Biomedical subjects

S Phaneuf

Publications and source records attributed to S Phaneuf.

45 records · Page 3Linked to original sources

PubMed1988

The effects of maitotoxin on phosphoinositides and calcium metabolism in a primary culture of aortic smooth muscle cells.

Maitotoxin, a potent marine toxin isolated from toxic tropical dinoflagellates and poisonous fishes induces contraction of different smooth muscle preparations. Actions of maitotoxin on phosphoinositides and calcium metabolism were studied using a primary culture of aortic smooth muscle cells. Maitotoxin induced a very large increase of cytosolic calcium concentration as evaluated by fura-2 acetoxymethyl ester fluorescence. This increase was concomitant with stimulation of inositol-phosphate accumulation and loss of viability of aortic smooth muscle cells. These responses to maitotoxin were abolished in Ca2+-free medium, and were mimicked by saponin. Calcium ionophores or K+ depolarisation did not induce inositol-phosphate formation. These results suggest that maitotoxin acts by altering smooth muscle cells permeability allowing a sustained calcium influx which is able to activate inositol-phosphate formation and which is lethal for the cells.

Angiotensin II↗

PubMed1988

Phorbol ester modulation of cyclic AMP accumulation in a primary culture of rat aortic smooth muscle cells.

Over the past few years, the importance of calcium and cyclic AMP in the regulation of vascular smooth muscle tone has been well documented. We used a primary culture of rat aortic myocytes to study the effect of protein kinase C on isoproterenol- and forskolin-stimulated cyclic AMP production. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) to these cells, but not an inactive analog, increased the stimulation of cyclic AMP production induced with isoproterenol or forskolin without changes in the apparent affinity of these compounds but did not affect the basal cAMP level. TPA also enhanced the cholera toxin-stimulated cyclic AMP accumulation. Isoproterenol and cholera toxin increased the forskolin apparent potency suggesting that interaction of activatory GTP-dependent protein with the catalytic subunit of adenylate cyclase facilitates forskolin interaction to the catalytic subunit. Treatment of myocytes with pertussis toxin had no effect on the basal level of cyclic AMP production and did not significantly modify isoproterenol- and forskolin-induced stimulation. Pertussis toxin treatment of cells did not affect the TPA-enhanced isoproterenol or forskolin stimulations suggesting that pertussis toxin and TPA actions would not share a common target of myocyte adenylate cyclase system. Our data would be in agreement with a possible direct interaction of protein kinase C with the catalytic subunit of adenylate cyclase system.

Adenylate Cyclase Toxin↗

PubMed1987

ATP stimulates inositol phosphates accumulation and calcium mobilization in a primary culture of rat aortic myocytes.

The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ concentration were studied in a primary culture of rat aortic myocytes. ATP increases the level of inositol phosphates, the putative second messenger for Ca2+ mobilization. No saturation of inositol phosphates accumulation is obtained (up to 10(-2) M ATP). Under the same conditions, ATP rapidly mobilizes intracellular Ca2+ in fura-2 loaded myocytes. The mobilization of intracellular Ca2+ is dose-dependent (maximal at 10(-4) M ATP), and is not affected by addition of EGTA. It is concluded that the receptors mediating the cytosolic increase of Ca2+ are of the P2-purinoceptor subtype. The physiological functions of these receptors are not presently known.

Adenosine Triphosphate↗

PubMed1986

Calcium binding properties of purified zymogen granule membrane of pig pancreas. Evidence for calcium binding proteins.

Ca2+ binding properties of purified zymogen granule membranes of pig pancreas have been measured: Binding increased linearly with Ca2+ concentration in the medium up to the micromolar range; in the millimolar range a sharp rise in binding capacity was observed. Binding increased with pH both at low and high concentrations of Ca2+. It was insensitive to Na+ and K+ ions at concentrations up to 100 mM. Mg2+ was inhibitory in the millimolar range whereas La2+ and Tb3+ were inhibitory in the micromolar range. The Ca2+ binding components of zymogen granule membranes were identified by two methods: (1) by measuring 45Ca2+ binding after counter-ion electrophoresis and (2) by Stain's-all (forms a complex with Ca2+ binding proteins absorbing maximally at 600 nm), after SDS-polyacrylamide gel electrophoresis. The first method, counter-ion electrophoresis, indicated that most of the 45Ca2+ was associated with an acidic band which could be subsequently subfractionated by SDS-polyacrylamide gel electrophoresis in five bands: 66, 57, 30, 27 and 22.5 kDa. The second method, Stain's-all, revealed six positive polypeptides after SDS-polyacrylamide gel electrophoresis of native zymogen granule membranes' two were unreactive after neuraminidase treatment (130 and 92 kDa, respectively), whereas four other bands were still reactive (66, 57, 43, 30 kDa, respectively.) Ca2+ binding was also measured on intact zymogen granules: the binding capacity was higher than for zymogen granule membranes. Among the Ca2+ binding proteins of the zymogen granule membrane only one is apparently located on the granule external surface: the 30 kDa polypeptide. If Ca2+ directly facilitates fusion of zymogen granules with plasma membrane by a Ca2+-protein interaction, then this protein is a presumptive candidate to play such a key role.

Animals↗

PubMed1985

Electrophoretic and cytological evidence for heterogeneity of pancreatic acinar cell responsiveness to carbachol, caerulein and secretin.

