Biomedical subjects

G Defaye

Publications and source records attributed to G Defaye.

At least 19 recordsLinked to original sources

PubMed2000

Expression and regulation of melanocortin receptor-5 (MC5-R) in the bovine adrenal cortex.

Among the five members of the melanocortin receptor (MC-R) family, MC2 and MC5 are expressed in peripheral tissues. The receptor MC2 (ACTH receptor) almost exclusively expressed in the adrenal cortex whereas MC5-R is expressed in several organs including the adrenal cortex. Both receptors bind ACTH and activate adenylate cyclase. The aim of this work was to study the spatial distribution of MC5-R among the different zones of the bovine adrenal cortex and to analyze the regulation of its expression by its own ligands, ACTH and alpha-MSH and by angiotensin II (AII). Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and RNase protection assay, MC5-R was detected only in the glomerulosa zone whereas MC2-R was present in both glomerulosa and fasciculata zones of adult adrenal cortex. Treatments by ACTH, alpha-MSH, or AII increased the MC5-R mRNA level in glomerulosa cells by factors 7, 5, and 4.5, respectively. However, although potentially regulated by hormones, MC5-R is expressed at a level at least 100 times less than MC2-R, suggesting that MC5-R expression might only be at trace levels in grown adults, but could be much higher during embryogenesis.

Adrenal Cortex↗

PubMed2000

Bilateral laparoscopic adrenalectomy for congenital adrenal hyperplasia with severe hypertension, resulting from two novel mutations in splice donor sites of CYP11B1.

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Sequencing of the CYP11B1 gene showed two new base substitutions, a conservative 954 G-->C transversion at the last base of exon 5 (T318T), and a IVS8 + 4A-->G transition in intron 8. In addition, two polymorphisms were found in exons 1 and 2. The genetically female patient was raised as a male because of severe pseudohermaphroditism. Glucocorticoid-suppressive treatment encountered difficulties in equilibration and compliance, resulting in uncontrolled hypertension with pronounced hypertrophic cardiomyopathy. At 42 yr of age the occurrence of central retinal vein occlusion with permanent loss of left eye vision led to the decision to perform bilateral laparoscopic adrenalectomy. Surgery was followed by normalization of blood pressure and good compliance with glucocorticoid and androgen substitutive therapies. In vitro, adrenal cells in culture and isolated mitochondria showed extremely low 11beta-hydroxylase activity. Analysis of adrenal CYP11B1 messenger ribonucleic acid (mRNA) by RT-PCR and sequencing showed the expression of a shorter mRNA that lacked exon 8 and did not contain either the exon 5 mutation or the exon 1 and 2 polymorphisms. This suggested that one CYP11B1 allele carried the intron 8 mutation, responsible for skipping exon 8. The other allele carried the exon 5 mutation, and its mRNA was not detectable. Western blot analysis showed weak expression of a shorter CYP11B immunoreactive band of 43 kDa, consistent with truncation of exon 8. Thus, bilateral adrenalectomy in this patient allowed effective treatment of severe hypertension and helped in understanding the mechanisms and physiopathological consequences of two novel mutations of CYP11B1.

Adrenal Glands↗

PubMed2000

Chronic stress effects on the rat adrenal cortex.

Under the influence of a chronic permanent stress, the adrenal function as well as the entire hypothalamic-pituitary-adrenal axis (HPAA) suffered an adaptation process that resulted in the normalization of the studied stress hormones (ACTH, corticosterone and aldosterone) with the exception of plasma renin activity which first diminished and at the end increased. ACTH receptors exhibited a dual response since after 14 days of permanent stress MC2-R showed a slight reduction while MC5-R was still up-regulated.

Adrenal Cortex↗

PubMed2000

Two novel mutations in splice donor sites of CYP11B1 in congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency.

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Genetic analysis showed two new base substitutions of CYP11B1, a conservative transition at the last base of exon 5, and a IVS8+4A-->G transition in intron 8. Difficulties with suppressive therapy resulted in severe hypertension. A laparoscopic adrenalectomy was decided which lead to normalization of blood pressure. In vitro, steroidogenesis by adrenal cells showed no measurable 11beta-hydroxylase activity. Analysis of CYP11B1 mRNA by RT-PCR and sequencing showed expression of a mRNA which lacked exon 8, presumably resulting from the intron 8 mutation. In addition a highly truncated mRNA was detected corresponding to exons 1, 2, 8, 9, with the loss of exons 3-7, presumably related to the exon 5 mutation. Western blot analysis showed a shorter CYP11B immunoreactive band of 43 kDa, consistent with truncation of exon 8. Thus adrenalectomy in this patient allowed effective treatment of severe hypertension and helped to understand the mechanisms of two novel mutations responsible for aberrant splicing of CYP11B1.

