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J Poggioli

Publications and source records attributed to J Poggioli.

At least 19 recordsLinked to original sources
PubMed1992

Plasma membrane Na(+)-H+ antiporter and H(+)-ATPase in the medullary thick ascending limb of rat kidney.

To characterize H+ transport mechanisms in a fresh suspension of rat medullary thick ascending limb (MTAL) tubules, we have monitored intracellular pH (pHi) with use of the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. First, a Na(+)-H+ antiporter was identified in bicarbonate-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered media at 25 degrees C. pHi recovery of Na-depleted acidified cells was dependent on extracellular sodium concentration, which was inhibited by amiloride in a manner consistent with simple competitive interaction with one external transport site (amiloride Ki = 1.5-2.1 x 10(-5) M); Na-induced pHi recovery of acidified cells was electroneutral since it was not affected by 5 or 100 mM extracellular potassium in the presence or absence of valinomycin. Second, at 37 degrees C, pHi recovery after acute intracellular acidification caused by 40 mM acetate addition to cell suspension was inhibited 36% by 200-400 nM bafilomycin A1, a macrolide antibiotic that specifically inhibits vacuolar-type H(+)-ATPase at submicromolar concentrations. In addition, amiloride-insensitive pHi recovery was inhibited by bafilomycin A1, 10(-3) M N-ethylmaleimide, and 10(-4) M preactivated omeprazole but not by 10(-5) M vanadate, 10(-4) M SCH 28080, or removal of extracellular potassium. Also, metabolic inhibition by absence of substrate, 10(-4) M KCN, or 5 x 10(-4) M iodoacetic acid inhibited amiloride-insensitive pHi recovery. The inhibitory effects of absence of metabolic substrate and iodoacetic acid were removed by reexposure to glucose and L-leucine and by exogenous ATP, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1992

Effects of angiotensin II and nonpeptide receptor antagonists on transduction pathways in rat proximal tubule.

Because the presence of the angiotensin II (ANG II)-dependent phosphoinositide hydrolysis has been questioned from studies in proximal cells in culture, we looked for this transduction pathway in suspension of freshly isolated rat proximal tubule fragments. ANG II-receptor activation induced a prompt (within 15 s) and sustained increase in [3H]inositol phosphates (IPs; inositol trisphosphate, inositol bisphosphate, and inositol monophosphate). In fura-2-loaded tubules, it elicited a rapid and biphasic rise in cytosolic free calcium ([Ca2+]i) with an early peak (within 15 s) followed by a plateau. The peak was maintained in the absence of extracellular calcium. ANG II-induced inositol trisphosphate and [Ca2+]i rises showed a similar dose dependency, with a 50% effective concentration (EC50) of 2.9 and 5.5 nM, respectively. We checked that ANG II inhibited basal (EC50 4.4 nM) and parathyroid hormone- and forskolin-stimulated cAMP production, the latter effect being inhibited by pertussis toxin pretreatment. The effects of ANG II on IPs and [Ca2+]i were inhibited by the ANG II receptor subtype 1 (AT1) antagonist losartan and not by the ANG II receptor subtype 2 (AT2) antagonists PD 123177 and PD 123319. The effect of ANG II on forskolin-stimulated cAMP was inhibited by losartan and not by PD 123319. In agreement with these results, specific binding of 125I-[Sar1,Ile8]ANG II was markedly inhibited by losartan, whereas PD 123319 had no effect. These results demonstrate that AT1 receptor subtypes are present in intact rat proximal tubule cells and are coupled to both IPs-Ca2+ and cAMP signaling pathways. No evidence for AT2 receptor subtype is found.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1992

Acute variations in extracellular pH modulate transduction pathways of PTH in rat proximal tubule.

An increase in circulating parathyroid hormone (PTH) has been shown to enhance the capacity for the kidney to excrete an acid as well an alkaline load, which suggests that changes in systemic acid-base status may modulate the effect of the hormone on bicarbonate absorption in proximal tubule. In the present study, we tested the possibility that acute variations in extracellular pH (pHe), obtained by modifying bicarbonate concentration at constant PCO2 (40 mmHg), may modulate the responses of intracellular messengers coupled to PTH receptors in a preparation of freshly isolated proximal tubule fragments. Variations in pHe, which induced parallel variations in intracellular pH (pHi), did not affect unstimulated values for adenosine 3',5'-cyclic monophosphate (cAMP) production, inositol trisphosphate accumulation, or cytosolic free Ca2+ concentration. In contrast, reducing pHe from 7.4 to 7.2 elicited a decrease of the PTH-induced cAMP production, whereas increasing pHe from 7.4 to 7.6 enhanced it. The ability for cholera toxin and forskolin (which both bypass PTH receptors) to stimulate cAMP formation was diminished at pHe 7.2 and enhanced at pHe 7.6 (the increase did not achieve statistical significance in the presence of forskolin), suggesting that variations in pHe and/or pHi may affect per se adenylyl cyclase activity. Conversely, reducing pHe from 7.4 to 7.2 enhanced the PTH-induced inositol trisphosphate accumulation and rise in cytosolic free Ca2+ whereas increasing pHe from 7.4 to 7.6 had opposite effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1991

