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F Pecker

Publications and source records attributed to F Pecker.

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

Activation of Na+/Ca2+ exchange by adenosine in ewe heart sarcolemma is mediated by a pertussis toxin-sensitive G protein.

We studied the effect of adenosine on Na+/Ca2+ exchange activity in ewe heart ventricular sarcolemmal vesicles. Adenosine was found to stimulate Na+/Ca2+ exchange activity in a dose-dependent manner from 0.1 nM to 10 microM, with maximal stimulation (40%) at 0.1 microM adenosine. The Vmax of Na+/Ca2+ exchange was increased, but the Km for Ca2+ was not altered. The effect of adenosine was specific since 1 microM adenine, inosine, and guanosine led to less than 15% stimulation, and adenosine diphosphate had no effect. Caffeine antagonized the activation of Na+/Ca2+ exchange by adenosine, and the order of potency of adenosine analogs was N6-(L-2-phenylisopropyl)adenosine = N6-cyclohexyladenosine = 5'-(N- ethylcarboxamido)adenosine much greater than N6-(D-2-phenylisopropyl)adenosine, indicating the involvement of A1 subclass receptors. The effect of adenosine was mimicked by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and blocked by pertussis toxin treatment. Taken together, these results suggest that A1 subclass receptors coupled to a pertussis toxin-sensitive G protein mediate the activation of Na+/Ca2+ exchange activity by adenosine. We conclude that the negative inotropic effect of adenosine in ventricular muscle, antagonistic toward cyclic AMP, may involve activation of Na+/Ca2+ exchange.

Adenosine↗
PubMed1990

Glucagon-(19-29) exerts a biphasic action on the liver plasma membrane Ca2+ pump which is mediated by G proteins.

We have recently shown that nanomolar concentrations of glucagon-(19-29), which can derive from native glucagon by proteolytic cleavage of the dibasic doublet Arg17-Arg18, inhibit the Ca2+ pump in liver plasma membrane vesicles independently of adenylyl cyclase activation (Mallat, A., Pavoine, C., Dufour, M., Lotersztajn, S., Bataille, D., and Pecker, F. (1987) Nature 325, 620-622). We report here that the regulation of the Ca2+ pump by glucagon-(19-29) is dependent on guanine nucleotides. In the presence of 10 microM guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) or 75 microM GTP, glucagon-(19-29) caused a biphasic regulation of the Ca2+ pump. ATP-dependent Ca2+ transport was inhibited in the presence of 10 pM to 1 nM glucagon-(19-29), while higher concentrations of the peptide (1-100 nM) reversed the inhibition caused by lower ones. GTP gamma S alone, at high concentrations (100 microM), reproduced the inhibitory effect of glucagon-(19-29) and induced a 40% inhibition of the basal activity of the Ca2+ pump which was reversed by low concentrations of glucagon-(19-29) (10 pM to 1 nM). Treatment of rats with cholera toxin resulted in a 70% increase in the basal activity of the Ca2+ pump, a loss of sensitivity to GTP gamma S and to the biphasic regulation by glucagon-(19-29). Treatment with pertussis toxin did not affect the response of the Ca2+ pump to GTP gamma S and glucagon-(19-29). We conclude that glucagon-(19-29) can exert a biphasic effect on the Ca2+ pump which is mediated by G protein(s) sensitive to cholera toxin.

Adenylate Cyclase Toxin↗
PubMed1990

Glucagon stimulates the cardiac Ca2+ current by activation of adenylyl cyclase and inhibition of phosphodiesterase.

Glucagon exerts positive inotropic and chronotropic effects in the heart. Like its glycogenolytic effect in liver cells, the cardiac effects of glucagon are often correlated with adenylyl cyclase stimulation. Therefore, cyclic AMP-dependent phosphorylation of L-type Ca2+ channels might be involved in the inotropic effect of glucagon. There have been no reports, however, of the effects of glucagon on the cardiac Ca2+ current (ICa). Also, the physiological effects of glucagon could involve mechanisms other than stimulation of adenylyl cyclase. Here we show that glucagon enhances ICa in frog and rat ventricular myocytes. The effect of glucagon in rats resulted from a stimulation of adenylyl cyclase. In frogs, however, the effect of glucagon on ICa was smaller and occurred at a concentration tenfold lower than in rats, and adenylyl cyclase was not modified. In addition, cAMP potentiated the effect of glucagon on ICa in frog ventricle, which correlated with the observed inhibition by glucagon of low-Km cAMP phosphodiesterase activity. Therefore, this is an example of a hormone that affects cardiac function in a similar way to a variety of synthetic cardiotonic compounds, such as milrinone and Ro-20-1724. Inhibition of phosphodiesterase activity by glucagon may be essential in animals in which glucagon increases cardiac contractility but does not effectively stimulate adenylyl cyclase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Inhibition of the calcium pump by human parathyroid hormone-(1-34) and human calcitonin in liver plasma membranes.

