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P Corvol

Publications and source records attributed to P Corvol.

At least 91 records · Page 5Linked to original sources
PubMed1998

Structural analysis and evaluation of the aldosterone synthase gene in hypertension.

Anomalies in either of the tightly linked genes encoding the enzymes CYP11B1 (11beta-hydroxylase) or CYP11B2 (aldosterone synthase) can lead to important changes in arterial pressure and are responsible for several monogenically inherited forms of hypertension. Mutations in these genes or their regulatory regions could thus contribute to genetic variation in susceptibility to essential hypertension. To test this hypothesis, we performed 2 complementary studies of the CYP11B1/CYP11B2 locus in essential hypertension. After characterizing a DNA contig containing the CYP11B1 gene and mapping the gene in the Centre d'Etudes du Polymorphisme Humain reference panel of families, we performed a linkage study with 292 hypertensive sibling pairs and a highly informative microsatellite marker near CYP11B1. We also analyzed the association of 2 frequent biallelic polymorphisms of the CYP11B2 gene, 1 in the promoter at position -344 (-344C/T) and the other, a common gene conversion in intron 2, with hypertension in 380 hypertensive patients and 293 normotensive individuals. Statistical analyses did not show significant linkage of the CYP11B1 microsatellite marker to hypertension. No positive association with hypertension was found with the gene conversion in intron 2, but a positive association with hypertension was found with the -344T allele. The hypertensive and normotensive samples differed significantly in both genotype (P=0.023) and allele frequencies (P=0.010). Our data suggest a modest contribution of the CYP11B2 gene to essential hypertension.

Adult↗
PubMed1998

Angiotensinogen, prorenin, and renin are Co-localized in the secretory granules of all glandular cells of the rat anterior pituitary: an immunoultrastructural study.

In addition to the circulating renin-angiotensin system (RAS), a local system has been postulated in the anterior pituitary because immunodetection of its components in various mammalian species. However, different cell types appear to be involved in different species, and there is no general consensus on the subcellular localization of prorenin, renin and angiotensinogen. In this ultrastructural study, we investigated and quantified the presence of these components using double or triple immunogold labeling methods, in all the immunologically identified glandular cell types of the rat anterior pituitary. In contrast to previous reports, all these components were identified not only in lactotropes and gonadotropes but also in somatotropes, corticotropes, and thyrotropes. The highest levels were detected in lactotropes and gonadotropes, and renin gave the greatest signal. Angiotensinogen, prorenin, and renin were co-localized in the secretory granules of all rat pituitary glandular cell types. The simultaneous detection of the substrate (angiotensinogen) and both its specific cleavage enzyme and its proenzyme within the same granule suggests intragranular processing of this component. Moreover, the localization of these three constituents in the secretory granules also suggests that, in the rat anterior pituitary, they follow the regulated secretory pathway.

Adrenocorticotropic Hormone↗
PubMed1998

Recombinant CYP11B genes encode enzymes that can catalyze conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol, and 18-oxocortisol.

CYP11B1 (11beta-hydroxylase) and CYP11B2 (aldosterone synthase) are 93% identical mitochondrial enzymes that both catalyze 11beta-hydroxylation of steroid hormones. CYP11B2 has the additional 18-hydroxylase and 18-oxidase activities required for conversion of 11-deoxycorticosterone to aldosterone. These two additional C18 conversions can be catalyzed by CYP11B1 if serine-288 and valine-320 are replaced by the corresponding CYP11B2 residues, glycine and alanine. Here we show that such a hybrid enzyme also catalyzes conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol, and 18-oxocortisol. These latter two steroids are present at elevated levels in individuals with glucocorticoid suppressible hyperaldosteronism (GSH) and some forms of primary aldosteronism. Their production by the recombinant CYP11B enzyme is enhanced by substitution of further amino acids encoded in exons 4, 5, and 6 of CYP11B2. A converted CYP11B1 gene, containing these exons from CYP11B2, would be regulated like CYP11B1, yet encode an enzyme with the activities of CYP11B2, thus causing GSH or essential hypertension. In a sample of 103 low renin hypertensive patients, 218 patients with primary aldosteronism, and 90 normotensive individuals, we found a high level of conversion of CYP11B genes and four cases of GSH caused by unequal crossing over but no gene conversions of the type expected to cause GSH.

