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J Poggioli

Publications and source records attributed to J Poggioli.

At least 37 records · Page 2Linked to original sources
PubMed1990

Mediation by GTP gamma S and Ca2+ of inositol trisphosphate generation in rat heart membranes.

The possibility, that a GTP-binding protein is involved in the transducing mechanism leading to the formation of inositol trisphosphate (InsP3) in heart was explored in rat heart ventricles. Accordingly, a crude membrane fraction was isolated from 3[H] inositol prelabelled rat heart ventricles. When incubated with the non-hydrolysable GTP analogues GTP gamma S and GMP-PNP, it produced InsP3 in a time- and concentration-dependent manner. GDP beta S and the aminoglycoside antibiotic neomycin were effective inhibitors of this activation. In the absence of GTP gamma S or GMP-PNP, no such formation occurred with Ca2+ concentration from 10 nM to 1 microM but formation tripled in relation to the control level when Ca2+ concentration was raised from 1 microM to 100 microM. GTP gamma S increased the Ca2+ sensitivity of InsP3 production towards more physiologically relevant concentrations occurring during diastole (100 nM). These findings strongly suggest the presence in heart of a particulate Ca2(+)-dependent phospholipase C, whose activity is regulated by guanine nucleotides. This Ca2(+)-dependent phospholipase C observed in a cell free system was evidenced also in a multicellular system when altering the free Ca2+ concentrations around the physiological range. The results support the possibility that the enzyme might be activated during each cardiac cycle and thus produce two potential activators of cardiac contraction, namely InsP3 and diglycerides.

Animals↗
PubMed1989

The separation of [32P]inositol phosphates by ion-pair chromatography: optimization of the method and biological applications.

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and [3H]inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.

Blood Platelets↗
PubMed1988

Activation of the phosphatidylinositol metabolic pathway by low molecular weight B cell growth factor.

The possible role of phosphatidylinositol breakdown in the induction of proliferation of human activated B cells by low molecular weight B cell growth factor (LMW-BCGF) was examined. LMW-BCGF was found to induce a rapid rise in the concentration of inositol trisphosphate (InsP3) in [3H]inositol-loaded B cell blasts, obtained by prior anti-mu antibody activation. A concomitant decrease in the concentration of phosphatidylinositol 4,5-bisphosphate could be detected at the same time. Maximum generation of InsP3 occurred within 15-30 s after the addition of the LMW-BCGF ligand to the activated B cells, then was followed by a slow decrease and return to control values. The amount of InsP3 generated by phosphatidylinositol hydrolysis was dependent on the concentration of LMW-BCGF. This effect was only detected in B cells already preactivated by a first signal such as anti-mu antibody and not in resting unstimulated B cells. In contrast, under similar conditions, interleukin 2, another B cell growth-promoting lymphokine, did not alter the rate of formation of the various phosphatidylinositol breakdown products. An augmentation of the [Ca2+]i concentration was also detected in activated B cells upon addition of LMW-BCGF and this increase could be blocked by TMB-8, a specific inhibitor of endoplasmic reticulum calcium release. Hydrolysis of phosphoinositides thus represents an essential component in the mechanism of transduction of the signal provided by LMW-BCGF.

B-Lymphocytes↗
PubMed1988

ATP and other adenine compounds increase mechanical activity and inositol trisphosphate production in rat heart.

1. The effects of adenosine 5'-triphosphate (ATP) and other adenine compounds were examined on rat papillary and right ventricular muscles in the presence of 10 microM-propranolol, 10 microM-atropine and 0.1 microM-prazosin or 10 microM-phentolamine. 2. Adenosine, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP and alpha,beta-methylene ATP (APCPP) produced small positive inotropic effects, sometimes preceded by transient negative effects. 3. 8-Phenyltheophylline (8-PT), a P1-purinoceptor antagonist antagonized the negative effects and increased the positive inotropy induced by ATP and adenosine. 4. In the presence of APCPP, a P2-purinergic agonist, ATP had only negative inotropic effects. 5. Adenosine and ATP increased inositol 1, 4, 5- and inositol 1, 3, 4-trisphosphate as well as inositol mono- and bisphosphate formation. Maximal effects were obtained at concentrations of 0.5 mM. 6. APCPP increased inositol phosphate formation while 8-PT did not prevent the effects of adenosine and ATP. 7. It is suggested that P2-purinoceptor activation induces both a positive inotropy and an increase in inositol-lipid metabolism in rat ventricular muscles.