Adrenal Cortex↗

PubMed1998

The flavoprotein component of the Escherichia coli sulfite reductase can act as a cytochrome P450c17 reductase.

The flavoprotein component (SiR-FP) of the E. coli sulfite reductase was found to support 17 alpha-hydroxylation of pregnenolone in the presence of cytochrome P450c17. Half maximum activity is obtained for a 1:1 ratio of SiR-FP, expressed as monomer concentration, to P450c17. When compared to bovine NADPH-cytochrome P450 reductase, SiR-FP is about 12-15 times less efficient. P450c17 was demonstrated to interact specifically with the FMN-binding domain of the protein and the N-terminal part of SiR-FP is suspected to play a role in electron transfer. A cluster of negatively charged residues was found in SiR-FP by amino acid sequence comparison with rat cytochrome P450 reductase. These results argue in favour of the flavodoxin origin of the FMN-binding domain of SiR-FP.

Amino Acid Sequence↗

PubMed1998

Transforming growth factor beta1 decreases cholesterol supply to mitochondria via repression of steroidogenic acute regulatory protein expression.

Transforming growth factor-betas (TGF-betas) constitute a family of dimeric proteins that affect growth and differentiation of many cell types. TGF-beta1 has also been proposed to be an autocrine regulator of adrenocortical steroidogenesis, acting mainly by decreasing the expression of cytochrome P450c17. Here, we demonstrate that TGF-beta1 has a second target in bovine adrenocortical cells, namely the steroidogenic acute regulatory protein (StAR). Indeed, supplying cells with steroid precursors revealed that TGF-beta1 inhibited two steps in the steroid synthesis pathway, one prior to pregnenolone production and another corresponding to P450c17. More specifically, TGF-beta1 inhibited pregnenolone production but neither the conversion of 25-hydroxycholesterol to pregnenolone nor P450scc activity. Thus, TGF-beta1 must decrease the cholesterol supply to P450scc. We therefore examined the effect of TGF-beta1 on the expression of StAR, a mitochondrial protein implicated in intramitochondrial cholesterol transport. TGF-beta1 decreased the steady state level of StAR mRNA in a time- and concentration-dependent manner. This inhibition occurs at the level of StAR transcription and depends on RNA and protein synthesis. It is likely that the TGF-beta1-induced decrease of StAR expression that we report here may be expanded to other steroidogenic cells in which a decrease of cholesterol accessibility to P450scc by TGF-beta1 has been hypothesized.

Animals↗

PubMed1998

Cushing's syndrome due to a gastric inhibitory polypeptide-dependent adrenal adenoma: insights into hormonal control of adrenocortical tumorigenesis.

We studied a patient with food-induced, ACTH-independent, Cushing's syndrome and a unilateral adrenocortical adenoma. In vivo cortisol secretion was stimulated by mixed, glucidic, lipidic, or proteic meals. Plasma ACTH levels were undetectable, but iv injection of ACTH stimulated cortisol secretion. Unilateral adrenalectomy was followed by hypocortisolism with loss of steroidogenic responses to both food and ACTH. In vitro, cortisol secretion by isolated tumor cells was stimulated by the gut hormone gastric inhibitory polypeptide (GIP) and ACTH, but not by another gut hormone, glucagon-like peptide-1 (GLP-1). Both peptides stimulated the production of cAMP but not of inositol 1,4,5-trisphosphate. In quiescent cells, GIP and ACTH stimulated [3H]thymidine incorporation and p42-p44 mitogen-activated protein kinase activity. GIP receptor messenger ribonucleic acid (RNA), assessed by RT-PCR, was highly expressed in the tumor, whereas it was undetectable in the adjacent hypotrophic adrenal tissue, in two adrenal tumors responsible for food-independent Cushing's syndrome, and in two hyperplastic adrenals associated with ACTH hypersecretion. In situ hybridization demonstrated that expression of GIP receptor RNA was confined to the adrenocortical tumor cells. Low levels of ACTH receptor messenger RNA were also detectable in the tumor. We conclude that abnormal expression of the GIP receptor allows adrenocortical cells to respond to food intake with an increase in cAMP that may participate in the stimulation of both cortisol secretion and proliferation of the tumor cells.

