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Biomedical subjects

F Pinet

Publications and source records attributed to F Pinet.

At least 19 recordsLinked to original sources
PubMed2000

Regulation of aminopeptidase A in human brain tumor vasculature: evidence for a role of transforming growth factor-beta.

Angiotensin peptides are potent vasoconstrictors, cell growth factors, and neuromodulators in normal and pathological situations. To assess the potential role of the angiotensins in brain tumor-associated vessels, the expression of the enzymes of the angiotensin cascade were evaluated in these tumors. The production of these bioactive peptides is dependent on the activities of exopeptidases, including several aminopeptidases and carboxypeptidases, producing angiotensin (Ang) I, II, III, IV and Ang 1-7. Human cerebral parenchymal and glioblastoma cells expressed renin, and tumor vasculature, but not glioblastoma cells, expressed angiotensin-converting enzyme. High aminopeptidase A (APA) activity, but no aminopeptidase N/B activity, was observed in human brain tumor vasculature, suggesting a predominant production of Ang III. Grafting of rat glioma cells in rat brains yielded tumors with high APA and low aminopeptidase N/B activities in tumor vessels, confirming human results. Tumor growth and APA activity in tumor vessels were not affected by chronic angiotensin-converting enzyme inhibition. The brain-derived EC219 endothelial cells expressed high APA activity, which was not involved in endothelial cell proliferation, but was down-regulated by exposure of cells to transforming growth factor-beta (TGF beta) or to TGF beta-secreting tumor cells, suggesting a role for this peptide in the control of APA activity in cerebral vasculature. Thus, APA is a potential marker of chronic dysfunction, involving loss of TGF beta function, of the metabolic blood-brain barrier, but not of neovascularization.

Aminopeptidases↗
PubMed2000

The endothelin system in normal human colon.

Endothelin (ET)-1 is a potent vasoconstrictor and mitogenic peptide that has a variety of biological effects in noncardiovascular tissues. The precise cellular distribution of the ET-1 system in the wall of the normal human colon was studied to identify the physiological role of ET in the gut. In situ hybridization revealed ET-converting enzyme-1 (ECE-1) mRNA in all vessels, the colon epithelium, and macrophages. Prepro-ET-1 (PPET-1) mRNA had a similar distribution except for a scattered signal in mucosal microvessels. ET(A) and ET(B) receptor mRNAs were mainly in the lamina propria, pericryptal myofibroblasts, microvessels, and mononuclear cells, with ET(A) mRNA more abundant than ET(B) mRNA. (125)I-ET-1 binding showed ET(B) along the crypts and in nerve fibers descending from the ganglionic plexus that contained PPET-1, ECE-1, and ET(B) transcripts, whereas glia contained ET(A) receptors. The finding of the entire ET system in the normal mucosa suggests its implication in some characteristic functions of the colon and its secretion as both a neuroactive and a vasoactive peptide.

Adult↗
PubMed1999

Dissection of silencer elements in first intron controlling the human renin gene.

OBJECTIVE: A silencer within the renin first intron (intron A) was identified using Calu-6 cells, a pulmonary carcinoma cell line which produced renin. In the present study, a dissection of the first intron was performed to determine precisely the cis-regulatory elements involved in the silencer transcriptional effects. MATERIALS AND METHODS: Intron A was completely sequenced to characterize potential binding sites for known transcription factors. Partial portions of intron A were subcloned upstream the 892 bp of the renin promoter and transfected in different models of renin-producing cells: primary culture of human chorionic cells, human Calu-6 cells and mouse As4.1 cells. RESULTS: There is significant DNA homology (67%) between the 3' and 5' ends of the human and rat renin first intron. Several transcription factor binding sites identified in human first intron, but not in rat intron, do not contribute to the reported silencer activity. Transfections of renin/ luciferase constructs containing partial portions of first intron inserted upstream of the 892 bp in both renin-producing cells do not allow the precise characterization of cis-elements involved in the silencer effect. CONCLUSIONS: The silencer located renin intron A is cell specific. The integrity of the human first intron seems necessary for its repressor activity on renin proximal promoter in renin-producing cells.

Animals↗
PubMed1999

Cellular distribution of endothelin-converting enzyme-1 in human tissues.