Mechanisms of H+/HCO3- transport in the medullary thick ascending limb of rat kidney.

The rat MTAL secretes protons into the tubular fluid and thus absorbs bicarbonate at substantial rates. Yet the cellular mechanisms of H+/HCO3- transport in the rat MTAL remain largely unsettled. We have performed intracellular pH recovery studies with use of the fluorescent probe BCECF in suspensions of rat MTAL fragments. Luminal H+ secretion occurs by two mechanisms (each responsible for 50% of the normal pHi recovery rate): (1) an electroneutral Na+/H+ antiporter that has an Na-Km of about 11 mM and is inhibited by amiloride (Ki = 2.8 x 10(-5) M); (2) a primary H+ pump that is inhibited by 10(-4) M NEM and 10(-4) M omeprazole, but not by 10(-4) M vanadate or removal of external K. These results suggest the presence of a vacuolar H(+)-ATPase rather than a H(+)-K(+)-ATPase. Basolateral HCO3 exit occurs predominantly by a Cl(-)- and Na(+)-independent electroneutral K+/HCO3- symporter, that has an HCO3-Km of about 17 mM, and is partially inhibited by 10(-4) M DIDS. Basolateral HCO3- efflux was not accompanied by variations of membrane potential monitored with the Em-sensitive fluorescent probe DIS-C3-5, and was not affected by maneuvers that depolarize the cells. It was strongly inhibited by cellular K depletion and dependent on transmembrane K gradient. We conclude that the rat MTAL should secrete protons through both Na+/H+ antiporter and H(+)-ATPase, and that basolateral HCO3- exit should occur through an electroneutral K+/HCO3- symporter.

Bicarbonates↗
PubMed1990

Monoclonal anti-CD23 antibodies induce a rise in [Ca2+]i and polyphosphoinositide hydrolysis in human activated B cells. Involvement of a Gp protein.

Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in human activated B lymphocytes using anti-CD23 mAb. B cell blasts expressing an increased amount of surface CD23 molecule were obtained by stimulation of normal peripheral blood B lymphocytes with Staphylococcus aureus strain Cowan I and IL-4. Anti-CD23 mAb were found to trigger polyphosphoinositide hydrolysis in these cells (and also in tumoral B cells expressing spontaneously CD23) and a rise in [Ca2+]i which could be attributed to mobilization from cytoplasmic pools. This increase in [Ca2+]i could be mimicked, with a comparable time-course, by the addition of InsP3 to permeabilized B cell blasts indicating that the increase in inositol phosphate accumulation induced by the antibodies was due to a preferential attack of phosphatidylinositol-bisphosphate by a specific phosphoinositidase C (PIC). In permeabilized cells, raising the free calcium concentration above 3 microM was found to induce polyphosphoinositides hydrolysis and to activate directly the PIC. Addition of 100 microM GTP-tetralithium salt, a non-hydrolyzable analogue of GTP, also resulted in an increased accumulation of inositol phosphates. A Ca2(+)-dependent PIC, linked to a GTP-binding protein (Gp protein), can thus be activated in B cell blasts. Addition of anti-CD23 antibodies to permeabilized B cells in the presence of a physiologic concentration of Ca2+ (100 nM) evoked, within 10 min, a rise in the various inositol phosphates. This ability of anti-CD23 antibodies to activate PIC was enhanced in the presence of GTP-tetralithium salt 100 microM. By contrast, preincubation with GDP-trilithium salt, a nonhydrolyzable analogue of GDP, caused a marked reduction in the release of inositol phosphates. Preincubation of B cell blasts with Pertussis toxin resulted in a total inhibition of the capacity of the toxin to ADP-ribosylate a 41-kDa protein, probably of the Gi type; in these conditions, no modification of anti-CD23-elicited polyphosphoinositide hydrolysis could be detected. These results suggest that the CD23 molecule may be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to Pertussis toxin.

Adenosine Diphosphate Ribose↗
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↗