Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump.

In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.     

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.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: calcium pumps

The regulation of cytosolic Ca2+ homeostasis is essential for cells, and particularly for vascular smooth muscle cells. In this regulation, there is a participation of different factors and mechanisms situated at different levels in the cell, among them Ca2+ pumps play an important role. Thus, Ca2+ pump, to extrude Ca2+; Na+/Ca2+ exchanger; and different Ca2+ channels for Ca2+ entry are placed in the plasma membrane. In addition, the inner and outer surfaces of the plasmalemma possess the ability to bind Ca2+ that can be released by different agonists. The sarcoplasmic reticulum has an active role in this Ca2+ regulation; its membrane has a Ca2+ pump that facilitates luminal Ca2+ accumulation, thus reducing the cytosolic free Ca2+ concentration. This pump can be inhibited by different agents. Physiologically, its activity is regulated by the protein phospholamban; thus, when it is in its unphosphorylated state such a Ca2+ pump is inhibited. The sarcoplasmic reticulum membrane also possesses receptors for 1,4,5-inositol trisphosphate and ryanodine, which upon activation facilitates Ca2+ release from this store. The sarcoplasmic reticulum and the plasmalemma form the superficial buffer barrier that is considered as an effective barrier for Ca2+ influx. The cytosol possesses different proteins and several inorganic compounds with a Ca2+ buffering capacity. The hypothesis of capacitative Ca2+ entry into smooth muscle across the plasma membrane after intracellular store depletion and its mechanisms of inhibition and activation is also commented.     

Calcium sequestration in the liver: Review article

Hepatic parenchymal cells maintain intracellular total and cytosolic free Ca2+ levels by: entry of Ca2+ through channels, extrusion of Ca2+ by an outwardly directed Ca2+ pump, and controlled sequestration into intracellular pools. The mechanism of Ca2+ inflow is poorly characterized. The plasma membrane Ca2+ channels seem to share some of the characteristics of Ca2+ channels in excitable cells, but also differ from them. The outwardly directed plasma membrane Ca2(+)-ATPase is a calmodulin independent, P-type enzyme. Ca2+ uptake into the endoplasmic reticulum is due to the activity of a different Ca2(+)-ATPase, which is similar in molecular weight and shares antigenic determinants with the sarcoplasmic reticulum enzyme. In addition, mitochondria and nuclei also take up calcium. The exact mechanism by which Ca2+ is released from intracellular organelles is not well known. Several mechanisms for Ca2+ release from the endoplasmic reticulum were reported, including IP3 and GTP-induced. The most effective identified way of eliciting Ca2+ release from microsomal fraction is by the oxidation of critical -SH groups. This mechanism is likely to be involved in the rise of cytosolic Ca2+ observed in many situations of hepatocellular injury. In addition to being sequestered into subcellular organelles, some of the intracellular Ca2+ is bound to specific Ca2+ binding proteins. Both calmodulin and members of the annexin family were identified in the liver. Stimulation of the liver with gluconeogenic hormones results in increased Ca2+ entry into the cell, the release of Ca2+ from intracellular pools, and an oscillatory increase in free cytosolic Ca2+ levels. Extensive research is still needed for the elucidation of the exact mechanisms by which these events occur.     

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.     

Interaction of metal cations with Ca2+-transport sites of the plasma membrane Ca2+ pump of secretory cells of gastric glands

Investigation of Ca2+ transport by calcium pump of the cell plasma membrane of the gastric glands isolated from guinea pigs and its inhibition by metal cations has been performed. The mainly competitive type of Ca2+ translocation inhibition by the calcium pump by metals cations (0.025-1.00 mM) was determined. Potency of inhibition increases in such an order (I50, mM): Ba2+ (0.336) < Sr2+ (0.251) < Mn2+ (0.099) < Co2+ (0.029) < Cd2+ (0.016). It was shown by one-factor dispersion analysis that potency of inhibition depends on ionic radii and hydration enthalpy of metal cations and also on stability constants of their complexes with oxygen-containing bioligands (acetic, aspartic and glutamic acid) (hx2 = 83.73-85.95). Dependence of the inhibition constants (I50) on ionic radii is most adequately described by the parabolic equation, such a dependence on hydration enthalpy and stability constants with oxygen-containing bioligands--by exponential or multiplicative equations. The conclusion has been made that selective Ca2+ translocation by the calcium pump and its inhibition by metal cations is determined by the interaction between energy of their interaction with cation-binding sites of the transport system and energy of hydration. Energetics of such interactions depends on the steric factors. The physicochemical model of the Ca2+ selective translocation by calcium pump and its inhibition by metal cations has been proposed.     