Case-Control Studies↗
PubMed1998

Pharmacological inactivation of the endothelin type A receptor in the early chick embryo: a model of mispatterning of the branchial arch derivatives.

In the present study, we have applied an antagonist treatment to the chick embryo in ovo in order to demonstrate and dissect the essential roles of the endothelin type A (ETA) receptor in the embryonic development. We have cloned, sequenced and expressed the cDNA of the chick ETA receptor and shown that its affinity for endothelin antagonists is very similar to that shown by its mammalian counterparts. We have studied the spatio-temporal expression pattern of this receptor by in situ hybridization and shown that there is a high level of its mRNA within the mesenchyme of the branchial arches at E3-E5, in keeping with the direct effect of endothelin-1 (ET-1) on the fate of this region of the embryo. Unlike the endothelin type B (ETB) receptor mRNA, ETA mRNA is not expressed in neural crest cells during emigration from the neural tube, but is detected in neural crest-derived ectomesenchyme of the branchial arches. Finally, the functional involvement of this receptor in craniofacial and cardiovascular organogenesis was assessed by selectively inactivating the ETA receptor with specific antagonists applied during the time period corresponding to the expression of the ETA receptor and colonisation of the branchial arches. Embryos treated by these antagonists show a severe reduction and dysmorphogenesis of the hypobranchial skeleton, as well as heart and aortic arch derivative defects. This phenotype is very similar to that obtained in mice by gene inactivations of ET-1 and ETA. These results are observed with ETA antagonists but not with an ETB antagonist, and are dependent on the dose of the antagonists used and on the time of application to the embryo. Altogether, these data strongly show that the ET-1/ETA pathway, in chicken as in mammals, is a major factor involved directly and functionally in morphogenesis of the face and heart. This experimental model of pharmacological inactivation of a gene product described in this study offers a simple and rapid alternative to gene inactivation in mouse. This strategy can be applied to other ligand-receptor systems and extended to compounds of various chemical and functional natures.

Amino Acid Sequence↗
PubMed1998

[Genetic study of renal artery fibromuscular dysplasia].

The aim of this study was to conduct a formal pedigree analysis of the involvement of the elastin gene in families. From 140 subjects with renal FMD documented on angiography, family cases with documented renal artery fibromuscular dysplasia (FMD) and to test pedigrees were constructed and familial cases defined by angiographic evidence of FMD in at least one sibling. Familial screening was made either by echodoppler for asymptomatic subjects or by digital intravenous angiography for hypertensive subjects. Linkage analysis at the elastin gene locus was performed in these families with two polymorphic markers: one diallelic RFLP located in exon 16 and one multiallelic CA repeat located in intron 17 of the elastin gene. Fourteen pedigrees (10%) were obtained including nine sibling pairs, four trios and one vertical transmission from a father to his daughter. Most affected subjects were females (84%) but familial cases were more frequently bilateral than sporadic cases (80% vs 49%, p = 0.07). Pedigrees analysis was compatible with an autosomal dominant mode of inheritance and suggested in these families an age and sex-dependent incomplete penetrance model. Linkage analysis resulted in a maximum two-point lod score of 0.06 at theta = 0.20 using the dinucleotide CA repeat. Analysis of the diallelic marker revealed similar frequencies in affected and non affected subjects. This study highlights the role of genetics factors in approximately 10% of FMD cases. The elastin gene does not seem to be involved in the pathogenesis of FMD.

Adult↗
PubMed1997

Cleavage of arginyl-arginine and lysyl-arginine from the C-terminus of pro-hormone peptides by human germinal angiotensin I-converting enzyme (ACE) and the C-domain of human somatic ACE.