Adenine Nucleotides↗
PubMed1987

Does the inositol tris/tetrakisphosphate pathway exist in rat heart?

Appearance of two isomers of inositol trisphosphate (InsP3) was observed when [3H]inositol prelabelled rat heart ventricles were stimulated for 10 and 30 s with noradrenaline. In contrast, inositol tetrakisphosphate (InsP4) could not be detected. However the existence of the inositol tris/tetrakisphosphate pathway was demonstrated by studying [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) metabolism in a soluble fraction of rat heart. There, [3H]Ins-1,4,5-P3 was phosphorylated to form [3H]Ins-1,3,4,5-P4. Raising [Ca2+] from 1 nM to 1 microM increased InsP3 kinase activity by 2-fold (EC50 for Ca2+ approx. 56 nM). This effect appeared to be due to an increase of the apparent Vmax of the enzyme while the apparent Km was unchanged.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

How far does phospholipase C activity depend on the cell calcium concentration? A study in intact cells.

The dependence of phospholipase C activity on the cytosolic Ca2+ concentration ([Ca2+]i) was studied in intact liver cells treated with the Ca2+-mobilizing hormone vasopressin, or not so treated. Phospholipase C (PLC) activity was estimated from the formation of [3H]inositol trisphosphate (InsP3) and the degradation of [3H]phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The [Ca2+]i of the cells was clamped from 29 to 1130 nM by quin2 loading. This wide concentration range was obtained by loading the hepatocytes with a high concentration of the Ca2+ indicator in low-Ca2+ medium or by using the Ca2+ ionophore ionomycin in medium containing Ca2+. In resting cells, in which [Ca2+]i was 193 nM, treatment with 0.1 microM-vasopressin which stimulates liver PLC maximally, tripled InsP3 content and raised [Ca2+]i to 2 microM within 15 s. Lowering [Ca2+]i partially decreased cell InsP3 content as well as the ability of vasopressin to stimulate InsP3 formation maximally. At 29 nM, the lowest Ca2+ concentration obtained in isolated liver cells, basal InsP3 content was 64% of that measured in control cells. Addition of vasopressin no longer affected [Ca2+]i, but significantly increased InsP3 by 200%, although less than in the controls (300%). The maintenance of the greater part of the PLC response at constant [Ca2+]i indicated that, in the liver, InsP3 formation does not result from an increase in [Ca2+]i. The effects of lowering [Ca2+]i were reversible. When low cell [Ca2+]i was restored to a normal value, resting InsP3 content and the ability of vasopressin to stimulate InsP3 formation maximally by 300% were also restored. Raising [Ca2+]i from 193 to 1130 nM had little effect on the InsP3 content or the vasopressin-mediated increase in InsP3. In agreement with the stimulation of PLC activity by vasopressin, cell [3H]PtdInsP2 and total PtdInsP2 were degraded by application of this hormone for 15 s. In contrast, when [Ca2+]i was lowered to 29 nM, basal [3H]PtdInsP2 and total PtdInsP2 were increased by about 30%, [3H]PtdInsP2 was further increased by vasopressin, but total PtdInsP2 was not changed. These results show that, in intact hepatocytes, PLC is little affected by [Ca2+]i concentrations above 193 nM, but is partially dependent on Ca2+ below that value. They suggest that, in addition to activating PLC activity, vasopressin might stimulate PtdInsP2 synthesis, presumably via phosphatidylinositol-phosphate kinase, and that this pathway might predominate in cells with low [Ca2+]i.

Aminoquinolines↗
PubMed1986

Inositol phosphate production following alpha 1-adrenergic, muscarinic or electrical stimulation in isolated rat heart.

A possible participation of polyphosphoinositide metabolism in the excitation-contraction coupling in heart was investigated. Isolated rat ventricles prelabelled with myo-[2-3H]inositol were stimulated by conditions that increase mechanical activity. Both noradrenaline and carbachol increased the basal level of IP3, IP2 and IP by the activation of alpha 1-adrenergic and muscarinic receptors, respectively. Electrical stimulation accelerated inositol lipid degradation by phospholipase C thus enhancing the IP3 level as compared to quiescent ventricles. It is proposed that IP3 may be involved in excitation-contraction coupling in cardiac tissue.

