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Biomedical subjects

P Marche

Publications and source records attributed to P Marche.

At least 55 records · Page 3Linked to original sources
PubMed1988

Enhanced turnover of phosphatidylcholine in platelets of hypertensive rats. Possible involvement of a phosphatidylcholine-specific phospholipase C.

In an attempt to determine the mechanism involved in the hyperreactivity of platelets in primary hypertension, the dynamic behavior of phospholipids was investigated in quiescent platelets of spontaneously hypertensive rats (SHR) compared to normotensive controls. By using 32Pi, [methyl-3H]choline or [3H]glycerol as the radioactive precursors, the labeling of phosphatidylcholine (PC) was shown to be markedly enhanced (10-20-times) in SHR compared to controls. This difference between SHR and controls could not be ascribed to differences either in the actual amount of PC or in the uptake of various labels, suggesting that PC turnover was markedly enhanced in platelets of SHR. The [methyl-3H]choline labeling of phosphocholine and of CDP-choline was twice as high in SHR as in controls; chase experiments showed that when the label disappeared from phosphocholine, it was rapidly converted to PC. The results indicated that in rat platelets, PC synthesis occurred mainly via the CDP-choline pathway, and suggested that CTP:phosphocholine cytidylyltransferase was the rate-limiting step; they also indicated that the activity of this enzyme and that of choline kinase might be enhanced in SHR platelets compared to Wistar-Kyoto rat (WKY) platelets, and may thus be responsible for the enhanced PC synthesis. From these results, the existence of a PC-specific phospholipase C activity involved in PC turnover in SHR platelets can be envisaged.

Animals↗
PubMed1988

Defective phosphoinositide metabolism in primary hypertension.

An increase in free cytosolic calcium content has been reported in essential hypertension. Since within the membrane, the phosphoinositides participate in the control of cell calcium homeostasis, we investigated whether impaired phosphoinositide metabolism could account for the calcium handling abnormality observed in hypertensives. In erythrocyte membranes of hypertensives the activity of kinases involved in polyphosphoinositide formation appears to be impaired and could be related to the alteration in calcium handling binding capacity and ATP-dependent calcium transport. In platelets of hypertensives, the hyperactivity of phospholipase C (observed even in the absence of calcium in the external medium) is likely to be responsible for the hypersensitivity of cells to various agonists. These observations are consistent with the hypothesis that in cells from hypertensives, a membrane defect linked to phosphoinositide metabolism is involved in the overall calcium handling defect.

Animals↗
PubMed1988

Histamine H1-receptors mediate phosphoinositide and calcium response in cultured smooth muscle cells--interaction with cicletanine (CIC).

Smooth muscle cells were cultured from guinea-pig aorta and labelled with 45Ca++ and 32Pi to investigate the possible effect of cicletanine, a new antihypertensive drug, on the release of intracellular Ca++ and the metabolism of phosphoinositide induced by histamine. In 45Ca++ labelled cells, histamine increased in a dose-dependent manner the 45Ca++ efflux in the first two minutes. Stimulation of 45Ca++ release was observed with H1-agonist [2-pyridylethylamine dihydrochloride (2-PEA)] but not with H2-agonist (dimaprit). In addition, histamine- or 2-PEA- induced 45Ca++ efflux was inhibited by the H1-antagonists (mepyramine and terfenadine) whereas the H2-antagonist (cimetidine) was without effect. Similar results were obtained in 32Pi labelled cells; both H1-agonists (histamine and 2-PEA) increased the labelling of phosphoinositides. This effect was completely blocked by mepyramine. These results demonstrate that the histamine-induced stimulation of 45Ca++ efflux and phosphoinositide metabolism are mediated through H1-receptors. In the above systems, cicletanine was as effective as the H1-antagonist (mepyramine) with an IC50 of 10(-6) M for both 45Ca++ efflux and phosphoinositide metabolism. Blockade of these systems by cicletanine may be part of the mechanism by which this drug produces relaxation of blood vessels and may account for its in vivo antihypertensive action.

Animals↗
PubMed1988

Cell membrane in hypertension.

