Chronic hypocalcemia of vitamin D deficiency leads to lower intracellular calcium concentrations in rat hepatocytes.

Several lines of evidence indicate that calcium deficiency is associated with cellular defects in many tissues and organs. Owing to the large in vivo gradient between ionized extra- and intracellular Ca2+ concentrations ([Ca2+]i), it is generally recognized that the prevailing circulating Ca2+ does not significantly affect resting cytosolic Ca2+. To probe the consequences of hypocalcemia on [Ca2+]i, a model of chronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hepatocytes were isolated from livers of hypocalcemic D-deficient, of normocalcemic D3-repleted, or of normal control rats presenting serum Ca2+ of 0.78 +/- 0.02, 1.24 +/- 0.03, or 1.25 +/- 0.01 mM, respectively (P < 0.0001). [Ca2+]i was measured in cell couplets using the fluorescent probe Fura-2. Hepatocytes of normocalcemic D3-repleted and of normal controls exhibited similar [Ca2+]i of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had significantly lower resting [Ca2+]i (172 +/- 10 nM; P < 0.0003). Stimulation of hepatocytes with the alpha 1-adrenoreceptor agonist phenylephrine illicited increases in cytosolic Ca2+ leading to similar [Ca2+]i and phosphorylase a (a Ca(2+)-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accompanied by a rapid decay in the sustained phase of the [Ca2+]i response. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers responded with a blunted maximal [Ca2+]i of 237.6 +/- 18.7 compared with 605.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocalcemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+]i homeostasis associated with chronic hypocalcemia revealed the presence of an unchanged plasma membrane Ca2+ ATPase but of a significant decrease in agonist-stimulated Ca2+ entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, thus indicate that, in rat hepatocytes, the in vivo calcium status significantly affects resting [Ca2+]i, and from this we raise the hypothesis that this lower than normal [Ca2+]i may be linked, in calcium disorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.

Calcium-binding properties and ATPase activities of rat liver plasma membranes.

Plasma membranes from rat liver purified according to the procedure of Neville bind calcium ions by a concentration-dependent, saturable process with at least two classes of binding sites. The higher affinity sites bind 45 nmol calcium/mg membrane protein with a K(D) of 3 microM. Adrenalectomy increases the number of the higher affinity sites and the corresponding K(D). Plasma membranes exhibit a (Na(+)-K(+))-independent-Mg(2+)-ATPase activity which is not activated by calcium between 0.1 microM and 10 mM CaCl(2). Calcium can, with less efficiency, substitute for magnesium as a cofactor for the (Na(+)-K(+))-independent ATPase. Both Mg(2+)- and Ca(2+)-ATPase activities are identical with respect to pH dependence, nucleotide specificity and sensitivity to inhibitors. But when calcium is substituted for magnesium, there is no detectable membrane phosphorylation from [gamma-(32)P] ATP as it is found in the presence of magnesium. The existence of high affinity binding sites for calcium in liver plasma membranes is compatible with a regulatory role of this ion in membrane enzymic mechanisms or in hormone actions. Plasma membranes obtained by the procedure of Neville are devoid of any Ca(2+)-activated-Mg(2+)-ATPase activity indicating the absence of the classical energy-dependent calcium ion transport. These results would suggest that the overall calcium-extruding activity of the liver cell is mediated by a mechanism involving no direct ATP hydrolysis at the membrane level.

Inhibition of the sarcolemmal Ca2+ pump in embryonic chick heart cells by mini-glucagon.