Endothelin-converting enzyme-1 (ECE-1) is the key enzyme of endothelin biosynthesis, catalyzing the final processing step. As shown by the targeted disruption of the ECE-1 gene, mature endothelins must be produced at specific sites for normal embryonic development. Therefore, it is important to know the exact pattern of ECE-1 gene expression. In this study we investigated the cellular distribution of ECE-1 in a variety of human tissues by in situ hybridization and immunohistochemistry. Widespread expression of the ECE-1 gene was noted, with a similar distribution pattern for mRNA and protein in normal human tissues, suggesting a major biological role for ECE-1. ECE-1 levels were particularly high in the cardiovascular, reproductive, and endocrine systems. There was strong and consistent labeling for ECE-1 in the vascular endothelial cells of all organs examined and in various nonvascular cells, especially some glandular cells. A large amount of ECE-1 protein and mRNA was detected in the Leydig cells of the testis and in the granulosa and theca cells of the ovary. In the adrenal gland, ECE-1 was detected in the cortex and medulla, with the strongest labeling in the zona glomerulosa. Therefore, ECE-1 may be involved in other systems, such as the regulation of hormone secretion, rather than exclusively generating ET-1 from its precursor. These results point out the potential side effects of ECE-1 inhibitors that are currently under development for treatment of cardiovascular diseases. (J Histochem Cytochem 47:447-461, 1999)

Adolescent↗
PubMed1999

Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms.

Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting endothelins. Three ECE-1 isoforms, differing by their N-terminal cytoplasmic tails, are generated from a single gene. When expressed in CHO cells, they display comparable enzymatic activity but whereas ECE-1a is strongly expressed at the cell surface, ECE-1b is exclusively intracellular and ECE-1c presents an intermediate distribution. In the present study these different localizations were further described at the ultrastructural level, by electron microscope immunocytochemistry. To characterize the motifs responsible for the intracellular localization of ECE-1b we constructed chimeric proteins and point mutants. Two di-leucine-based motifs, contained in the N-terminal part of ECE-1b, were thus identified. One of these motifs (LV), displayed by both ECE-1b and ECE-1c, accounts for the reduced surface expression of ECE-1c as compared to ECE-1a. Mutation of both motifs (LL and LV) induces a very strong appearance of ECE-1b at the cell surface indicating that their presence in the N-terminal extremity of ECE-1b is critical for its exclusively intracellular localization.

Amino Acid Motifs↗
PubMed1998

A novel distal enhancer confers chorionic expression on the human renin gene.

Renin catalyzes the rate-limiting step of the renin-angiotensin system, which regulates blood pressure and electrolyte homeostasis. To determine cell-specific human renin gene control elements, the transcriptional activity of promoter regions up to position -8876 was studied in renin-expressing cells. A positive regulatory region conferring approximately 57-fold higher transcriptional activity to the human renin gene promoter in chorionic cells was identified between nucleotides -5777 and -5552. It had the orientation-independent activity typical of classical enhancers. It also conferred approximately 59-fold higher transcriptional levels from the heterologous simian virus 40 (SV40) promoter in chorionic cells and approximately 6-fold higher transcriptional levels in Calu-6 and As4.1 cells, whereas no effect was measured in non-renin-expressing cells. DNase I footprinting showed that this enhancer contains three binding sites for chorionic cell nuclear extracts. Functional analysis suggested that the activity of the enhancer is regulated by differential mechanisms in the three renin-expressing cells involving a complex arrangement of AP-1 motifs binding cell-specific members of the basic leucine zipper family of transcription factors. Thus, our results demonstrate that this enhancer plays a key role in the expression of the human renin gene in the chorion and may also be involved in its regulated expression in other tissues.

Animals↗
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

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

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

A live-cell assay for studying extracellular and intracellular endothelin-converting enzyme activity.

Endothelin-1 (ET-1) is formed from its precursor preproET-1 via the cleavage of the intermediate bigET-1 by endothelin-converting enzyme (ECE-1). However, the subcellular site at which this step occurs is not clear: It could occur intravesicularly along the secretory pathway or bigET-1 might be released and processed extracellularly. To address this point, we have developed an integrated autocrine system that uses a recombinant Chinese hamster ovary (CHO) luciferase reporter cell line that permanently expresses the human ET(A) receptor. Into these cells we transiently transfected human ECE-1a cDNA, either together with the human preproET-1 cDNA (as an endogenous source of bigET-1), or alone (in which case exogenous bigET-1 was added). Phosphoramidon inhibited the conversion of exogenous bigET-1 (IC50 = 5 to 30 micromol/L) much better than that of endogenous bigET-1 (IC50 > 1 mmol/L). Both conversions showed similar high yields (20% to 100%) that depended on the amount of ECE-1a expressed. Thus, ECE-1a has two equally relevant activities in this recombinant system for CHO cells: (1) an intracellular, probably intravesicular activity, corresponding to the ECE-1a-mediated step of ET-1 biosynthesis and (2) an extracellular activity at the plasma membrane. If this is also the case for endothelial cells, ECE-1a inhibitors would have to cross the plasma and vesicle membranes to be effective. The present system could be useful for screening such inhibitors.