Several alterations in membrane transport systems are observed in rat and human hypertension. Na+ flux changes are numerous, and cellular homeostasis to Na+ loading is impaired. Transmembrane Ca2+ movements are also numerous but clearly defined by a reduction in Ca2+ binders, a hypersensitivity of membrane phospholipase C, possible increased Ca2+ leak and reduced sensitivity of the Ca2+-pump to calmodulin. The resulting Ca2+ increase within arterial cells can be responsible for increased contractility and tone, leading to hypertension. These functional alterations in membrane transport can be secondary to a few well-defined membrane defects of genetic origin or to a diffuse structural perturbation in membranes involving lipid changes.

Animals↗
PubMed1988

Growth rate and phospholipase C activity in cardiac and aortic spontaneously hypertensive rat cells.

The cellular proliferation rate and phospholipase C sensitivity have been compared in various cardiovascular cells cultured from spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY). Aortic smooth muscle cells from SHR showed an enhanced proliferation rate under the culture conditions (in the presence of 10% fetal calf serum) and hypersensitivity to the mitogenic action of angiotensin II and serotonin. Phospholipase C activity (determined by measuring the formation of tritiated inositol phosphates from [3H]-myoinositol) triggered by angiotensin II was also higher in these cells than in those from WKY. Cardiac fibroblasts from SHR proliferated more quickly than those from WKY, which suggests that the increased growth rate is a general feature of SHR cardiovascular cells. Furthermore, as cardiac cells were obtained from newborn animals, this impairment appears to be an intrinsic characteristic of the SHR cells. In contrast, no difference in the phospholipase C reactivity to phenylephrine was found in cardiomyocytes from SHR and WKY. The amount of tritiated inositol monophosphate measured in unstimulated cells, however, was significantly higher in cultures from WKY, which may suggest a difference in the phosphoinositide cycle between both strains of cells. Like the impairment in the proliferation rate, alterations in phosphoinositide metabolism may be a common feature of cardiovascular SHR cells.

Angiotensin II↗
PubMed1988

Impaired phospholipase C activity is involved in the hyperreactivity of platelets in primary hypertension.

Phospholipase C activity, which influences the control of platelet physiological responses, was investigated in platelets of human essential hypertensives and of spontaneously hypertensive rats (SHR) compared with appropriate normotensive controls, in order to determine whether this enzyme activity could account for the enhanced platelet responses exhibited by hypertensive subjects. After 32P-labelling of cells, the enzyme activity was estimated by measuring the variations in 32P-phosphatidic acid. In resting platelets no difference was observed between hypertensives and normotensives. In contrast, the thrombin-induced increase in 32P-phosphatidic acid in platelets of human hypertensives and of SHR was 30% higher than in controls, suggesting hypersensitivity to phospholipase C in hypertensives. Since, as revealed by phorbol-stimulated phosphorylation of 47-kilodalton protein, intrinsic protein kinase C activity is similar in SHR and controls, our data strengthen the hypothesis than hypersensitivity to phospholipase C influences the hyperreactivity of platelets in primary hypertension.

Animals↗
PubMed1988

Relationship between enhanced phosphoinositide turnover and cellular responses in platelets from spontaneously hypertensive rats.

Thrombin-induced aggregation and serotonin release were markedly enhanced in platelets from spontaneously hypertensive rats (SHR) when compared to normotensive Wistar-Kyoto (WKY) controls. Since phosphoinositides are involved in calcium-mediated platelet responses, the metabolism of these lipids was investigated in SHR and WKY rats by using 32P-labeled quiescent platelets. In unstimulated cells, both the rate and extent of 32P incorporation into individual inositol-containing phospholipids and phosphatidic acid (PA) were identical in SHR and WKY rats. This suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced phosphoinositide metabolism, when monitored as changes in 32P-PA, was significantly higher in SHR than in WKY rats. Following thrombin stimulation, 32P-PA formation likely reflects the initial agonist-receptor interaction; therefore, our results suggest that phospholipase C activity is enhanced in SHR platelets. Thus, it can be postulated that the observed hypersensitivity of SHR phospholipase C may play a role in the overall alteration of cell calcium handling and hence in the SHR platelet response.