Animals↗
PubMed1986

Effect of cyclic AMP-dependent hormones and Ca2+-mobilizing hormones on the Ca2+ influx and polyphosphoinositide metabolism in isolated rat hepatocytes.

The effect of the interaction between the Ca2+-mobilizing hormone adrenaline, used as alpha-adrenergic agonist, and cyclic AMP-dependent hormones, including beta-adrenergic agonists and glucagon, on the initial 45Ca2+ uptake rate and polyphosphoinositide metabolism were investigated in isolated rat hepatocytes. Each hormone alone increased the initial 45Ca2+ uptake rate. When adrenaline was added without inhibitor, it induced a rise in the initial 45Ca2+ uptake rate larger than the sum of the rises elicited by its alpha and beta components singly. Similarly, when adrenaline was used as an alpha-agonist and added together with glucagon, it enhanced the initial 45Ca2+ uptake rate synergistically. Kinetic analysis of the initial 45Ca2+ uptake rate measured at different Ca2+ concentrations suggested that the increased influx elicited by the combination of adrenaline as alpha-adrenergic agonist and glucagon reflects an activation of the rate of Ca2+ transport via a homogeneous population of Ca2+ channels or carriers. Dose-response curves for the alpha-adrenergic action of adrenaline or glucagon applied in the presence of increasing doses of glucagon or adrenaline showed that each hormone increases the maximal response to the other without affecting its ED50. Measurement of polyphosphoinositide hydrolysis and of the inositol phosphates formed in the presence of adrenaline or vasopressin and/or glucagon showed that Ca2+-mobilizing hormones and glucagon had no synergistic effects on inositol 1,4,5-trisphosphate production. It is therefore proposed that the synergistic action of glucagon and Ca2+-mobilizing hormones on Ca2+ influx occurs at a step that takes place close to the Ca2+ channels or carriers themselves. The Ca2+ gating involved might be mainly controlled by two products, one of them arising from the polyphosphoinositide metabolism, and the other from the increase in internal cyclic AMP.

Animals↗
PubMed1985

Synergistic stimulation of the Ca2+ influx in rat hepatocytes by glucagon and the Ca2+-linked hormones vasopressin and angiotensin II.

Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin II, and the effects on the initial 45Ca2+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly increased the initial 45Ca2+ uptake rate. When glucagon was added together with vasopressin or angiotensin II--both of which when added separately increase the initial 45Ca2+ uptake rate but did not affect the cellular content of cyclic AMP--the measured initial 45Ca2+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca2+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or forskolin, suggesting that cyclic AMP augments both the resting Ca2+ and the vasopressin- or angiotensin II-stimulated influx. Measurement of the initial 45Ca2+ uptake rate as a function of the extracellular Ca2+ concentration indicated that the increase in the Ca2+ influx resulting from single or combined glucagon and vasopressin administration occurred through a homogeneous population of Ca2+ gates. These hormones were found to raise both the apparent Km for external Ca2+ and the apparent Vmax of the Ca2+ influx. The maximal increase in these two parameters was observed when the two hormones were added together. This suggests that glucagon and vasopressin synergistically stimulate the same Ca2+ gating mechanism. The dose-response curves for the action of glucagon or vasopressin applied in the presence of increasing concentrations of vasopressin or glucagon, respectively, showed that each hormone increases the maximal response to the other without affecting its ED50. It is proposed that glucagon and the Ca2+-linked hormones control the cellular concentration of two intermediates which are both necessary to allow Ca2+ entry into the cells.

Angiotensin II↗
PubMed1985

A regulatory calcium-binding site for calcium channel in isolated rat hepatocytes.