The effect of mini-glucagon, the metabolite (19-29) of glucagon was examined on the sarcolemmal (SL) Ca2+ pump activity measured in situ, in single quiescent embryonic chick heart ventricular cells loaded with Fura-2. The method consisted in triggering limited cytosolic Ca2+ concentration ([Ca2+]i) pulses by the addition of the Ca2+ ionophore 4-bromo-A23187. [Ca2+]i decays, imposed by the addition of EGTA, were monitored in conditions in which only the SL Ca2+ pump could ensure [Ca2+]i removal, i.e. in the presence of the sarcoplasmic reticular (SR) Ca2+ pump specific inhibitor, thapsigargin, substituting NaCI by LiCI in the external medium in order to quench the Na+/Ca2+ exchanger, and under null Ca2+ gradient. Mini-glucagon elicited a dose-dependent inhibition of the SL Ca2+ pump, maximal 80% inhibition being observed with 1 nM mini-glucagon. In addition to its effect on the SL Ca2+ pump, mini-glucagon evoked a delayed onset of a [Ca2+]i oscillatory response in cells incubated in normal conditions. Both effects of mini-glucagon were mimicked by vanadate tested at 2 microM, a concentration at which it acts as a specific inhibitor of the SL Ca2+ pump. These results define the contribution of the cardiac sarcolemmal Ca2+ pump to Ca2+ homeostasis in situ and its role as a target for mini-glucagon action.

The liver plasma membrane Ca2+ pump: hormonal sensitivity.

The liver plasma membrane Ca2+ pump is supposed to extrude cytosolic calcium out of the cell. This system has now been well defined on the basis of its plasma membrane origin, its high affinity Ca2+ -stimulated ATPase activity, its Ca2+ transport activity, its phosphorylated intermediate. The liver calcium pump appears to be a target of hormonal action since it has been shown that glucagon and calcium mobilizing hormones namely alpha 1-adrenergic agonists, vasopressin, angiotensin II inhibit this system. The present review details the mechanism of calcium pump inhibition by glucagon and points out its difference from the inhibition process induced by calcium mobilizing hormones. We conclude that the inhibitory action of the Ca2+ mobilizing hormones and glucagon on the liver plasma membrane Ca2+ pump might play a key role in the actions of these hormones by prolonging the elevation in cytosolic free Ca2+.

[Role of guanidylic nucleotides in the adenylate cyclase activity of the rat liver]. / Rôle des nucléotides guanidyliques dans l'activité adénylate cyclasique du foie de rat

Glucagon and adrenaline exert their action upon the liver via the cyclic AMP synthetizing system located in the plasma membrane. The enzyme adenylate cyclase is further regulated by guanyl nucleotides. It has been recently shown that the rat liver plasma membrane system could respond to GTP by simultaneous increase in the cyclase activity in response to glucagon and by the dissociation of this hormone from its binding sites (1). Unambiguous relationship between the activating effect of GTP upon the cyclase and its action upon glucagon binding has not been determined yet (2). This problem was approached using the in vitro action of epinephrine as a model. When 1 to 100 muM GTP or DGP were added to rat liver plasma membranes isolated from adrenalectomized animals, they increased markedly the response of the cyclase system to epinephrine. These effects could be observed in the absence of an ATP-regenerating system and were mimicked by 5'-guanylyl diphosphonate; GTP and GDP were the most active compounds followed by ITP, CTP and by a series of guanyl derivatives. UTP, as well as guanosine, GMP, cyclic GMP and ppGpp were inactive. Guanyl nucleotides did not increase the affinity of the cyclase system for the activating hormones, but enhanced the affinity for ATP-Mg and also the Vmax of the reaction. Finally, GTP, ATP, CTP, UTP but not GDP displaced epinephrine bound to plasma membranes by a mere chelation phenomenon. It is concluded that 1) guanyl nucleotides do not act primarily by influencing the binding of hormones to the membranes; 2) they act directly upon the catalytic subunit of the cyclase; 3) the low concentrations of GTP required for its action strongly suggest that this nucleotide plays a role in the physiological regulation of the intrahepatic cyclic AMP level.

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.

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.

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.

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+.

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.

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.

Beta(2)-adrenergic receptor agonists increase intracellular free Ca(2+) concentration cycling in ventricular cardiomyocytes through p38 and p42/44 MAPK-mediated cytosolic phospholipase A(2) activation.