Adenoma↗

PubMed1997

Bovine adrenocortical cells in culture synthesize an ouabain-like compound.

Ouabain or a closely related isomer, and 'ouabain-like compound' (OLC), has been identified in plasma, by Hamlyn et al., using several physico-chemical and biological methods. Using a radioimmunoassay, the same authors later characterized an identical compound in adrenal cortex tissue and culture medium from adrenocortical cells. Nevertheless, other groups, using different immunosera, were not able to detect OLC in adrenal cortex and adrenocortical cells medium. In this report, we confirm the presence of OLC in bovine adrenal cortex and in fasciculata cells culture medium. The compound that we obtained has the same chromatographic properties as ouabain on HPLC using two types of elution systems. It presents the same mass spectrum and is able to bind to erythrocytes membranes Na(+)-K(+)-ATPase. In primary cultures of adrenocortical cells, its biosynthesis is increased after addition of pregnenolone or progesterone suggesting that these compounds may represent intermediate substrates in the biosynthetic pathway. Rhamnose readily enters the adrenocortical cell and increases slightly the biosynthesis of OLC. The present studies confirm that bovine adrenocortical cells in primary culture release an OLC with no differences with authentic ouabain using, HPLC, mass spectrometry and radioreceptor assay and suggest that OLC may be a product related to the adrenocortical steroidogenic pathway.

Adrenal Cortex↗

PubMed1997

ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells.

Angiotensin II (ANG II) has long been known for its pressor and growth-promoting effects, which are both mediated by the AT1 receptor. By contrast, the AT2 receptor has recently been reported to mediate inhibition of proliferation through as yet undefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks the cells in G1 phase. Consistent with this, ANG II inhibits cyclin D1 expression and cyclin D1-associated kinase activity. The antimitogenic effect of ANG II is partly mimicked by the AT2-selective agonist CGP-42112. It is also blocked partly and in an additive fashion by the AT1- and AT2-selective antagonists losartan and PD-123319, indicating the contribution of both receptor subtypes to this response. AT1-dependent antiproliferation is selectively blocked by the cyclooxygenase inhibitor indomethacin and restored by prostaglandin E2, whereas AT2-receptor-mediated inhibition of growth is suppressed by the tyrosine phosphatase inhibitors orthovanadate and bpV(pic). Both pathways are, however, pertussis toxin sensitive. We hypothesize that, in fasciculata cells, the AT1 receptor inhibits bFGF-induced proliferation by stimulating prostaglandin synthesis, whereas the AT2 receptor mediates its effect through a pathway that requires protein tyrosine phosphatase activation.

Adrenal Cortex↗

PubMed1996

Specific inhibition of the last steps of aldosterone biosynthesis by 18-vinylprogesterone in bovine adrenocortical cells.

18-Vinylprogesterone (18-VP), designed for mechanism-based specific inhibition of the last steps of the aldosterone biosynthesis, was used to characterize the mechanism of the 11 - and 18-hydroxylase activities of bovine cytochrome P450(11beta). In the present work, its action was studied by observations on a primary culture of bovine adrenocortical cells. First, we investigated the effects of 18-VP on the different enzymatic steps of the biosynthesis of cortisol and aldosterone. The production of cortisol, baseline or hormone-stimulated (ACTH or AII), was inhibited by 18-VP in a dose-dependent manner with a maximal inhibition at 5 microM. Supply of different exogenous substrates to support steroidogenesis revealed an inhibition of the last step of cortisol or corticosterone biosynthesis. We then used specific blockers to measure individual activities and conclude that 11beta-hydroxylation was the only enzymatic activity affected. Aldosterone, as well as 18-hydroxycorticosterone, was also measured following addition of corticosterone. The 18-hydroxylation of corticosterone was inhibited by 18-VP, with 50% inhibition occurring at 0.04 microM compared with the 50% inhibition value of 0.3 microM obtained for 11-hydroxylation. Surprisingly, 18-ethynyl-progesterone (18-EP), which has a structure very similar to 18-VP, only weakly inhibits 11beta-hydroxylation. The inhibition of aldosterone formation was also much lower with 18-EP than with 18-VP. These studies demonstrate that 18-VP inhibits only the later steps of aldosterone biosynthesis and more specifically 18- than 11-hydroxylation activity.

Adrenal Cortex↗

PubMed1996

ACTH angiotensin II and TGF beta participate in the regulation of steroidogenesis in bovine adrenal glomerulosa cells.