Animals↗
PubMed1996

Transcriptional induction of the human renin gene by cyclic AMP requires cyclic AMP response element-binding protein (CREB) and a factor binding a pituitary-specific trans-acting factor (Pit-1) motif.

To delineate the cis-acting elements of the proximal promoter responsible for cyclic AMP (cAMP)-induced human renin gene transcription, 5'-flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected in chorionic cells. Forskolin treatment induced the expression of luciferase by 2.4-fold when the reporter plasmid contained the promoter region (-582 to + 16). Mutation or deletion of the cAMP response element (CRE) diminished (1.7-fold) but did not abolish cAMP-induced transcription, demonstrating that the (-582 to -145) region containing the CRE and the region (-145 to -38) containing a Pit-1 (pituitary-specific trans-acting factor) site were both necessary for cAMP maximal induction. To study the molecular events mediating the cAMP induction, DNase I footprinting and electromobility shift assays (EMSAs) were performed with renin-producing chorionic cell and kidney cortex cell nuclear extracts, showing that the CRE-binding protein (CREB) interacts with the CRE and that tissue-specific factors, distinct from Pit-1, specifically bind the renin Pit-1 motif. Taken together, these results demonstrate that the cAMP response of the human renin gene may involve CREB binding the CRE and tissue-specific factors, different from Pit-1, that interact with the Pit-1 response DNA elements.

Base Sequence↗
PubMed1996

Evidence that renal and chorionic tissues contain similar nuclear binding proteins that recognize the human renin promoter.

This study examines whether the human renal cortex, the major renin producing site, contains nuclear factors that bind to the human renin proximal promoter. Footprint analysis of the human renin promoter region showed that human renal cortex cell nuclear extracts interacted with 6 putative cis-elements (the Ets domain-protein, a Pit-1 like binding site, a CRE sequence, an ARP-1 like binding site, an AGE3 like region, and a unknown consensus region, designated element C). Transient DNA transfection studies on chorionic cells implicated the CRE and Pit-1 consensus sites in the regulation of renin gene transcription by cAMP. Electromobility shift assays showed that renal proteins bind specifically to these sequences, and that one of them is CREB; two others seem to be Ets-1 and ARP-1. These results raise the possibility that the human renal cortex and human chorionic cells have the same trans-acting factors that bind to the proximal human renin promoter.

Base Sequence↗
PubMed1996

Regulation of renin release is impaired after nitric oxide inhibition.

The aim of the present study was dual: first to establish that a preparation of afferent arterioles freshly isolated from the rat kidney is a suitable model to study renin release and synthesis, and second to investigate the effect(s) of nitric oxide (NO) inhibition on renin release in this model. Purification of renal microvessels was based on iron oxide infusion into the kidneys and separation of the afferent arterioles from glomeruli and connective tissue with a magnet. These microvessels express preprorenin mRNA, contain renin granules and release renin as evidenced by RT-PCR, immunocytochemistry and measurement of renin activity, respectively. Renin secretion was increased in isolated afferent arterioles after in vivo treatment with the diuretic furosemide (+300%) or in vitro treatment with the adenylyl cyclase activator forskolin (+50%), indicating that this vascular preparation responds appropriately to regulators of the renin-angiotensin system. Furthermore, in afferent arterioles isolated from control rats, renin release was positively correlated with total renin content (r = 0.85). In afferent arterioles isolated from rats chronically treated with the NO-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), forskolin was ineffective in modifying renin release despite stimulation of cAMP levels. In addition, the correlation between renin release and tissue renin content was disrupted. Similar results were obtained when cortical slices were used instead of afferent arterioles, suggesting that this defect in the regulation of renin release is independent of the presence of macula densa cells. To verify that the lack of regulation of renin release after L-NAME treatment was due to NO inhibition, the NO donor 3-morpholino-syndonimin-hydrochloride (SIN-1) was administered in afferent arterioles or cortical slices from kidneys of L-NAME-treated rats. In both preparations, SIN-1 reversed the L-NAME effect and re-established the responsiveness of renin release to forskolin and the relationship between renin release and renin content. These data indicate that the adenylyl cyclase-mediated mechanism regulating renin release is impaired when NO synthesis is inhibited.

Animals↗
PubMed1995

Renin gene expression in the aging kidney: effect of sodium restriction.

The activity of the renin-angiotensin system as well as the ability of the kidney to retain sodium following salt restriction are reduced with age. The relationship between these age-related changes in renal function and the renin gene expression was presently investigated. The concentrations of renin and its mRNA were measured in kidney of 10- and 30-month-old control female WAG/Rij rats and of animals which were salt restricted for 4 days. In the senescent rats, the kidney renin concentration, like the plasma concentration of angiotensin II, was half that in adult rats. The intrarenal content of renin mRNA did not differ between 10- and 30-month-old animals, suggesting that the transcriptional rate of the renin gene is unchanged with age. During the early phase of adaptation to sodium depletion, the systemic angiotensin II concentration was not modified in either age groups. Four-days salt restriction did not significantly change the renal storage of renin. In contrast, this short term salt restriction induced a 2.3-fold increase in the renin mRNA in adult kidney, and a 1.9-fold increase in the senescent kidney. These data suggest that the age-related decrease in renal concentration of renin is linked to a modification in the rate of translation of renin mRNA, or to an alteration in the protein maturation. The difference in adaptation to the early phase of salt restriction with age should not be linked to changes in renin gene transcription, but more likely to a change in the tissue response to the local renin-angiotensin system.

Adaptation, Physiological↗
PubMed1995

Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells.

A quantitative reverse transcriptase-polymerase chain reaction for mouse renin mRNA was utilized to study the influence of classic second messenger molecules on renin mRNA levels in primary cultures of juxtaglomerular (JG) cells isolated from the kidneys of C57/B16 mice. We found that forskolin (3 microM), an activator of adenylate cyclase led to proportional increases of renin secretion and renin mRNA levels. The nitric oxide (NO) donor, sodium nitroprusside (100 microM), stimulated both renin secretion and renin gene expression, the effect on secretion being stronger than that on renin mRNA levels. An increase of the extracellular concentration of calcium from 0.5 to 3 mM led to a transient inhibition of renin secretion, followed by a marked stimulation of secretion and to a continuous suppression of renin mRNA levels. These were also decreased by the calcium ionophore A 23187 (1 microM). The membrane permeable 8-bromo-cyclic GMP (100 microM) inhibited basal renin secretion without an effect on renin mRNA levels. The phorbol ester phorbol-12-myristate-13-acetate (1 to 100 nM), which was used to stimulate protein kinase C activity, had no significant effects on renin secretion and renin mRNA levels, neither alone nor in combination with forskolin. These findings suggest that cAMP, NO and calcium are effective regulators of renin gene expression in renal JG cells, in a way that cAMP and NO are stimulators and calcium acts as an inhibitor. Moreover, in these acute experiments there appears to be no obligatory link between the secretion and the expression of renin, suggesting that both parameters are separately regulated.

Animals↗
PubMed1994

A mutant renin gene in familial elevation of prorenin.

A case of familial elevation of plasma prorenin levels was discovered during an epidemiological survey of a Dutch population. Trypsin-activated prorenin was elevated in the 58-year-old father, his son, and one of his sisters. All family members were normotensive and had normal plasma renin activities. Exon sequencing of the renin gene of the proband and of his son after polymerase chain reaction amplification identified a point mutation in the last exon of the gene (exon 10). A cytosine to thymine transition creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus. All family members presenting high levels of plasma prorenin were heterozygous for the mutation. Expression vectors carrying normal or mutated renin cDNA were transiently transfected into AtT-20 cells to test in vitro the functional consequences of this mutation. Measurements of renin activity and pulse-chase experiments indicated that the truncated renin is inactive and not secreted from transfected cells. We hypothesize that the abnormal gene product of the mutated allele alters renin sorting and propose that plasma prorenin elevation may result from a compensatory mechanism.

Animals↗