Biomedical subjects

M Lombès

Publications and source records attributed to M Lombès.

27 records · Page 2Linked to original sources

PubMed1995

Prerequisite for cardiac aldosterone action. Mineralocorticoid receptor and 11 beta-hydroxysteroid dehydrogenase in the human heart.

BACKGROUND: It has been proposed that aldosterone exerts direct effects on heart function, most notably on the development of myocardial fibrosis during ventricular hypertrophy in rat. Initial events in aldosterone action entail its binding to mineralocorticoid receptor (MR). Because MR displays similar affinities for aldosterone and glucocorticoids, the in vivo aldosterone selectivity of MR requires the presence of an enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD), which metabolizes glucocorticoids into inactive derivatives. Although evidence exists for the presence of MR in rodent heart, no data are available for humans; moreover, the existence of cardiac 11-HSD is controversial. METHODS AND RESULTS: The heart samples used originated from tissue removed during cardiac surgery in nontransplant patients or from endocavitary biopsies done for the follow-up of heart transplantation. The expression of MR was examined at the mRNA and protein level by in situ hybridization with cRNA probes specific for human MR mRNA and by immunodetection with two specific anti-MR antibodies. 11-HSD catalytic activity was determined by measurement of the metabolic rate of tritiated corticosteroids by cardiac samples. In nontransplanted hearts, an in situ hybridization signal equivalent to that found in the whole kidney was present on cardiomyocytes. Specific immunolabeling of cardiomyocytes with anti-MR antibodies demonstrated the presence of the MR protein. Cardiac 11-HSD activity was detected (243 +/- 26 fmol.30 min-1.mg protein-1) and was dependent on the cofactor NAD, not NADP, suggesting that it corresponds to the form of the enzyme specifically responsible for MR protection. In transplanted hearts that presented severe alterations, MR immunodetection was weaker and irregular, with no specific hybridization signal. CONCLUSIONS: Our results demonstrate that MR is coexpressed with 11-HSD in human heart, which thus possesses the cellular machinery required for direct aldosterone action.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1995

A key enzyme in the biosynthesis of neurosteroids, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD), is expressed in rat brain.

In rat brain, the presence of pregnenolone and progesterone, not attributable to peripheral glandular sources, has been demonstrated and thus the two compounds can be classified as neurosteroids. In vitro experiments have shown the conversion of pregnenolone, a 3 beta-hydroxy-delta 5-ene steroid, into progesterone, a delta 4-oxo steroid, thus demonstrating a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) enzymatic activity. The conversion of 3 beta-hydroxy-delta 5-derivatives into the corresponding delta 4-oxo steroids by 3 beta-HSD is an essential step in the biosynthesis of all steroid hormones in endocrine glands. To date, four isoforms of 3 beta-HSD have been characterized in the rat. We report here the selective expression of a 3 beta-HSD isoform in rat brain. An in situ hybridization study, using an oligonucleotide common to the 4 known isoforms, demonstrated 3 beta-HSD mRNA in neurons of the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, habenula, hippocampus and cerebellum. The cerebellum showed the highest level of 3 beta-HSD mRNA corresponding to a transcript of 1.8 kb. Nucleotide sequencing of PCR-amplified cDNA fragments from cerebellar mRNA indicated the expression of an isoform of 3 beta-HSD cDNA very closely related to the isoform I expressed in the adrenals and gonads. Further evidence for the expression of 3 beta-HSD gene in the brain was demonstrated utilizing anti-peptide 3 beta-HSD antibodies which revealed an immunoreactive protein of approximately 45 kDa in the cerebellum. Our results demonstrate for the first time the expression of the enzyme 3 beta-HSD in the brain, at both the mRNA and protein levels. Since several neuroactive neurosteroids are substrates or products of the 3 beta-HSD enzymatic activity, our findings offer new possibilities to study the regulatory mechanisms governing their biosynthesis in the brain.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Aldosterone: intracellular receptors in human heart.

It has been suggested that aldosterone exerts direct effects on heart functioning, in particular by inducing cardiac fibrosis. We examined human heart tissue for the expression of aldosterone receptors (mineralocorticoid receptors, MRs) and of the MR-protecting enzyme, 11 beta hydroxysteroid dehydrogenase (11 beta HSD). In situ hybridization using cRNA probes specific for human MRs revealed the presence of mRNA encoding for MRs in cardiomyocytes. Immunohistochemistry with specific antibodies against the MR exhibited the expression of MR protein in cardiomyocytes. In contrast, intramyocardial small blood vessels showed no evidence of immunolabelling. A distinct 11 beta HSD activity, which was nicotinamide-adenine dinucleotide dependent, was also demonstrated in human cardiac tissue. These results illustrate that all the components required for a specific aldosterone effect are present in the human heart.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

Differential intracellular localization of human mineralocorticosteroid receptor on binding of agonists and antagonists.