Mammalian germinal angiotensin I-converting enzyme (gACE) is a single-domain dipeptidyl carboxypeptidase found exclusively in male germ cells, which has almost identical sequence and enzymic properties with the C-domain of the two-domain somatic ACE. Mutant mice that do not express gACE are infertile, suggesting a role for the enzyme in the processing of undefined peptides involved in fertilization. A number of spermatid peptides [e.g. cholecystokinin (CCK) and gastrin] are processed from pro-hormones by endo- and exo-proteolytic cleavages which might generate substrates for gACE. We have shown that peptide hormone intermediates with Lys/Arg-Arg at the C-terminus are high-affinity substrates for human gACE. gACE from human sperm cleaved Arg-Arg from the C-terminus of the CCK5-GRR (GWMDFGRR), a peptide corresponding to the C-terminus of a CCK-gastrin prohormone intermediate. Hydrolysis of CCK5-GRR by recombinant human C-domain ACE was Cl- dependent, with maximal activity achieved in 5-10 mM NaCl at pH 6.4. C-Domain ACE cleaved Lys/Arg-Arg from the C-terminus of dynorphin-(1-7), a pro-TRH peptide KRQHPGKR, and two insect peptides FSPRLGKR and FSPRLGRR. C-Domain ACE displayed high affinity towards all these substrates with Vmax/Km values between 14 and 113 times greater than the Vmax/Km for the conversion of the best known ACE substrate, angiotensin I, into angiotensin II. In conclusion, we have identified a new class of substrates for human gACE, and we suggest that gACE might be an alternative to carboxypeptidase E for the trimming of basic dipeptides from the C-terminus of intermediates generated from pro-hormones by subtilisin-like convertases in human male germ cells.

Chlorides↗
PubMed1997

Construction, expression and characterization of a soluble form of human endothelin-converting-enzyme-1.

Endothelin-converting-enzyme-1 (ECE-1) belongs to the family of zinc metallopeptidases and is responsible for generating endothelin (ET) peptides from their inactive precursors the big endothelins (bigET). The enzyme is a type II integral membrane protein consisting of a short amino-terminal cytosolic domain of 56 amino acids, a single transmembrane domain and a large putative extracellular domain containing the catalytic site. Recombinant and native ECE-1 are expressed as a dimer. We have constructed a soluble form of ECE, named sECE*, by fusing the cleavable signal peptide of pro-opiomelanocortin in frame to the complete extracellular domain of human ECE-1. Stable expression of this construct in CHO cells resulted in the secretion of a fully active enzyme. In contrast to membrane-bound ECE, sECE* was expressed as a monomer, highly glycosylated, as assessed by gel filtration and Western blot. However, recombinant sECE* converted bigET-1 with similar specific activity as ECE-1a. This activity was completely inhibited by phosphoramidon, but not by thiorphan and captopril. sECE* was active in a broad range of pH, showing an optimum of 6.6-6.8 for bigET-1. Thus, the extracellular domain alone is sufficient for conferring full ECE-1 activity, inhibitors recognition and substrate specificity.

Animals↗
PubMed1997

A tyrosine residue essential for catalytic activity in aminopeptidase A.

Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.

Amino Acid Substitution↗
PubMed1997

The C-terminal third intracellular loop of the rat AT1A angiotensin receptor plays a key role in G protein coupling specificity and transduction of the mitogenic signal.

To identify the role(s) of the third intracellular loop of the angiotensin II (AngII) type 1A (AT1A) receptor in G protein coupling specificity and receptor activation, several chimerae were constructed and characterized. The cDNA sequence encoding the C-terminal segment of the third intracellular loop of the AT1A receptor (residues 234-240) was replaced with the homologous regions of the alpha1B adrenergic (alpha1B-AR), the beta2 adrenergic (beta2-AR), and the AngII type 2 (AT2) receptors. These chimeric receptors were stably expressed in Chinese hamster ovary cells, and their pharmacological and functional properties were characterized, including AngII-induced inositol phosphate and cyclic AMP (cAMP) productions, [3H]thymidine incorporation into DNA, and internalization. The affinities of these chimeric receptors for [Sar1]AngII, [Sar1,Ile8]AngII, and losartan were essentially normal; however, the affinity of these mutants was increased by a factor of 10-40 for the AT2-specific ligand CGP42112A. The functional properties of the alpha1B-AR chimera were essentially identical to those of the wild type AT1A receptor. On the other hand, replacement with the beta2-AR segment produced a partial reduction of the inositol phosphate production, a measurable AngII-induced cAMP accumulation, a reduced internalization, and a total impairment to transduce the mitogenic effect of AngII. The AT2 chimera presented a normal internalization, but was inactive in all the other functional tests. In conclusion, the distal segment of the third intracellular loop of the rat AT1A receptor plays a pivotal role in coupling selectivity and receptor signaling via G protein(s) as well as in the activation of the specific signaling pathways involved in the mitogenic actions of AngII.