Animals↗
PubMed1988

Phosphoinositides and cicletanine.

In vascular smooth muscle cells, the activation of histaminergic receptors of the H1 subtype induces an increase in cytosolic free calcium. This has been shown to involve the metabolism of inositol-containing lipids. Since according to its pharmalogical characteristics, cicletanine could be considered as an H1 antagonist, it was of interest to check whether the drug behaved as such with regard to phosphoinositide metabolism. Phosphoinositide metabolism was studied by measuring the 32P labelling of phosphatidylinositol (PI) and of its phosphorylated derivatives in cultured smooth muscle cells isolated from guinea-pig aorta and incubated in the presence or absence of various histaminergic agonists and/or antagonists. The results clearly demonstrate that the increase in 32P-PI could be selectively induced by treatment of cells with histamine or 2-pyridylethylamine dihydrochloride (2-PEA) (an H1 agonist) but not with the H2 agonist dimaprit. In addition, histamine- or 2-PEA-induced 32P-PI increase was inhibited by pretreatment of cells with the H1 antagonist mepyramine, whereas the H2 antagonist cimetidine was without effect. These data demonstrate that the histamine-induced 32P-PI increase is mediated through H1 receptors. Under the same experimental conditions, cicletanine was as effective as mepyramine with an IC50 value of 10(-6) M. The development of tension in arterial smooth muscle is under the control of variations of calcium concentration in the intracellular space. Therefore, the blockade by cicletanine of histamine-enhanced phosphoinositide turnover may participate in the mechanism by which the drug exerts its antihypertensive action.

Angiotensin II↗
PubMed1987

Signal transduction in normal and pathological thrombin-stimulated human platelets.

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.

Blood Platelet Disorders↗
PubMed1987

Aminoglycoside-induced alterations of phosphoinositide metabolism.

There exists a strong interaction between aminoglycosides and phosphoinositides, and these membrane lipids are even considered as the drug receptors. To shed some light on the role of such an interaction in the drug nephrotoxicity, we have investigated the influence of aminoglycosides on phosphoinositide metabolism in kidney proximal tubules where these compounds accumulate. Experiments were carried out by measuring 32P labelling of phosphatidylinositol 4-phosphate (PI-P) and of phosphatidylinositol 4,5-bisphosphate (PI-P2) after incubation of homogenates of isolated proximal tubules with [gamma-32P] ATP. The treatment of rabbits with neomycin, gentamicin and amikacin (50, 50 and 300 mg/kg/day, respectively for seven days) promoted a decrease in 32P-PI-P2 and an increase in 32P-PI-P, when compared to the respective values observed in tubules from untreated rabbits. Under these conditions, the extent of modifications in lipid labelling was similar with the three drugs tested. In in vitro experiments, the exogenous addition of the above aminoglycosides to the incubation medium containing tubule homogenates from untreated rabbits also produced, in a dose dependent manner, a decrease in 32P-PI-P2 and an increase in 32P-PI-P. In the in vitro experiments, however, amikacin and gentamicin appeared to be less potent than neomycin. The results indicated moreover that phosphoinositide metabolism was more sensitive to the in vivo (vs. in vitro) action of the drugs. Phosphoinositides are involved in Ca2+ transport and/or mobilization processes, and aminoglycosides are known to interfere with the Ca2+ binding to membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amikacin↗
PubMed1987

Abnormal phosphoinositide metabolism and protein phosphorylation in platelets from a patient with the grey platelet syndrome.