Loading isolated rat hepatocytes with high concentrations of the fluorescent Ca2+-chelator quin-2 in the absence of extracellular Ca2+ decreases by about 3-fold the cytosolic Ca2+ concentration ([Ca2+]i). In these low [Ca2+]i cells, the initial 45Ca2+ uptake rate, assumed to represent the Ca2+ influx, is stimulated to a level close to that promoted by maximal doses of vasopressin and angiotensin II in control cells. The subsequent addition of Ca2+ to the quin-2-loaded hepatocytes results in a rapid increase in [Ca2+]i and a return of Ca2+ influx towards the basal level usually observed in nonloaded cells. This indicates that the Ca2+ influx is dependent on [Ca2+]i but not on the quin-2 load itself. In the low [Ca2+]i cells, both the apparent Km and the apparent Vmax of the Ca2+ influx are increased as compared to the controls, indicating that the properties of the channels activated by lowering [Ca2+]i are apparently identical to those initiated by the hormones (Mauger, J.-P., Poggioli, J., Guesdon, F., and Claret, M. (1984) Biochem. J. 221, 121-127). It is proposed that in the isolated rat hepatocytes there is an inverse relationship between the Ca2+ influx and [Ca2+]i. Under resting conditions, [Ca2+]i might be high enough to partially inhibit the Ca2+ influx via a Ca2+ binding to an inhibitory site presumably located at the inner membrane surface. The role of the site in the hormonal action is discussed.

Aminoquinolines↗
PubMed1984

Noradrenaline, vasopressin and angiotensin increase Ca2+ influx by opening a common pool of Ca2+ channels in isolated rat liver cells.

The effects of the Ca2+-mobilizing hormones noradrenaline, vasopressin and angiotensin on the unidirectional influx of Ca2+ were investigated in isolated rat liver cells by measuring the initial rate of 45Ca2+ uptake. The three hormones increased Ca2+ influx, with EC50 values (concentrations giving half-maximal effect) of 0.15 microM, 0.44 nM and 0.8 nM for noradrenaline, vasopressin and angiotensin respectively. The actions of noradrenaline and angiotensin were evident within seconds after their addition to the cells, whereas the increase in Ca2+ influx initiated by vasopressin was slightly delayed (by 5-15s). The activation of Ca2+ influx was maintained as long as the receptor was occupied by the hormone. The measurement of the resting and hormone-stimulated Ca2+ influxes at different external Ca2+ concentrations revealed Michaelis-Menten-type kinetics compatible with a saturable channel model. Noradrenaline, vasopressin and angiotensin increased both Km and Vmax. of Ca2+ influx. It is proposed that the hormones increase the rate of translocation of Ca2+ through a common pool of Ca2+ channels without changing the number of available channels or their affinity for Ca2+.

Angiotensin II↗
PubMed1983

Alpha-adrenergically mediated changes in membrane lipid fluidity and Ca2/ binding in isolated rat liver plasma membranes.

Noradrenaline (0.1-5 microM, in the presence of 5 microM propranolol to block beta-receptors), ATP (100 microM) and angiotensin II (0.1 microM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the alpha-antagonists phenoxybenzamine (50 microM) and phentolamine (1 microM). The response to a maximal dose of noradrenaline (5 microM) and that to ATP (100 microM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 microM) along with the foreign amphiphile Na+-oleate (1-30 microM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 microM) and angiotensin II (0.1 microM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the alpha-antagonist phentolamine (5 microM). It is proposed that noradrenaline, via alpha-adrenergic receptors and other Ca2+ -mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.

Adenosine Triphosphate↗
PubMed1983

Norepinephrine-induced loss of phosphatidylinositol from isolated rat liver plasma membrane. Effects of divalent cations.

Norepinephrine at 5 microM induces a rapid (60 s) and specific loss of phosphatidylinositol (PtdIns) when added to isolated rat liver plasma membranes. The hormone action is inhibited by the alpha-adrenergic antagonist phentolamine (20 microM). Depletion of Mg2+ and Ca2+ singly or in combination from the incubation buffer mimicks the hormone effect on PtdIns breakdown. No further effect on PtdIns degradation could be measured when norepinephrine was added to the cation-depleted buffers. Addition of the Ca2+ ionophore A23187 to the isolated membranes has no effect. It is concluded that PtdIns degradation can be provoked in isolated rat liver plasma membrane through alpha-adrenergic receptor activation and that this effect is dependent on divalent cations in the sense that loss of cations from the membrane allows degradation to commence.

Animals↗
PubMed1983

Mechanism of action of noradrenaline on the sodium-potassium pump in isolated rat liver cells.