Incubation of rat pancreatic lobules for 90 min with optimal concentrations of caerulein, carbachol or secretin caused the release of about 30% of the amylase content. Combination of secretin with carbachol or caerulein increased the amylase output to about 40%. With secretin, as with carbachol or caerulein, heterogeneity of cellular responsiveness was observed, some acini being partially or completely depleted of their zymogen granules, whereas others appeared to be resting. When secretin was combined with carbachol or caerulein, granule depletion, originally confined to small groups of neighbouring acini, spread to form large areas of degranulated cells, sometimes comprising a whole section of a lobule. In dispersed acini, under the same conditions, carbachol caused the release of about 60% of the amylase content, and secretin 40%. When both secretagogues were combined, a significant increase to 78% was observed. Under these conditions, there was some important cellular damage, as indicated by the release of 20% of the amylase content and between 6 and 12% of lactate dehydrogenase into the media, in the absence of stimulus. These results were corroborated by cytological observations. On the basis of their secretory response two groups of acini can be distinguished, those that respond to carbachol, caerulein or secretin and those that respond to the combination of secretin with carbachol or caerulein. Electrophoretic patterns of secretory proteins released by lobules stimulated by these different types of secretagogues were essentially similar. The pattern was quite different, however, in the absence of a stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗

PubMed1984

Cytological effects of ionophore-induced stimulation on the exocrine pancreas of the rat.

Rat-pancreas lobules were incubated with the ionophore A-23187 in the presence of Ca2+. After 90 min, some of the acini were partially or almost completely depleted of their zymogen granules while others had the appearance of resting acini. With few exceptions, the cells of a given acinus were degranulated to a comparable level. Slight dispersion of the zymogen granules was noticed in cells incubated in a Ca2+-free medium containing EGTA with or without A-23187. In the presence of Ca2+ the secretory response obtained with the ionophore was comparable to that observed with 10(-5)M urecholine. The results obtained provide cytological evidence that the secretory response is only partially determined at the membrane-receptor level and that other mechanisms intervene between cytosol Ca2+ increase and exocytosis.

Amylases↗

PubMed1980

Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas.

An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.

Animals↗

PubMedDate not supplied

Desensitization of oxytocin receptors in human myometrium.

In the present study, we investigated the possible mechanisms by which oxytocin might regulate oxytocin receptor (OTR) density. Exposure of cultured myometrial cells to oxytocin for a prolonged period caused desensitization: the steady-state level of oxytocin binding was 210 x 10(3) binding sites/cell, but this was time-dependently reduced to 20.1 x 10(3) sites/cell by exposing the cells to oxytocin for up to 20 h. In contrast, Western blotting data showed that the total amount of OTR protein was not affected by oxytocin treatment for up to 24 h. Flow cytometry experiments demonstrated that OTRs were not internalized during this treatment. However, RNase protection assays and Northern analysis showed that in cultured myometrial cells OTR mRNA was reduced by oxytocin treatment to reach a new low steady-state concentration. Analysis of this mRNA in myometrial biopsies from 17 patients undergoing emergency Caesarean section showed how it decreased with advancing labour. Samples obtained after 12 h of labour contained approximately 50 times less OTR mRNA than samples obtained from patients in labour for less than 12 h. We speculate that this decrease in OTR mRNA represents in-vivo OTR desensitization.

Cells, Cultured↗

PubMedDate not supplied

Prostaglandin E receptors in myometrial cells.

Prostaglandins (PGs) exert their effects via binding to specific cell surface receptors and influencing second messenger systems through G-proteins. PGE2 may interact with at least four receptor subtypes (EP1, EP2, EP3, EP4), each showing different pharmacological profiles. The second messengers calcium, inositol phosphates (InsPs) and cyclic nucleotides play decisive roles in uterine contractility. The question in this investigation was, which EP receptors, G-proteins and second messenger systems transmit PGE2 induced signals in human myometrium. We have measured changes in InsPs and cAMP formation and also in intracellular calcium concentration ([Ca2+]i) induced by PGE2 and receptor subtype selective analogues in cultured human myometrial cells. PGE2 increased cAMP level and this effect was shared by the EP2 receptor subtype selective agonist Butaprost and by Misoprostol (EP3 > EP2 > EP1). Sulprostone (EP3 > EP1) did not stimulate adenylyl cyclase activity per se, but inhibited forskolin-stimulated adenylyl cyclase in a pertussis toxin (PT) sensitive way. PGE2, GR63799X (EP3 selective), Sulprostone and Misoprostol activated phospholipase-C (PLC), this effect was resistant to PT treatment. PGE2 also elevated [Ca2+]i from the resting level of 60-90 nM up to 350 nM. Low concentrations (1-300 nM) of PGE2 increased [Ca2+]i without PLC activation. The selective EP1 inhibitor AH6809, Nifedipine, Verapamil and PT treatment inhibited this effect of PGE2. In cultured human myometrial cells PGE2 interacts with EP1 receptors, which elevate [Ca2+]i independently from PLC, but involving a Gi protein and plasmamembrane calcium channels; EP2 receptors which stimulate adenylyl cyclase; EP3A receptors, which inhibit adenylyl cyclase activity through Gi activation and EP3D receptors which activate PLC through a PT-insensitive pathway and also elevate [Ca2+]i.

Calcium↗