Amino Acid Sequence↗
PubMed1997

The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice.

Angiotensin-converting enzyme (ACE) generates the vasoconstrictor angiotensin II, which plays a critical role in maintenance of blood pressure in mammals. Although significant ACE activity is found in plasma, the majority of the enzyme is bound to tissues such as the vascular endothelium. We used targeted homologous recombination to create mice expressing a form of ACE that lacks the COOH-terminal half of the molecule. This modified ACE protein is catalytically active but entirely secreted from cells. Mice that express only this modified ACE have significant plasma ACE activity but no tissue-bound enzyme. These animals have low blood pressure, renal vascular thickening, and a urine concentrating defect. The phenotype is very similar to that of completely ACE-deficient mice previously reported, except that the renal pathology is less severe. These studies strongly support the concept that the tissue-bound ACE is essential to the control of blood pressure and the structure and function of the kidney.

Alleles↗
PubMed1997

Regulation of human renin secretion and gene transcription in Calu-6 cells.

Calu-6 cells were characterized for studying the transcriptional regulation of the human renin gene. Analysis of cis-acting elements of the renin promoter showed the highest activity within the first 582 bp in serum-free conditions and of the 892 bp in the presence of serum. cAMP activates renin mRNA synthesis parallel to renin production (20-fold increase) as well renin promoter activity (2-fold). cAMP response element and the (-77 to -67) element are both necessary for activation of the renin promoter but do not act independently. Functional analysis of Intron A revealed the presence of a silencer specific to renin-producing cells.

Animals↗
PubMed1997

Cleavage-secretion of angiotensin I-converting enzyme in yeast.

Angiotensin I-converting enzyme (ACE) is a type I transmembrane protein composed of two domains (N and C domains) which undergoes a post-translational proteolytic cleavage in mammalian cells to release the soluble ectodomain. The protease involved in ACE cleavage-secretion (ACE-secretase) is not well characterised and eludes isolation: the presence of a yeast homologue, thus more amenable to genetic manipulation, would facilitate its identification. We have expressed a secreted form of the ACE C domain, lacking the C-terminal membrane anchor (C domain(deltaCOOH)), and the membrane-anchored C domain (C domain) in the yeast Pichia pastoris by fusion to prepro-alpha-factor. Immunofluorescent labelling localises the ACE C domain to the periphery of yeast cells but not C domain(deltaCOOH), however, expression of both C domain and C domain(deltaCOOH) produced soluble enzymes in the culture medium. Immunocharacterisation of the two soluble forms of the C domain indicates a proteolytic cleavage of the membrane-bound C domain to produce the soluble counterpart. Thus ACE undergoes a proteolytic cleavage in yeast.

Amino Acid Sequence↗
PubMed1997

A sensitive reverse transcriptase polymerase chain reaction assay for measuring the effects of dehydration and gestation on rat amounts of vasopressin and ocytocin mRNAs.

This study describes a competitive reverse transcriptase polymerase chain reaction (RT-PCR) method for assaying the amounts of vasopressin (AVP) and ocytocin (OT) mRNAs in the rat hypothalamus and uterus. Despite the low concentrations of these mRNAs, the RT-PCR method readily measured both AVP and OT mRNAs in the same sample. A common internal standard for both reactions was designed to quantify the reaction. Both AVP and OT mRNAs were readily quantified in a 75 ng sample of total RNA from the hypothalamus. Water deprivation stimulated AVP mRNA production 3-fold and OT mRNA production 1.7-fold in the hypothalamus. Gestation only influenced the amount of OT mRNA in the hypothalamus (3-fold increase) and uterus (38-fold increase). The amount of AVP mRNA in the hypothalamus remained unchanged and no AVP mRNA was detected in the uteri of either non-pregnant or pregnant rats. This competitive RT-PCR is a powerful tool that provides rapid and precise assays of AVP and OT mRNAs.