Washed platelets isolated from one patient suffering from the inherited grey platelet syndrome were studied during thrombin-induced activation. The agonist-induced changes in (i) morphology, (ii) typical functional cell responses, (iii) membrane phospholipid metabolism and protein phosphorylation were studied and compared with the changes obtained with normal platelets. The morphology of the platelets as visualized by electron microscopy confirmed the almost total absence of intracellular alpha-granules and marked vacuolization. During thrombin stimulation the morphological changes were clearly delayed as compared to normal platelets, the granule centralization and aggregation occurred only 15 s after thrombin addition instead of 5 s in normal platelets. After 15 s, however, even though no alpha-granules were observed, a ring-like structure occurred centrally, indicating that they are not a prerequisite for this reaction. The whole release reaction, i.e. liberation of [14C]serotonin from dense granules and beta-N-acetylglucosaminidase activity from lysosomes, and the thromboxane synthesis were delayed and remained lower than in normal platelets. No thrombin-induced phosphatidyl 4,5-bisphosphate breakdown was measurable on 32P-prelabelled platelets although [32P]phosphatidate formation occurred normally. Phosphorylation time courses of myosin light chain (P20) and of protein P43 (mol wt 43,000) markedly differed from those of controls, being less than half of the normal during the first 15 s and remaining subnormal even after complete aggregation. These results suggest that in platelets devoid of alpha-granules a deficient transmembrane signalling system is likely responsible for the impaired physiological responses.

Blood Platelet Disorders↗
PubMed1987

Hypersensitivity of phospholipase C in platelets of spontaneously hypertensive rats.

Thrombin-induced aggregation and serotonin release were markedly enhanced in platelets from spontaneously hypertensive rats (SHR) when compared with those from normotensive Wistar-Kyoto rats (WKY). Since phosphoinositides are involved in calcium-mediated platelet responses, the metabolism of these lipids was investigated in SHR and WKY by using 32P-labeled quiescent platelets. In unstimulated cells, both the rate and extent of 32P incorporation into individual inositol-containing phospholipids and phosphatidic acid were identical in SHR and WKY. This finding suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced phosphoinositide metabolism, when monitored as changes in [32P]phosphatidic acid, was significantly higher in SHR than in WKY. For example, a 20-second exposure to thrombin, 0.3 U/ml, induced the formation of 1.6 times more [32P]phosphatidic acid in SHR than in WKY. These results provide evidence for a leftward shift of the dose-response and time-course curves of thrombin-induced [32P]phosphatidic acid formation in SHR. Moreover, the extent of the difference between SHR and WKY was independent of the extracellular calcium concentration. Following thrombin stimulation, [32P]phosphatidic acid formation likely reflects the initial agonist-receptor interaction; therefore, these results suggest that phospholipase C activity is enhanced in platelets of SHR and that the hypersensitivity of phospholipase C in SHR may play a role in the overall alteration of cell calcium handling and, hence, in the platelet responses of SHR.

Animals↗
PubMed1987

[Phospholipase C control of the hyperreactivity of platelets of the spontaneously hypertensive rat].

With respect to their biological responses the platelets of essentially hypertensive patients and of spontaneously hypertensive rats (SHR) are hyper-reactive to a variety of agonists. Platelet responses are mainly controlled by Ca2+ influx and/or mobilization and most of the platelet-activating substances act by stimulating the metabolism of phosphoinositides. Thus it is the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C that initiates platelet activation by thrombin. Assuming that an altered metabolism of phosphoinositides might participate in the hyper-reactivity of platelets from hypertensives, we studied phosphoinositide metabolism in washed platelets from SHR in relationship with the physiological cellular responses. This was carried out by measuring the variations in 32P-labeled lipids following incubation of platelets with 32P-orthophosphate. The results were compared with those obtained from washed platelets isolated from normotensive control rats of the Wistar-Kyoto strain (WKY). In unstimulated cells, both the rate and extent of 32P-incorporation into individual phospholipids were identical in SHR and WKY. This suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced lipid metabolism, when monitored as changes in 32P-phosphatidic acid (PA), was significantly higher in SHR than WKY and the extent of the difference was independent of the extracellular calcium concentration. Since following thrombin stimulation, 32P-PA formation likely reflects phospholipase C activity, our results suggest that this enzyme activity is enhanced in SHR platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Structural and functional alterations of the cell membrane in the prehypertensive rat of the Okamoto Aoki strain.