Noradrenaline, which mobilizes Ca from intracellular stores, stimulated the Na-K pump in isolated rat liver cells. This resulted in transient decreases in internal Na content and external K concentration. The effect of the hormones was observed in the presence of the beta-adrenergic antagonist propranolol and was blocked by the alpha-antagonist phenoxybenzamine. Prazosin appeared to be 1000 times more potent than yohimbine in suppressing the cell response to the hormone, suggesting that the effect is mediated by an activation of alpha 1-adrenergic receptors. Externally applied ATP and the Ca ionophore A23187 which, in common with alpha-agonists, deplete internal Ca stores in this tissue, similarly stimulated the Na-K pump and transiently decreased internal Na and external K. The effects of noradrenaline and ATP were not additive. Moreover, the cell response to ATP was observed in the presence of the alpha-antagonist phenoxybenzamine, indicating that though acting via separate receptors, noradrenaline and ATP use a common mechanism to alter the carrier. The effect of noradrenaline and A23187 on the Na-K pump was not dependent on the presence of extracellular Ca. In contrast, when the hepatocytes were incubated in Ca-free medium for long periods (cell Ca depletion) the activity of the Na-K pump was increased to a level corresponding to that induced by maximal doses of noradrenaline. In these conditions, noradrenaline and A23187 did not increase the pump activity further. In cells in which the Na content was raised, leading to a 3-fold increase in the Na-K pump activity, noradrenaline continued to be able to stimulate the pump. Again long-term incubations in Ca-free medium increased the pump activity and the effect of noradrenaline was greatly reduced. It is proposed that in isolated rat liver cells alpha-agonists and applied ATP influence the Na-K pump by releasing Ca bound to plasma membranes, thus removing the inhibitory effect of this ion on the Na pump.

Adenosine Triphosphate↗
PubMed1983

Effects of antimycin A on receptor-activated calcium mobilization and phosphoinositide metabolism in rat parotid gland.

The effects of the mitochondrial poison, antimycin A, on responses of parotid acinar cells to cholinergic stimuli were examined. Antimycin A (10 microM) partially inhibited the agonist-induced increase in 86Rb efflux. Specifically, the initial transient phase of the response, believed to arise from intracellular calcium release, was partially inhibited, while the sustained phase of the response, believed to result from calcium entering from the extracellular space, was completely blocked. The stimulation of 45Ca influx by a cholinergic agonist was also completely blocked. Antimycin A (10 microM) caused a rapid loss of [32P] polyphosphoinositides. Stimulation of [32P]phosphatidylinositol breakdown and [32P] phosphatidate synthesis by methacholine was blocked by antimycin A. Breakdown of [32P]phosphatidylinositol-4,5-bisphosphate in response to cholinergic stimulation was partially inhibited. These results indicated that the activation by cholinergic agonists of cellular calcium mobilization as well as effects on phosphoinositide metabolism are similarly inhibited by antimycin A. Furthermore, this presumably indicates a role for ATP in receptor-activated calcium mobilization and phosphoinositide turnover.

Animals↗
PubMed1982

Net calcium fluxes in rat parotid acinar cells: evidence for a hormone-sensitive calcium pool in or near the plasma membrane.

The effects of receptor stimulation on net fluxes of 45Ca in parotid acinar cells were investigated. When cellular 45Ca content was near steady-state, muscarinic receptor activation by carbachol caused a net efflux of 45Ca (not always reproducible) followed by a net influx. In the presence of excess ethylene glycol bis (beta-aminoethyl ether) N,N,N,N-tetraacetic acid, net efflux invariably occurred, but the influx phase was prevented. When the muscarinic receptor antagonist atropine was added to the medium during the influx phase, an abrupt transient influx occurred followed by a return of net influx to the prestimulation level. When cellular responses believed to reflect intracellular ionized Ca (k + permeability, protein secretion) were examined under similar conditions, atropine invariably had an inhibitory effect. The Ca taken up in response to atropine apparently replenishes the hormone-sensitive pool of cellular Ca since it can be released subsequently by adrenoceptor activation. Taken together, these observations suggest that when atropine is administered to cholinergically activated cells, the hormone-sensitive Ca pool rapidly refills from the extracellular fluid without a concomitant increase in ionized intracellular Ca. Thus, it is suggested that this Ca pool is most likely associated with the plasma membrane.

Animals↗