Characterization and Distribution of Transferrin Receptors in the Rat Brain

The mechanism of calcium transport across the plasma membrane of chromaffin cells was studied using plasma membrane vesicles prepared from cells of adrenal medulla. Purification of the plasma membrane was about 30-fold, based on the alpha-bungarotoxin binding activity. The isolated membrane vesicles have both Na+/Ca2+ exchange and calcium pump activities. The Na+/Ca2+ exchange activity increased with the free calcium concentration and was not saturated at 1 mM, the highest concentration tried. The K1/2 of the calcium pump for calcium is 0.06 microM. Part of the Na+/Ca2+ exchange activity was inhibited by preincubation of the membrane vesicles with veratridine and the effect of veratridine was reversed by tetrodotoxin. The calcium taken up by the calcium pump was released by 0.005% saponin, but was not affected by oxalate. The calcium taken up by the calcium pump was released by exchanging with the external sodium, which suggests that the two calcium transport systems are located on the same population of membrane vesicles. The above evidence indicates that both calcium transport activities are located on the plasma membrane and not on contaminating organelle membranes. The significance of the two calcium transport systems in regulation of cytosolic calcium concentration of chromaffin cells is discussed.     

The origin, quantitation, and kinetics of intracellular calcium mobilization by vasopressin and phenylephrine in hepatocytes

The addition of phenylephrine or vasopressin to isolated hepatocytes resulted in an efflux of calcium. The intracellular source of this calcium was determined by measuring the calcium released upon the sequential additions of an uncoupling agent and the Ca2+ ionophore A23187 to control and hormone-treated cells. The release promoted by these agents was used as an estimate of the calcium content of the mitochondria and endoplasmic reticulum, respectively. The validity and limitations of this method are critically evaluated. The source of the calcium mobilized by the hormones was found to depend on the intracellular calcium distribution. When the amount of total cell-releasable Ca2+ was low (less than 0.9 nmol/mg cell dry weight), the endoplasmic reticulum represented the major cellular calcium pool and was also the predominant pool mobilized by the hormone. As the cell calcium content was increased, the endoplasmic reticulum attained its maximum capacity and the mitochondria sequestered increasing amounts of calcium. Under these conditions, the hormones mobilized calcium from the mitochondria with minimal effects on the endoplasmic reticulum calcium pool. These results suggest that more than one hormone-induced Ca2+-releasing agent may be formed. Both the total amount and the rate of calcium released from the cell under the influence of hormones was independent of the cell calcium content. The appearance of hormone-releasable Ca2+ in the extracellular medium showed a lag period of 5 to 10 s, during which a rapid increase of phosphorylase activity was observed. In contrast, the mobilization of a comparable amount of calcium by carbonyl cyanide p-trifluoromethoxyphenylhydrazone showed no significant lag, but the activation of phosphorylase was slower. A kinetic analysis of the hormone-releasable Ca2+ indicated a rapid onset with a peak increase of cytosolic free Ca2+ between 5 and 10 s prior to release of Ca2+ from the cell. The results suggest that an early action of the hormone is the inhibition of the plasma membrane Ca2+ efflux pump.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum.

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Comparison of calcitonin, epinephrine and glucagon effects on plasma membrane Ca-ATPase activity and calcium content in liver of rats

The effects of calcitonin (CT), epinephrine and glucagon on the plasma membrane Ca-ATPase activity and the calcium content in the liver were investigated 30 min after a single subcutaneous administration of hormones to rats. Ca-ATPase activity in the plasma membrane fraction was significantly decreased by CT (80 MRC mU/100 g BW), while it was not significantly lowered by insulin (100 mU/100 g BW), epinephrine (100 micrograms/100 g BW), glucagon (50 micrograms/100 g BW), or parathyroid hormone (25 U/100 g BW). The calcium content in the liver was markedly increased by CT, while it was not significantly elevated by epinephrine or glucagon. Meanwhile, the decrease of Ca-ATPase activity in the plasma membrane fraction produced by CT was significantly prevented by simultaneous administration of epinephrine or glucagon, and also the increase in liver calcium was noticeably interfered with. The present results suggests that the action of CT on liver calcium may differ from that of epinephrine or glucagon which causes an increase in cyclic AMP in the liver cells.     