The effect of human parathyroid hormone-(1-34) (hPTH) and human calcitonin (hCT) on the activity of the Ca2(+)-extrusion pump in liver plasma membranes was studied. Both hormones were found to be potent inhibitors of Ca2+ transport and the related high-affinity (Ca2(+)-Mg2+)-ATPase activity, causing maximal inhibition of 25-30% at concentrations of 100 nM. Half-maximal inhibition was observed with 20 nM-hPTH and with 0.5 nM-hCT. By comparison, salmon calcitonin and intact bovine parathyroid hormone-(1-84) were inhibitory only at 10 microM. The effects of hCT and hPTH on the Ca2+ pump activity were not mimicked by cyclic AMP. Also, 10 microM of either hPTH-(1-34) or hCT did not alter the 45Ca2+ influx rate into isolated hepatocytes. We conclude that inhibition of Ca2+ efflux, rather than the stimulation of Ca2+ influx, may play a functional role in the control of hepatic calcium homeostasis by hPTH-(1-34) and hCT.

Biological Transport, Active↗
PubMed1989

[Glucagon is processed to the (19-29) fragment at the level of the hepatocyte membrane].

Upon incubation with hepatic plasma membranes, glucagon is processed into its (19-29) C-terminal fragment. This suggests that, in physiological conditions, glucagon is processed in a target tissue at the level of its Arg17-Arg18 basic doublet, leading to the production of a fragment which is known to display an original biological specificity, namely the modulation of the calcium pump present in hepatocyte plasma membrane.

Animals↗
PubMed1987

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles.

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.

Adenosine Triphosphate↗
PubMed1987

Cholera toxin blocks glucagon-mediated inhibition of the liver plasma membrane (Ca2+-Mg2+)-ATPase.

We have previously shown that liver plasma membrane (Ca2+-Mg2+)-ATPase activity is inhibited by glucagon. To investigate the possible involvement of a GTP-binding (G) protein in this regulation, we have examined the effects of pertussis toxin and cholera toxin on inhibition of (Ca2+-Mg2+)-ATPase by glucagon. Treatment of liver plasma membranes with pertussis toxin did not affect the sensitivity of (Ca2+-Mg2+)-ATPase to the hormone. In contrast, treatment of plasma membranes or prior injection of animals with cholera toxin prevented inhibition of the (Ca2+-Mg2+)-ATPase by glucagon. Even though adenylate cyclase activity was increased by cholera toxin treatment, addition of cyclic AMP did not mimic the effect of cholera toxin in blocking glucagon-mediated inhibition of (Ca2+-Mg2+)-ATPase activity. These data suggest that a cholera toxin-sensitive protein, perhaps Gs or a Gs-like protein, is involved in the regulation of liver (Ca2+-Mg2+)-ATPase activity. The results emphasize the possible role of Gs-like proteins in regulation of enzymes other than adenylate cyclase and suggest that the study of (Ca2+-Mg2+)-ATPase may provide a useful enzymatic system to examine such regulation.

Adenylate Cyclase Toxin↗
PubMed1985

The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action.

Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.

Animals↗
PubMed1985

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes.

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.

Adenosine Triphosphate↗
PubMed1984

Inhibition by glucagon of the calcium pump in liver plasma membranes.

The ATP-dependent calcium transport in plasma membrane vesicles prepared from rat liver was inhibited by 0.1 to 10 microM glucagon. Inhibition of the high affinity (Ca2+-Mg2+)-ATPase was observed concomitantly. This effect was neither mimicked by cyclic AMP nor by dibutyryl cyclic AMP. A study of the structure-activity relationships of six glucagon derivatives demonstrated the specificity of glucagon action since only one or two analogs markedly altered the (Ca2+-Mg2+)-ATPase activity. The study also demonstrated the total absence of correlation between adenylate cyclase activation and (Ca2+-Mg2+)-ATPase inhibition induced by these glucagon derivatives. The decrease in the maximal velocities induced by glucagon of both calcium transport and (Ca2+-Mg2+)-ATPase activity were related to a reduction in the rate of dephosphorylation of the Ca-dependent phosphorylated intermediate of the enzyme. This phosphorylated intermediate was characterized as a 32P-labeled 110,000-dalton protein which accumulated to 50 to 150% over the basal level in the presence of glucagon. The present results demonstrate a novel aspect of the role of glucagon as a calcium-mobilizing agent.