Plasma membrane properties of 3 to 4-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were investigated in both excitable cells, i.e. cardiomyocytes and platelets, and non-excitable cells, i.e. erythrocytes. Cardiac sarcolemma from SHR exhibited: lowered capacity of high-affinity Ca2+-binding sites; higher Ca2+ pump activity; higher Na+K+-ATPase activity due to increased density of Na+ pump units; suppression of the above three effects by the Ca2+-calmodulin complex, and increased Na+Ca2+ exchange. No difference in platelet cytosolic free Ca2+ concentration was observed between SHR and WKY. In both substrains, erythrocyte intracellular Na+ content was similar in spite of reduced Na+ and K+ net fluxes. Isolated membranes from SHR erythrocytes were also characterized by: lowered phosphoinositide turnover; decreased ATP-dependent Ca2+-transport, and lowered capacity of high affinity Ca2+-binding sites. Structural alterations detected by fluorescence polarization of diphenylhexatriene were observed in SHR cardiac sarcolemma, erythrocyte and brain synaptosomal membranes. Membrane organization and activity of transport systems controlling the intracellular Na+ and Ca2+ level were thus already modified in the prehypertensive animals whereas the resulting intracellular ion contents were still unaltered.

Animals↗
PubMed1985

Influence of adrenoceptors on thrombin-induced phosphoinositide metabolism in rat platelets.

Stimulation of washed rat platelets with thrombin resulted in an increased turnover of phosphoinositides. Adrenaline and isoproterenol both inhibited thrombin-induced phosphatidic acid formation in a dose-dependent manner. Inhibitory responses of both compounds were blocked by a beta-adrenoceptor antagonist. However, isoproterenol was a more potent inhibitor than adrenaline. Addition of a selective alpha2-adrenoceptor antagonist potentiated the inhibitory effect of adrenaline up to the level observed with isoproterenol. Prestimulation of beta-adrenoceptors with isoproterenol, followed by addition of adrenaline (or noradrenaline) markedly diminished the inhibitory effect induced by the full beta-adrenoceptor agonist. Our results indicate that, in rat platelets, catecholamines are able to counteract, via alpha2-receptors, the beta-adrenoceptor-mediated inhibition of thrombin-induced phosphatidic acid formation. This suggests that catecholamines, by controlling cAMP level, may modulate phospholipase C activity and thereby platelet reactivity.

Alprostadil↗
PubMed1985

Influence of alpha- and beta-adrenoceptors on thrombin-induced serotonin release in rat platelets.

This work was designed to investigate the influence of rat platelet adrenoceptors on the early thrombin-induced serotonin release. In washed platelets prelabeled with [3H]-serotonin, adrenaline and isoproterenol both inhibited, in a dose-dependent manner, the early thrombin-induced secretion of serotonin. Inhibitory responses of both adrenaline and isoproterenol were blocked in the presence of beta-adrenoceptor antagonists, suggesting that the catecholamine acted solely through beta-adrenoceptors. However, isoproterenol inhibited the thrombin-induced serotonin release to a much greater extent than the catecholamine, suggesting that the alpha 2-component of adrenaline might account for the difference observed between the two compounds. Our observation that selective alpha 2-adrenoceptor antagonists as yohimbine and rauwolscine potentiated the inhibitory effect of adrenaline to a level close to that observed with isoproterenol, lends support to the above hypothesis. This latter result suggested that, conversely, alpha 2-adrenergic compounds might exert a counteracting effect on a full beta-adrenoceptor mediated inhibition. Although synthetic alpha 2-adrenergic agents failed to influence isoproterenol inhibitory effect, our study shows that prestimulation of beta-adrenoceptors by isoproterenol, followed by addition of adrenaline or noradrenaline markedly diminished the inhibitory effect of isoproterenol to a level close to that which characterized the inhibition observed with catecholamines, when tested alone. Our work favours the hypothesis that, in rat platelets, early after platelet stimulation, catecholamines might counteract a beta-adrenoceptor- mediated inhibition, through alpha 2-adrenoceptor sites.

Adrenergic alpha-Antagonists↗