The effect of aldosterone and antimineralocorticoids on the intracellular localization of human mineralocorticosteroid receptor (hMR) was studied using a new monoclonal anti-peptide antibody FD4. This antibody was directed against the peptide hMR-(412-422). As demonstrated by ultracentrifugation analysis, immunoprecipitation assays and Western blot, FD4 recognized both the native and denatured form of the receptor overexpressed in the baculovirus expression system. In whole-cell assays, the amount of hMR recovered in high-salt extracts was significantly lower after exposure to the antimineralocorticoid ZK91587 than to aldosterone, suggesting a lack of nuclear MR translocation. FD4 was also used for immunohistochemical studies on hMR-expressing High Five cells. In the absence of hormone, immunoreactive hMR was detected almost exclusively in the cytoplasmic compartment of cells. After aldosterone exposure, intense nuclear immunostaining appeared in a time-dependent manner, consistent with stable nuclear localization of the receptor. Immunohistochemistry showed that antimineralocorticosteroids (ZK91587, SC9420, 18-vinylprogesterone) predominantly maintained a cytoplasmic distribution of hMR and inhibited its aldosterone-dependent nuclear localization. Thus, in our model, the nuclear/cytoplasmic partition of hMR is drastically different in the presence of antagonists from that in the presence of aldosterone. This phenomenon may contribute to their mechanism of action by preventing productive interaction of antagonist-receptor complex with specific DNA sequences in aldosterone target cells.

Aldosterone↗

PubMed1994

Aldosterone-induced apical Na+ and K+ conductances are located predominantly in surface cells in rat distal colon.

Aldosterone is a major regulator of Na(+)-absorptive and K(+)-secretory processes in the distal segment of mammalian colon. In this study, the distribution of aldosterone-sensitive cell types in isolated rat distal colon was determined using site-directed intracellular microelectrodes, specific Na(+)- and K(+)-channel blockers, and aldosterone-receptor binding techniques. Electrophysiological data indicated that aldosterone induced parallel apical membrane Na+ and K+ conductances, mainly in surface cells and to a significantly lesser degree in crypt cells. Scatchard analyses of aldosterone-receptor binding in cytosolic fractions revealed the maximum number of specific binding sites in whole mucosal homogenate and in the upper one-third and lower two-thirds of isolated crypt units to be 74.9 +/- 2.0, 59.8 +/- 2.4, and 59.3 +/- 3.2 fmol/mg protein, respectively, indicating the presence of aldosterone receptors in the crypt cell population. We conclude that in rat distal colon aldosterone-induced Na+ and K+ conductances (and by inference, electrogenic Na(+)-absorptive and K(+)-secretory processes) are located predominantly in the surface cell population and to a lesser extent in crypt cells, which also contain aldosterone receptors. This spectrum of aldosterone-induced Na+ and K+ conductances may reflect varying stages of differentiation along the surface cell-crypt cell axis.

Aldosterone↗

PubMed1993

Characterization of the interaction of the human mineralocorticosteroid receptor with hormone response elements.

Although the mineralocorticosteroid receptor (MR) belongs to the superfamily of hormone-dependent transcription factors, the molecular mechanism by which it regulates gene expression is poorly understood. Binding of the MR to target gene promoters has never been characterized, and specific mineralocorticosteroid response elements (MREs) remain to be identified. The human MR (hMR) was overexpressed in Sf21 insect cells using the baculovirus system. The high degree of similarity between the glucocorticosteroid receptor (GR) and the MR prompted us to examine the DNA-binding properties of the recombinant MR with glucocorticosteroid-regulated genes. Gel shift mobility assays demonstrated that the recombinant receptor interacted with oligonucleotides containing perfect and imperfect palindromic sequences of GRE. A monoclonal anti-hMR antibody (FD4) induced a supershift of protein-DNA complexes and identified the MR in Western blot analysis. In vitro DNAse I protection assays with the hormone-regulated murine mammary tumour virus promoter showed that recombinant hMR generated four footprints whose limits encompassed the GRE motifs. By means of these two complementary approaches, no difference between the interaction of free, agonist- or antagonist-bound MR and DNA was detected. We provide evidence that hMR functions as a sequence-specific DNA-binding protein.