Bovine zona glomerulosa cells, on the first day of culture, produce aldosterone as their major steroid with no detectable cortisol secretion. Continuous incubation with ACTH had no effect on aldosterone production nor on aldosterone synthase activity. This treatment resulted in a dose and time dependent rise in 17 alpha-hydroxylase activity, in parallel with an increase in cytochrome P-450(17 alpha) (CYP17) protein and mRNA. We have previously shown that TGF beta 1 is a potent inhibitor of differentiated functions of bovine fasciculata-reticularis cells and that CYP17 and AII receptors are the major targets explaining this effect. The present study examined whether 17 alpha-hydroxylase activity in glomerulosa cells could be regulated by angiotensin II (AII) and transforming growth factor-beta 1 (TGF beta 1). AII inhibits the induction of CYP17 by ACTH in a dose dependent manner. TGF beta 1 also blocks almost completely the stimulatory effect of ACTH. In order to suppress the endogenous action of TGF beta 1, incubations were performed with an anti-TGF beta antibody. This specific antibody induces the expression of CYP17 resulting in increased activity and mRNA levels. These results show that AII is able to modulate the expression of CYP17 in adrenal glomerulosa cells following ACTH stimulation. Furthermore, TGF beta 1 exerts an autocrine effect on the differentiation of glomerulosa cells through a regulatory loop repressing CYP17 activity.

Adrenocorticotropic Hormone↗

PubMed1996

Transforming growth factors-beta s: a multifunctional cytokine family. Implication in the regulation of adrenocortical cell endocrine functions.

Knowledge of the structure of the first recognized transforming growth factor-beta (TGF-beta 1) has led to the identification of more than two dozen structurally related peptides which appear of crucial importance in the regulation of cell proliferation, cell differentiation and embryogenesis. TGF-beta 1 and its close homologs (TGF-beta 2-5) are multifunctional peptides whose effects on cell functions are dependent upon the cell type, the environment and the presence of other growth factors. TGF-beta 1 is produced and secreted as a latent macromolecular complex. One of the major steps in the control of TGF-beta activity may thus be its release (activation) from its latent form upon the effect of local factors. Adrenocortical cells may be taken as an example in which autocrine production of TGF-beta may be a component of a negative regulatory loop in balance with the positive effect of a systemic hormone (ACTH) in controlling the expression of the cell steroidogenic differentiated functions. In this system, latent TGF-beta can be activated by an ACTH-induced secreted protein (CISP), a member of the thrombospondin family. This points to the importance of the functional interaction between TGF-beta s and extracellular matrix components in the local regulation of cell activities.

Adrenal Cortex↗

PubMed1995

Contribution of apoptosis to the phenotypic changes of adrenocortical cells in primary culture.

The aim of this study was to evaluate the occurrence and physiological consequences of apoptosis in primary cultures of bovine adrenocortical cells (of fasciculata-reticularis origin). Under ACTH-free culture conditions, we observed apoptotic cells in the cell layer and the accumulation of apoptotic bodies in the culture medium. These were hardly detectable in ACTH-supplemented cultures. Under ACTH-free conditions, the DNA content of apoptotic bodies collected over 48 h represented up to 10-15% of that of the cell layer at the onset of the culture (as compared to 3% in ACTH-supplemented cultures). Past the fourth day of culture in the absence of ACTh, most cells lacked several markers of their originating fasciculata-reticularis phenotype and progressively evolved to an undifferentiated phenotype. The vast majority of the apoptotic bodies released during the first 4 days of culture were immunoreactive for P450 17 alpha. Inversely, during the same period of time, the proliferating cells (PCNA-positive) did not appear to express P450 17 alpha. Therefore, apoptosis could contribute, together with dedifferentiation, to the phenotype shift observed in ACTH-depleted cultures of adrenal fasciculata-reticularis cells. These observations also characterize this endocrine cell system as an in vitro model for the study of hormone-repressed apoptosis.

Adrenal Cortex↗

PubMed1995

Modulation of hydroxylase and lyase activities of bovine cytochrome P-450(17) alpha in adrenal and testicular microsomes by a tissue-specific local membrane environment.