Effect of ethanol on the activity of the energy-dependent Ca2+-transporting system of myometrial cells

In order of estimating some regularities of ethanol direct (effectory) effect to transmembrane calcium metabolism in the myometrium the action of this substance on the energy-dependent Ca(2+)-transporting systems of the uterine myocytes subcellular structures has been studied. The systems of Mg2+, ATP-dependent Ca2+ transport regarding their sensitivity to ethanol inhibitory effect were displayed as satisfying the following sequences: endoplasmic reticulum calcium pump > plasma membrane solubilized Ca2+, Mg2+, ATP-ase > mitochondrial Ca(2+)-accumulating system = plasma membrane calcium pump. Alongside with the latter, the oxytocin-insensitive component of Mg2+, ATP-dependent Ca2+ accumulation in the endoplasmic reticulum was defined to be less resistant to inhibitory effect of ethanol if compared with the oxytocin-sensitive one. On the base of the data received some mechanisms of ethanol effectory action on the intracellular calcium homeostasis in the myometrium cells are under the discussion.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs.

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

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.     

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.     

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity: role of intracellular calcium

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity to isolated hepatocytes was studied. MPTP was more toxic to hepatocytes than its major metabolite, 1-methyl-4-phenylpyridine (MPP+); this may, in part, be explained by the lesser permeability of the hepatocyte plasma membrane to the cation compared to its parent compound, MPTP. Loss of cell viability was preceded by plasma membrane bleb formation and disturbance of intracellular Ca2+ homeostasis. MPTP caused a rapid depletion of the mitochondrial Ca2+ pool which was followed by a marked and sustained elevation of cytosolic free Ca2+ concentration. This increase of cytosolic Ca2+ level appeared to be associated with the impairment of the cell's Ca2+ extrusion system since the plasma membrane Ca2+-ATPase was markedly inhibited in MPTP-treated hepatocytes. Preincubation of hepatocytes with inhibitors of monoamine oxidase type B, but not A, protected the cells from MPTP-induced cytotoxicity. Moreover, the monoamine oxidase B inhibitor, pargyline, prevented the rise in cytosolic free Ca2+ concentration and partially protected the plasma membrane Ca2+-ATPase from inhibition by MPTP. As observed with MPTP, MPP+ caused an extensive loss of mitochondrial Ca2+ and significantly decreased the rate of Ca2+ efflux from hepatocytes. However, MPP+ was without effect on the plasma membrane Ca2+-ATPase. In conclusion, our studies demonstrate that MPTP caused a substantial elevation of cytosolic Ca2+ which preceded loss of cell viability and we propose that calcium ions are of major importance in the mechanism of MPTP- and MPP+-induced toxicity in hepatocytes.     

Cytosolic Concentration of Ca2+ Regulates the Plasma Membrane H+-ATPase in Guard Cells of Fava Bean

Opening of the stomata is driven by the light-activated plasma membrane proton pumping ATPase, although the activation and inactivation mechanism of the enzyme is not known. In this study, we show that the H+-ATPase in guard cells is reversibly inhibited by Ca2+ at physiological concentrations. Isolated microsomal membranes of guard cell protoplasts from fava bean exhibited vanadate-sensitive, ATP-dependent proton pumping. The activity was inhibited almost completely by 1 [mu]M Ca2+ with a half-inhibitory concentration at 0.3 [mu]M and was restored immediately by the addition of 1,2-bis(2-aminophenoxy)ethane N,N,N[prime],N[prime]-tetraacetic acid, a calcium chelating reagent. Similar reversible inhibition by Ca2+ was shown by the generation of electrical potential in the membranes. Activity of ATP hydrolysis was inhibited similarly by Ca2+ in the same membrane preparations. The addition of 1,2-bis(2-aminophenoxy)ethane N,N,N[prime],N[prime]-tetraacetic acid and EGTA, Ca2+ chelators, to epidermal peels of fava bean induced stomatal opening in the dark, and the opening was suppressed by vanadate. This suggests that the lowered cytosolic Ca2+ activated the proton pump in vivo and that the activated pump elicited stomatal opening. Inhibition of H+-ATPase by Ca2+ may depolarize the membrane potential and could be a key step in the process of stomatal closing through activation of the anion channels. Furthermore, similar inhibition of the proton pumping and ATP hydrolysis by Ca2+ was found in isolated plasma membranes of mesophyll cells of fava bean. These results suggest that Ca2+ regulates the activity of plasma membrane H+-ATPases in higher plant cells, thereby modulating stomatal movement and other cellular processes in plants.