Animals↗

PubMed1992

A novel monoclonal anti-rabbit hsp90 antibody: usefulness for studies on hsp90-steroid receptor interaction.

The M(r) 90,000 protein associated with steroid receptors in their non-transformed state has been identified as a heat shock protein (hsp90) but the relationship between hsp90 binding and receptor function is still poorly understood. In this work, we have obtained and characterized one monoclonal anti-rabbit hsp90 antibody (7C10), among more than 2000 wells plated. This antibody was able to complex both free and rabbit uterine progesterone receptor-associated hsp90 as demonstrated by sedimentation analysis on sucrose gradients. As assessed by ELISA, 7C10 displayed a high binding affinity for hsp90 (approximately 4 nM). A standardized and specific competitive binding assay was developed for accurate quantification of hsp90 in rabbit tissues including reticulocyte lysate. 7C10 also permitted immunolocalization of hsp90 in various rabbit tissues. In Western blot, the monoclonal antibody recognized a single polypeptide band of M(r) approximately 90,000 in crude or purified rabbit preparations but failed to cross-react with any other mammalian or avian hsp90. These findings suggest that hsp90, a highly conserved protein, is a weak immunogen and elicits a strict species specific immunological response. Owing to its high affinity and specificity for rabbit hsp90, the monoclonal antibody 7C10 was used for purification and total depletion of hsp90 from the reticulocyte lysate, an efficient system for in vitro receptor translation and reconstitution studies. Thus, 7C10 represents a new powerful tool to further investigate the importance of hsp90 in steroid hormone receptor function.

Animals↗

PubMed1992

Immunohistochemical and biochemical evidence for a cardiovascular mineralocorticoid receptor.

The presence of mineralocorticoid receptors (MRs) and their physicochemical characteristics were investigated in the heart and blood vessels of rabbits. Immunohistochemical methods using the monoclonal anti-idiotypic antibody H10E, which interacts with the steroid binding domain of MRs, revealed the presence of immunoreactive material in the heart and large blood vessels. In the heart, a positive staining was observed in myocytes and endothelial cells of atria and ventricles. In vessels, MRs were detected in the aorta and pulmonary artery. They were localized in endothelial and vascular smooth muscle cells. No staining was present in the small vascular bed, arterioles, and capillaries. In all these studies, the mineralocorticoid specificity of the staining was assessed by in situ competition experiments with aldosterone and RU486, a glucocorticoid antagonist. The presence of MRs in the heart and vessels was further demonstrated by specific aldosterone binding to one class of high affinity binding sites in the cytosol of the adrenalectomized rabbit heart (Kd, 0.25 nM; maximum MR concentration, 15-20 fmol/mg protein), whose mineralocorticoid specificity has been clearly established by competition studies. Sedimentation gradient analyses revealed that the cardiovascular MR is an 8.5S hetero-oligomer that includes the heat shock protein 90. The physicochemical characteristics of the cardiovascular MRs are virtually identical to those of the renal MRs. Altogether, our results clearly demonstrate the presence of MRs in the cardiovascular system. This supports the possibility of direct aldosterone actions in the heart and blood vessels.

Adrenalectomy↗

PubMed1990

Immunohistochemical localization of renal mineralocorticoid receptor by using an anti-idiotypic antibody that is an internal image of aldosterone.

A monoclonal antibody (H10E), generated by an auto-anti-idiotypic procedure and directed at the aldosterone-binding site of mineralocorticoid receptor (MR), was used in immunohistochemical studies to localize MR in rabbit kidney preparations. In agreement with earlier physiological and biochemical observations, MR was detected in connecting and cortical collecting tubules. Additionally, MR was detected in the distal tubules, the medullary and papillary collecting ducts, and in the epithelial cells lining the papilla. The internal image properties of the antibody were exploited to assess the specificity of MR detection by means of competition studies with hormones and antihormones. Immunostaining was completely abolished by preincubation with aldosterone but not with RU 486, a steroid antagonist that does not bind MR. On the cellular level, immunostaining occurred in the cytoplasm and, in the majority of cells, in the nucleus as well. The nucleocytoplasmic distribution of MR was unaffected by adrenalectomy or by administration of aldosterone. The availability of this specific monoclonal antibody makes it feasible to study MR expression in other target tissues and in pathological disorders.

Adrenalectomy↗