Animals↗
PubMed1982

A membrane-bound protein inhibitor of the high affinity Ca ATPase in rat liver plasma membranes.

The Ca ATPase from rat liver plasma membranes has been recently characterized and partially purified in our laboratory and was shown to depend on a membrane-bound protein activator (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). In the present study, we report that a factor derived from ammonium sulfate washings of rat liver plasma membranes inhibits the partially purified enzyme activity measured in the presence of activator. This factor is a protein as judged by its sensitivity to heat and trypsin. A molecular weight of 29,000 was determined by sucrose gradient centrifugation and gel chromatography. The action of the inhibitor is due to a decrease in the maximal velocity of the enzyme reaction and is reversed by an excess of the activator associated with the enzyme. An important point in the mode of action of this inhibitor is its absolute dependence on magnesium, which most probably explains the difficulty in detecting the plasma membrane Ca ATPase when MgCl2 is added to the assay medium.

Animals↗
PubMed1981

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin.

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.

Adenosine Triphosphatases↗
PubMed1979

The adenylate cyclase system in human liver: characterization, subcellular distribution and hormonal sensitivity in normal or cirrhotic adult, and in foetal liver.

1. Adenylate cyclase (EC 4.6.1.1) activity was characterized in human liver, and its subcellular distribution compared with that of three other potential enzyme markers of the pericellular membrane: leucine aminopeptidase (EC 3.4.11.1), gamma-glutamyltransferase (EC 2.3.2.2) and 5'-nucleotidase (EC 3.1.3.5). Although these three enzyme activities were detected in each of the subcellular fractions studied, 85% of the total adenylate cyclase activity was found in the 1000 g pellet ('nuclear' fraction) with a threefold increase in specific activity as compared with the homogenate. No adenylate cyclase activity existed in the 150 000 g supernatant fraction. 2. In the 'nuclear' fraction, adenylate cyclase activity was increased in a dose-dependent fashion by glucagon with a half-maximal stimulation at 10 nmol/l and a maximal four- to seven-fold increase at 1 mumol/l. Catecholamines activated adenylate cyclase 2.5- to three-fold, with an order of potency (protokylol greater than isoprenaline greater than adrenaline greater than noradrenaline) typical of a beta 2-adrenoreceptor. Prostaglandin E1 and NaF also stimulated cyclase two- and four-fold respectively. Insulin, serotonin, dopamine, thyroid-stimulating hormone and ACTH had no effect. Adenosine provoked a weak inhibition at 0.1 mmol/l. Finally guanosine triphosphate and 5'-guanylyl imidodiphosphate induced a marked increase in basal activity, four- and eight-fold respectively, but both reduced the relative increase in enzyme activity due to glucagon or adrenaline. 3. Cyclase from foetal liver (12--16 weeks old) and cirrhotic adult liver appeared to behave similarly to that from normal liver; however, foetal cyclase was more active, and cirrhotic enzyme less active than normal adult liver. Both systems responded to catecholamines via a beta 2-adrenoreceptor. 4. These results validate the use of rat liver adenylate cyclase as a tool for pharmacological and physiological studies.

Adenosine↗
PubMed1977

Activation of epinephrine-sensitive adenylate cyclase in rat liver by cytosolic protein-nucleotide complex.

The cytosolic fraction from rat liver enhanced the basal and glucagon-sensitive adenylate cyclase (EC 4.6.1.1) of hepatic plasma membranes and revealed its (R)-(-)-epinephrine sensitivity. Such phenomena were usually obtained by the addition of low concentrations of GTP to the medium employed for the cyclase assay. Comparative studies of the behavior of the cytosolic factor and GTP in response to various treatments were performed. We present evidence that the stimulatory activity of the soluble factor was reduced after treatment by alkaline phosphatase, by the nucleotide phosphohydrolases present in the plasma membranes, and by trypsin. These results strongly suggest that the soluble activator is a nucleotide-protein complex and further demonstrate that GTP may be of physiological significance in the regulation of the adenylate cyclase system.

Adenylyl Cyclases↗