We have recently reported that arachidonic acid mediates beta(2)-adrenergic receptor (AR) stimulation of [Ca(2+)](i) cycling and cell contraction in embryonic chick ventricular cardiomyocytes (Pavoine, C., Magne, S., Sauvadet, A., and Pecker, F. (1999) J. Biol. Chem. 274, 628-637). In the present work, we demonstrate that beta(2)-AR agonists trigger arachidonic acid release via translocation and activation of cytosolic phospholipase A(2) (cPLA(2)) and increase caffeine-releasable Ca(2+) pools from Fura-2-loaded cells. We also show that beta(2)-AR agonists trigger a rapid and dose-dependent phosphorylation of both p38 and p42/44 MAPKs. Translocation and activation of cPLA(2), as well as Ca(2+) accumulation in sarcoplasmic reticulum stores sensitive to caffeine and amplification of [Ca(2+)](i) cycling in response to beta(2)-AR agonists, were blocked by inhibitors of the p38 or p42/44 MAPK pathway (SB203580 and PD98059, respectively), suggesting a role of both MAPK subtypes in beta(2)-AR stimulation. In contrast, beta(1)-AR stimulation of [Ca(2+)](i) cycling was rather limited by the MAPKs, clearly proving the divergence between beta(2)-AR and beta(1)-AR signaling systems. This study presents the first evidence for the coupling of beta(2)-AR to cardiac cPLA(2) and points out the key role of the MAPK pathway in the intracellular signaling elicited by positive inotropic beta(2)-AR agonists in heart.

[Calcium signal and contraction]. / Signal calcium et contraction.

The calcium ion plays a unique role as a messenger and a cofactor in cardiac contraction. This role relies on the strict control by the cell of Ca homeostasis, the components of which are described in this review. During the few last years, tools for the measurement of free intracellular Ca in living cells have been developed which include: probes (aequorin, Fura 2, Indo 1, Fluo 3...), tools for the loading of the cells (microinjection and AM-probes) and systems to analyze the signal (photometers, microfluorimeters, confocal microscopy). Those tools allowed the analysis of calcium signal in cardiomyocytes. In the cardiac cell, activation of a Ca influx through L type Ca channels is usually considered as the pathway initializing Ca mobilization and leading to contraction. It has now been demonstrated that this pathway is activated by beta 1-adrenergic agonists via cyclic AMP. However, amplification of contraction may involve other targets. Thus, the positive inotropic effect of beta 2-adrenergic agonists is also associated with a rise in cytosolic Ca but is not linked to cyclic AMP increase. The alpha 1-adrenergic pathway involves a sensitization of myofilaments for Ca, and increases contraction without an increase in cytosolic Ca. Finally, the positive inotropic effect of glucagon combines the cyclic AMP pathway with a cyclic AMP independent pathway triggered by the metabolite mini-glucagon.

Xanthine affects [Ca2+]i and contractile responses of ventricular cardiocytes to electrical stimulation.

Xanthine, a major purine by-product of ATP, accumulates during myocardial ischemia. In the present study, we show that xanthine (0.5-1 mM) impaired the occurrence of cytosolic Ca2+ concentration ([Ca2+]i) transients, visualized in fura 2-loaded cells, and twitches of contraction in ventricular cardiocytes in response to electrical stimulation. This effect of xanthine was independent of superoxide anion production. That it was a result of decreased membrane excitability was supported by the following: 1) it was reversed by increasing either the amplitude of the stimulus voltage required to stimulate cardiocytes or the extracellular concentration of NaCl; and 2) xanthine reversed the depolarization following electrical stimulation in cardiocytes loaded with the voltage-sensitive dye bis-oxonol. P2 purinergic-agonists, including ATP (10 microM), but not P1 purinergic agonists reproduced the effects seen with xanthine. In addition, a lack of additivity between xanthine and ATP at maximal concentrations was observed. We conclude that xanthine, through activation of a P2 purinoceptor, may contribute to myocardial arrhythmia occurring during ischemia-reperfusion injury.