In steroidogenic tissues, cytochrome P-450(17) alpha catalyzes both steroid 17 alpha-hydroxylation and 17,20-lyase reactions. The ratio of the two activities, hydroxylase over lyase (H/L) depends upon the tissue of origin; this ratio is low in the testis whereas it is high in the adrenal cortex. To examine the factors responsible for this specific regulation, two approaches were followed: (i) the purified enzyme was incorporated into liposomes made of microsomal lipids of testis or adrenal cortex; and (ii) the effects of disorganization of the microsomal membrane on the activities were observed. The results show that the cytochrome 17,20-lyase activity is stimulated by the presence of lipids from testicular origin. In the adrenal microsomes, this activity appears to be dependent upon the local membrane organization. Specific component(s) associated with the neutral fraction of the microsome lipid extract may be responsible for the repression of lyase activity in the adrenal.

Adrenal Glands↗

PubMed1995

Organization of 3 beta-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria.

We have previously reported the co-localization [Cherradi et al., Endocrinology 134 (1994) 1358-1364] of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) and cytochrome P450scc (cyt. P450scc) in the inner membrane and in the intermembrane contact sites of adrenocortical mitochondria. This observation raises the question of a possible functional association between the two proteins. Isolated bovine adrenocortical mitochondria are able to convert cholesterol to progesterone without the need of exogenous cofactors. An association of 3 beta-HSD and cyt. P450scc is observed during the purification of 3 beta-HSD from mitochondria. The behaviour of 3 beta-HSD on a column of Heparin-Sepharose is modified by the presence of cyt. P450scc. Immunoprecipitations from mitochondria with either anti-cyt. P450scc or anti 3 beta-HSD antibodies result in a co-precipitation of the two proteins. Both proteins engaged in these immunocomplexes are catalytically active. The interaction was further demonstrated by the surface plasmon resonance method using purified components. An affinity demonstrated by the surface plasmon resonance method using purified components. An affinity constant of 0.12 microM between 3 beta-HSD and P450scc was obtained. These observations suggest that P450scc and 3 beta-HSD may associate into a molecular complex in the mitochondrial compartment and may constitute a functional steroidogenic unit, thus opening new possibilities in the regulation of the production of progesterone and its flow in the adrenocortical cell.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Metabolism of malate in bovine adrenocortical mitochondria studied by 13C-NMR spectroscopy.

13C-NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron-transport system responsible for steroid hydroxylation. [13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin. The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.

Adrenal Cortex↗

PubMed1994

Suramin: an inhibitor of the final steps of the mineralocorticoid pathway?

The authors used incubated adrenal mitochondria to study the in vitro effect of suramin, an antiparasitic drug, on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that, under conditions preserving membrane integrity, the "impermeance" of suramin meant that concentrations similar to the plasma-levels reached in treated patients induced only slight inhibition of the final intramitochondrial steps in aldosterone synthesis. However, suramin strongly inhibited mitochondrial respiration. The inhibition of two intramitochondrial mechanisms (respiration and steroid synthesis) suggests that the effect of suramin involves partial inhibition of metabolic intermediate carriers. The inhibition of the activity of various extramitochondrial enzymes involved in intermediate metabolism, suggests that the inhibition of steroid biosynthesis can be explained only on the basis of an extramitochondrial action of suramin. The action of suramin must, therefore, primarily and directly affect extramitochondrial steroid synthesis and only indirectly affect intramitochondrial steroid synthesis as a result of an impact on the reducing equivalent supply. However, even if suramin does not bind to cytochrome P450 11 beta which catalyzes the final steps of aldosterone biosynthesis pathway, this does not imply that suramin has no direct effect on steroid synthesis within the mitochondria, in addition to its toxic effects, particularly if the cell structure is disrupted (as is often the case in tumor tissues).

18-Hydroxycorticosterone↗

PubMed1994

Growth factors and pericellular components as a local signalling relay in the action of systemic hormones.

Recent years have shown that peptidic signals, originally identified as growth factors, may represent a local signalling system involved in the regulation of cell function under the dependence of systemic hormones. As an example, (i) adrenocortical cells are highly sensitive to transforming growth factor-beta (TGF beta) which is a powerful inhibitor of their differentiated functions; (ii) adrenocortical cells produce and secrete TGF beta, which is present in the adrenal cortex in situ; (iii) adrenocorticotropin (ACTH), which is the best known positive effector of adrenocortical functions induces an up-regulation of TGF beta receptors in adrenocortical cells. Altogether, these observations suggest that TGF beta may be a local signalling system acting in balance with the systemic hormone ACTH to eventually tune the ability of adrenocortical cells to produce corticosteroids.

Adrenal Cortex↗