Animals↗
PubMed1997

Expression of angiotensin type-1 (AT1) and type-2 (AT2) receptor mRNAs in the adult rat brain: a functional neuroanatomical review.

The discovery that all components of the renin-angiotensin system (RAS) are present in the central nervous system led investigators to postulate the existence of a local brain RAS. Supporting this, angiotensin immunoreactive neurons have been visualized in the brain. Two major pathways were described: a forebrain pathway which connects circumventricular organs to the median preoptic nucleus, paraventricular nucleus, and supraoptic nucleus, and a second pathway connecting the hypothalamus to the medulla oblongata. Blood-brain barrier deficient circumventricular organs are rich in angiotensin II receptors. By activating these receptors, circulating angiotensin II may act on central cardiovascular centers via angiotensinergic neurons, providing a link between peripheral and central angiotensin II systems. Among the effector peptides of the brain RAS, angiotensin II and angiotensin III have the same affinity for the two pharmacologically well-defined receptors: type 1 (AT1) and type 2 (AT2). When injected in the brain, these peptides increase blood pressure, water intake, and anterior and posterior pituitary hormone release and may modify memory and learning. The cloning of AT1 and AT2 receptor cDNAs has revealed that these receptors belong to the seven transmembrane domain receptor family. In rodents, two AT1 receptor subtypes, AT1A and AT1B, have been isolated. Using specific riboprobes for in situ hybridization histochemistry, recent studies mapped the distribution of AT1A, AT1B, and AT2 receptor mRNAs in the adult rat and found a predominant expression of AT1A and AT2 mRNA in the brain and of AT1B in the pituitary. Very limited overlap was found between the brain expression of AT1A and AT2 mRNAs. In several functional entities of the brain, such as the preoptic region, the hypothalamus, the olivocerebellary system, and the brainstem baroreflex arc, the colocalization of receptor mRNA, binding sites, and angiotensin immunoreactive nerve terminals suggests local synthesis and expression of angiotensin II receptors. In other areas, such as the bed nucleus of the stria terminalis, the median eminence, or certain parts of the nucleus of the solitary tract, angiotensin II receptors are likely of extrinsic origin. The neuronal expression of AT1A and AT2 receptors was demonstrated in the subfornical organ, the hypothalamus, and the lateral septum. By using double label in situ hybridization, AT1A receptor expression was localized in corticotropin releasing hormone but not in vasopressin containing neurons in the hypothalamus. The information is discussed together with functional data concerning the role of brain angiotensins, in an attempt to provide a better understanding of the physiological and functional roles of each receptor subtype.

Angiotensin I↗
PubMed1997

Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats.

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.

Aminopeptidases↗
PubMed1997

Haplotypes of angiotensinogen in essential hypertension.

The M235T polymorphism of the angiotensinogen gene (AGT) has been associated with essential and pregnancy-induced hypertension. Generation of haplotypes can help to resolve whether the T235 allele itself predisposes to the development of hypertension or acts as a marker of an unknown causal molecular variant. We identified 10 diallelic polymorphisms at the AGT locus and genotyped both a series of 477 probands of hypertensive families and 364 controls, all French Caucasians, as well as a series of 92 hypertensives and 122 controls from Japan. Despite a large ethnic difference in gene frequency, a significant association of T235 with hypertension was observed both in Cancasians (.46 vs. .38, P = .004) and in Japanese (.91 vs. .76, P = .002). In both groups, the G-->A substitution located at position -6 upstream of the initial transcription site occurred at the same frequency and in complete linkage disequilibrium with the T235 allele. No other polymorphism was found to be consistently associated with hypertension. Five informative haplotypes subdividing the T235 allele were generated. Whereas two of them were associated with hypertension in Caucasians, none of these two haplotypes (H3 and H4) reached statistical significance in Japanese. The analysis of the AGT-GT repeat revealed marked linkage disequilibriums between each of the diallelic polymorphisms and some (GT)n alleles, with similar patterns in the two populations. The strong disequilibrium between M235 and (GT)16 explained the increased frequency of that particular allele in French controls compared with hypertensives (.42 vs. .36, P < .01). The haplotype combining the M235T and G-6A polymorphisms appears as the ancestral allele of the human AGT gene and as the one associated with hypertension.

Age of Onset↗