of the mitochondrial membrane surrounding P-45011βin the zonae fasciculata-reticularis is essential to keep the aldosterone synthesizing activity of the cytochrome in these zones latent. In rat adrenal cortex, two distinct molecules belonging to a P-45011βfamily exist. One is 11β-hydroxylase, and the other aldosterone synthase.

ISSN:0743-5800    

DOI:10.1080/07435809109027194

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Aldosterone, the major mineralocorticoid hormone, is produced exclusively in the zona glomerulosa of the mammalian adrenal cortex. In the rat species, this zonal specificity of aldosterone biosynthes is appears to be due mainly to the existence of a second form of cytochrome P-45011βwhich differs from the major form of the enzyme (molecular weight 51,000) by(1) a lower molecular weight (49,000)(2) a broader range of catalytic activities, which include corticosterone methyl oxidation 1 and 2(3) an exclusive occurrence in the zona glomerulosa, and(4) a crucial dependence on sodium and potassium intake.The 49K form of the enzyme can be induced by potassium ions in vivo (potassium repletion of potassium-deficient rats) or in vitro (primary cell culture). The biosynthesis of this protein is controlled most likely at the level of transcription. According to indirect evidence, ACTH induces only the 51K form of the enzyme in vitro. Prolonged treatment of rats with a high dose of ACTH has a repressive effect on the 49K form of the enzyme.

ISSN:0743-5800    

DOI:10.1080/07435809109027195

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor