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C Frelin

Publications and source records attributed to C Frelin.

At least 37 records · Page 2Linked to original sources
PubMed1998

Heart and lung VEGF mRNA expression in rats with monocrotaline- or hypoxia-induced pulmonary hypertension.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is upregulated during exposure to hypoxia. In this study, we analyzed heart and lung VEGF mRNA expression and examined pulmonary vascular remodeling as well as myocardial capillary density in two rat models of pulmonary hypertension involving exposure to chronic hypoxia (CH) and treatment with monocrotaline (MCT), respectively. The rats were studied after 0.5, 1, 3, 15, and 30 days of exposure to 10% O2 or 1, 6, and 30 days after a subcutaneous MCT injection (60 mg/kg). Both CH and MCT induced pulmonary hypertension and hypertrophy of the right ventricle (RV) with increased RV weight and atrial natriuretic peptide mRNA expression. VEGF mRNA expression as assessed by Northern blot analysis was potently induced after 12 h of hypoxia in both the right and left ventricles. After prolonged exposure to hypoxia, VEGF mRNA returned to baseline in the left ventricle (LV) but remained increased in the RV, where it peaked after 30 days. In MCT rats, VEGF mRNA was unchanged in the LV but decreased by 50% in the RV and by 90% in the lungs after 30 days. VEGF mRNA remained unchanged in the lungs from CH rats. Pulmonary vascular remodeling was more pronounced in MCT than in CH rats. The number of capillaries per RV myocyte was increased in rats exposed to 30 days of hypoxia, whereas it remained unchanged in MCT rats despite a similar degree of RV hypertrophy. Our results suggest that the sustained increase in VEGF expression in the hypertrophied RV during CH may account for the increased number of capillaries per myocyte. In contrast, reduced VEGF expression in the lungs and RV of MCT rats may aggravate pulmonary vascular remodeling and compromise RV myocardial perfusion.

Animals↗
PubMed1998

ATP derivatives are antagonists of the P2Y1 receptor: similarities to the platelet ADP receptor.

Pharmacological properties of the human P2Y1 receptor transfected in Jurkat cells and of the endogenous receptor in rat brain capillary endothelial cells were analyzed under conditions in which the purity of adenine triphosphate nucleotides was controlled by creatine phosphate/creatine phosphokinase (CP/CPK). ATP, a partial agonist of the receptor, was inactive in the presence of CP/CPK. Results further indicated that ATP was a competitive antagonist of ADP actions. Ki values were 23.0 +/- 1.5 microM in endothelial cells and 14.3 +/- 0.3 microM in Jurkat cells. Solutions prepared from commercially available 2-methylthio-ATP (2-MeSATP) or 2-chloro-ATP (2-ClATP) contained approximately 10% of ADP derivatives. ADP derivatives were removed from the solution by treatment with CP/CPK. Purified 2-MeSATP and 2-ClATP antagonized platelet aggregation induced by ADP. They did not activate P2Y1 receptors but prevented ADP actions in a competitive manner. Ki values for 2-MeSATP were 36. 5 microM in endothelial cells and 5.7 +/- 0.4 microM in Jurkat cells, and Ki values for 2-ClATP were 27.5 microM in endothelial cells and 2.3 +/- 0.3 microM in Jurkat cells. EDTA potentiated actions of ADP and ATP on endothelial cells by 2.4- and 3.6-fold, respectively. In conclusion, the rat and human P2Y1 receptors are ADP-specific receptors that recognize ADP and 2-methylthio-ADP, whereas ATP, 2-MeSATP, and 2-ClATP are competitive antagonists. The results further point to the close pharmacological similarity of the P2Y1 receptor and the platelet ADP receptor.

Adenosine Diphosphate↗
PubMed1997

Cardiac expressions of HIF-1 alpha and HLF/EPAS, two basic loop helix/PAS domain transcription factors involved in adaptative responses to hypoxic stresses.

Expression of many mammalian genes is regulated by oxygen tension. HIF-1 alpha and HLF/EPAS are two basic helix-loop-helix/PAS domain transcription factors that bind to hypoxia sensitive elements in the promoters /enhancers of hypoxia sensitive genes such as vascular endothelial growth factor (VEGF). This paper describes the structure of rat HIF-1 alpha and analyses expressions HIF-1 alpha and of HLF/EPAS mRNAs in lung and cardiac tissues from the rat. HLF/EPAS mRNAs appear at birth in the two tissues and are maintained at high levels throughout adult life. HIF-1 alpha mRNAs are expressed at a constant level during lung development. Their abundance increase transiently at birth in cardiac tissues. Cultured cardiomyocytes from new born rats only express HIF-1 alpha mRNAs. HIF-1 alpha mRNA expression is increased by phorbol myristate acetate but not by anoxia or cobalt. The results indicate (i) that HIF-1 alpha and HLF/EPAS are expressed in a cell specific manner and (ii) that the hypoxic induction of VEGF mRNA expression by isolated cardiomyocytes is independent of HLF/EPAS. Finally, they suggest that protein kinase C may prime hypoxia induced gene regulation by inducing expression of HIF-1 alpha mRNAs.

Amino Acid Sequence↗
PubMed1997

Endothelin-1 as a mediator of endothelial cell-pericyte interactions in bovine brain capillaries.

Endothelial cells and pericytes are closely associated in brain capillaries. Together with astrocytic foot processes, they form the blood-brain barrier. Capillaries were isolated from bovine brain cortex. Pure populations of endothelial cells and pericytes were isolated and cultured in vitro. Polarized monolayers of endothelial cells preferentially secreted immunoreactive endothelin-1 (Et-1) at their abluminal (brain-facing) membrane. They did not express receptors for Et-1. Pericytes expressed BQ-123-sensitive ETA receptors for endothelins as evidenced by 125I-Et-1 binding experiments. These receptors were coupled to phospholipase C as demonstrated by intracellular calcium measurements using indo-1-loaded cells. Addition of Et-1 to pericytes induced marked changes in the cell morphology that were associated with a reorganization of F-actin and intermediate filaments. It is concluded that Et-1 is a paracrine mediator at the bovine blood-brain barrier and that capillary pericytes are target cells for endothelium-derived Et-1.

Animals↗
PubMed1996

Interaction of endothelin-1 with cloned bovine ETA receptors: biochemical parameters and functional consequences.

This paper defines the properties of interaction of endothelin-1 (Et-1) with cloned bovine ETA receptors. The Kd value of Et-1/ETA receptor complexes was estimated in membrane preparations to 20 pM using kinetic experiments and saturation experiments performed under quasi equilibrium conditions. Competition experiments yield a wide range of apparent Kd(Et-1) values from 20 pM to 1 nM which were in fact measures of the receptor concentrations rather than of Kd values. This resulted from the fact that complex second-order rate kinetics rather than pseudo-first-order kinetics control the association of Et-1 to its receptor when the receptor concentration is larger than Kd(Et-1). Et-1 induced a production of inositol phosphates with an apparent affinity of 2.3 nM, 100 times higher than the Kd(Et-1) value determined previously. Numerical simulation suggested that under time-limited conditions, sub-nanomolar rather than picomolar concentrations of Et-1 are necessary to occupy an important fraction of picomolar sites. It is concluded that bovine ETA receptors have a single affinity state for Et-1 (Kd = 20 pM) and that this affinity state can account for nanomolar actions of Et-1 in intact cells. It is suggested that the sensitivity of a preparation to Et-1 is a cell property rather than a receptor property. It is also suggested that the main advantage of high-affinity Et-1 binding is to promote autocrine actions rather than a high potency of the peptide.

Animals↗
PubMed1996

Et-1 and Et-3 actions mediated by cloned ETA endothelin receptors exhibit different sensitivities to BQ-123.

Et-1 and Et-3 activate phospholipase C in fibroblasts expressing cloned ETA receptors of bovine, rat and human origins. BQ-123 competitively antagonizes both responses but Et-3 actions are 10 times more sensitive to BQ-123 than Et-1 actions. It is suggested that differential sensitivity to BQ-123 is an intrinsic property of Et-1 and Et-3 activated ETA receptors and that there is no need to postulate the existence of new ETA receptor isoforms to account for singular actions of BQ-123.

Animals↗
PubMed1996

Properties of an endothelin-3-sensitive Eta-like endothelin receptor in brain capillary endothelial cells.

[125I]Et-1 binding experiments showed the presence in rat brain capillary endothelial cells of typical ETA receptors that recognized Et-1, BQ-123 and FR139317 but not IRL1620, [Ala1,3.11,15]ET-1 or Et-3. [125I]Et-3 binding experiments showed that the same cells expressed high affinity Et-3 binding sites (Kd=1 nM) that recognized Et-1] (Kd=1 nM), BQ-123 (Kd = 5 nM) and FR139317 (Kd=2 nM) but not low concentrations of IRL1620 or of [Ala 1,3,11,15]Et-1. Et-3 activated N+/H+ exchange activity in a manner that was sensitive to BQ-123 (Ki=6 nM) and FR 139317 (Ki = 0.6 nM). The results suggest the existence of a high affinity Et-3 receptor that is insensitive to ETB receptor agonists and sensitive to ETA receptor antagonists and that is functionally coupled to Na+/H+ exchange activity.

Animals↗
PubMed1996

Differential regulation of cardiac heme oxygenase-1 and vascular endothelial growth factor mRNA expressions by hemin, heavy metals, heat shock and anoxia.

Increasing attention has been paid to the effects of hypoxia, heavy metals and heat shocks on gene expression and to the similarities in their actions. This paper compares mRNA levels of two putative hypoxia, heavy metal and heat shock sensitive genes: heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) in myocyte-enriched cultures of neonatal rat heart cells. HO-1 mRNA expression is stimulated by hemin, Cd2+, Co2+ and heat shocks but not by Ni2+ or Mn2+. It is stimulated by long (13h) but not by short (4h) periods of anoxia. Conversely, VEGF mRNA expression is stimulated by short as well as long periods of anoxia, by Cd2+, Co2+, Ni2+ and Mn2+ but not by hemin or heat shocks. The results suggest that heavy metals, anoxia and heat shocks exert their effects on VEGF and HO-1 mRNA expression through separate though potentially overlapping mechanisms. Increased expressions of HO-1 and VEGF may be both cardioprotective under hypoxic/ischemic conditions.

Animals↗
PubMed1996

Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.

1. The P2-purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca2+ concentration ([Ca2+]i) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques. 2. The P2-purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P2Y1-receptor. 3. Short applications of the purinoceptor agonists induced a transient rise in [Ca2+]i in an all-or-nothing manner. The rank order of potency of the analogues of ATP and ADP, determined by measuring the percentage of responding cells was 2-methylthioATP = 2-chloro-ATP > ADP > ATP, with concentrations giving [Ca2+]i response in 50% of cells ranging between 3 nM and 0.6 microM. The concentration-response curves to ADP and ATP were shifted to the right by 10 microM pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 4. Although the rise in [Ca2+]i induced by stimulation of the ileal P2v-purinoceptor was inhibited by heparin (5 mg ml-1), we were not able to detect stimulation of phospholipase C under conditions (37 degrees C) where muscarinic cholinoceptor activation markedly increased inositol phosphate (InsP) accumulation. However, the carbachol (CCh)-induced increase in InsP accumulation was suppressed when the agonist was applied at 20 degrees C while a CCh-induced [Ca2+]i rise similar to that obtained in response to the P2-purinoceptor agonist was still observed. 5. Our results indicate that the rat ileal myocytes express a PPADS-sensitive P2-purinoceptor similar to the P2Y1-receptor subtype. Although there is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca2+]i by activation of the inositol 1,4,5-trisphosphate receptor-channel of the intracellular Ca2+ store, indicating that they couple to phospholipase C.

Adenosine Triphosphate↗
PubMed1996

The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization.

1. Brain capillary endothelial cells responded to uridine 5'-triphosphate (UTP) and adenosine 5'-triphosphate (ATP) by activation of phospholipase C and by large changes in [Ca2+]i. These cells expressed mRNA sequences identical to the sequence of the P2Y2-purinoceptor of rat pituitaries. 2. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) at 100 microM did not prevent UTP and ATP induced accumulations of total [3H]-inositol (poly)phosphates. It inhibited UTP and ATP induced intracellular Ca2+ mobilization (IC50 = 30 microM) by non competitive mechanism. 3. PPADS (100 microM) inhibited endothelin-1 induced accumulation of total [3H]-inositol (poly)phosphates by less than 20% and prevented most of endothelin-1 induced intracellular Ca2+ mobilization (IC50 = 30 microM). 4. PPADS (100 microM) had no action on ionomycin induced intracellular Ca2+ mobilization. 5. Microinjection of inositol (1,4,5)trisphosphate (InsP3) into Xenopus oocytes induced large Ca2+ activated Cl- currents that were prevented by heparin and by PPADS. 6. It is concluded that PPADS does not recognize rat P2Y2-purinoceptors and prevents UTP and ATP induced intracellular Ca2+ mobilization by a non-specific mechanism that could involve the inhibition of InsP3 channels.

Adenosine Triphosphate↗
PubMed1996

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase.

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

Adenylyl Cyclase Inhibitors↗
PubMed1995

Properties and functions of a neuromedin-B-preferring bombesin receptor in brain microvascular endothelial cells.

Endothelial cells were isolated from rat brain microvessels and grown in vitro. They expressed a high density of [125I-Tyr4]bombesin receptor (Bmax = 0.9 pmol/mg protein) with an apparent Kd value of 10 nM. The pharmacological profile of inhibition of the specific [125I-Tyr4]bombesin binding [bombesin = neuromedin B > gastrin releasing peptide (GRP)] was consistent with the presence of a neuromedin-B-preferring receptor. Addition of bombesin, neuromedin B and GRP increased the activity of phospholipase C as measured by the production of total inositol phosphates and from intracellular Ca2+ measurements. They increase 86Rb+ uptake by the Na+, K+, 2Cl- cotransporter and by a charybdotoxin-sensitive, Ca(2+)-activated K+ channel and 22Na+ uptake by the Na+/H+ exchanger. The pharmacological profiles of activation of phospholipase C, Na+, K+, 2Cl- cotransport and Na+/H+ exchange by bombesin-like peptide were consistent with an involvement of the neuromedin-B-preferring receptor characterized in binding experiments. It is suggested that one of the actions of neuromedin B in brain vessels could be to control K+ secretion by the blood/brain barrier.

Animals↗
PubMed1995

Contributions of NO synthase and heme oxygenase to cGMP formation by cytokine and hemin treated brain capillary endothelial cells.

Two mechanisms contribute to cGMP formation by soluble guanylyl cyclase (i) NO production by NO synthase and (ii) CO production by heme oxygenase. We analyze here the contributions of these two pathways to IL1, TNF, lipopolysaccharide and hemin treated brain capillary endothelial cells. Cytokines and LPS induced cGMP formation in manners that were completely prevented by LY 83,583, methylene blue and by cyclosporin A. They were partially inhibited by inhibitor of NO synthase. Cyclosporin A acts by a posttranscriptional mechanism. Cells constitutively expressed mRNAs for heme oxygenase-1. Expression was enhanced by hemin but not by IL1 or lipopolysaccharide. Induction of heme oxygenase-1 and its inhibition by Sn protoporphyrin IX had no effect on cGMP levels.

Amino Acid Oxidoreductases↗
PubMed1995

Palytoxin. Recent electrophysiological and pharmacological evidence for several mechanisms of action.

1. Palytoxin is one of the most potent toxins known so far. It acts as an haemolysin and alters the functioning of excitable cells. 2. A primary action of palytoxin in excitable cells is to induce the activity of a small conductance (9-25 pS), non-selective cationic channel which then triggers secondary activations of voltage dependent Ca2+ channels and of Na+/Ca2+ exchange. This results in neurotransmitter release by nerve terminals and contractions of striated and smooth muscle cells. 3. Palytoxin induced channels are blocked by amiloride derivatives such as 3,4 dichlorobenzamil. They are also blocked by ouabain but at concentrations higher than those required to inhibit the (Na+,K+)ATPase. 4. A second and independent action of palytoxin is to open a membrane conductive pathway for H+ that drives H+ inside the cells and secondarily activates Na+/H+ exchange activity. 5. A third action of PTX in chick cardiomyocytes is to raise [Ca2+]i in a manner independent of its depolarizing action or of its action on intracellular pH. 6. It is suggested that PTX probably has more than one site of action in excitable cells and that it may act as an agonist for a family of low conductance channels that conduct Na+/K+, H+ and Ca2+ions.

Acrylamides↗
PubMed1995

A charybdotoxin-sensitive, Ca(2+)-activated K+ channel with inward rectifying properties in brain microvascular endothelial cells: properties and activation by endothelins.

A charybdotoxin-sensitive, Ca(2+)-activated K+ channel was identified in cultured rat brain capillary endothelial cells by using conventional single-channel recording techniques and 86(Rb+)-influx and efflux experiments. Channel activity was dependent on the presence of Ca2+ on the cytosolic face of the membrane with a threshold concentration of 100 nM. It was inhibited by charybdotoxin (IC50 30 nM) and quinine (IC50 0.1 mM) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin-1 (EC50 0.7 nM) and endothelin-3 (EC50 7-10 nM). The actions of endothelins were prevented by BQ-123 (Ki = 8 nM) in a competitive fashion, hence suggesting the involvement of an ETA-receptor subtype. The channel activity was unaffected by cyclic AMP- or cyclic GMP-elevating agents. The possible role of the intermediate conductance, Ca(2+)-activated K+ channels for mediating K+ movements across the blood-brain barrier is discussed.

Amino Acid Sequence↗
PubMed1995

Sensitization by calyculin A of brain capillary endothelial cells to endothelin-1.

1. Cultured brain capillary endothelial cells of the rat respond to endothelin-1 (ET-1) by an increased activity of the Na+,K+,2Cl-, cotransporter and a mobilization of intracellular Ca2+ stores. 2. Calyculin A (1-30 nM), but not okadaic acid, sensitizes up to 100 fold the Na+,K+,2Cl- cotransporter to the action of ET-1. 3. Calyculin A (30 nM) does not modify the binding properties of ET-1 to ETA receptors. 4. Calyculin A (30 nM) inhibits ET-1 induced intracellular Ca2+ mobilization. 5. It is concluded that inhibition of protein phosphatase 1 selectively modifies the repertoire of intracellular actions of ET-1 and favours actions that are unrelated to the phospholipase C signalling cascade.

Animals↗
PubMed1995

ATP, a partial agonist of atypical P2Y purinoceptors in rat brain microvascular endothelial cells.

1. Purinoceptor responses were analyzed in B10 cells, a clonal population of rat brain capillary endothelial cells. 2. B10 cells lack P2U receptors as evidenced by the lack of UTP responses and the failure to amplify P2U-related sequences by polymerase chain reaction. 3. B10 cells responded to adenine nucleotides by large increases in [Ca2+]i. Half maximum effective concentrations were 2-methylthio-ATP: 180 nM > 2-chloro-ATP: 310 nM = ADP: 330 nM > adenosine 5'-O-(3-thiotrisphosphate): 2.3 microM = ATP: 2.7 microM. The maximum response to ATP was only 55% of that to ADP while that to ATP derivatives was 75%. 4. The actions of adenine nucleotides were not associated with a measurable activation of phospholipase C. 5. Cross desensitizations of the actions of ADP and ATP were observed. 6. In additivity experiments, ADP superposed its action on top of that of ATP and ATP partially inhibited the action of ADP. 7. It is concluded that ATP acts as a partial agonist of the P2Y-like receptor of brain capillary endothelial cells.

Adenosine Diphosphate↗