Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca²⁺]_i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca²⁺]_i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca²⁺]_i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca²⁺]_i, which was sensitive to antioxidants and removal of external Ca²⁺, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca²⁺-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca²⁺ uptake. This suggests that hydrogen peroxide promotes Ca²⁺ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca²⁺ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca²⁺]_i and altered cholecystokinin-evoked [Ca²⁺]_i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca²⁺]_i also upregulated the activity of the plasma membrane Ca²⁺-ATPase in a Ca²⁺-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca²⁺-ATPase. This may be important in facilitating "Ca²⁺ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump     

Cytochrome P450 may regulate plasma membrane Ca2+ permeability according to the filling state of the intracellular Ca2+ stores.

The filling state of the intracellular Ca2+ stores of rat thymocytes regulates plasma membrane permeability to Mn2+, used here as a Ca2+ surrogate for plasma membrane Ca2+ channels. Emptying of the Ca2+ stores accelerated Mn2+ entry about 10-fold, and refilling with Ca2+ restored low Mn2+ permeability. The acceleration of Mn2+ entry observed in cells with empty intracellular Ca2+ stores was prevented by cytochrome P450 inhibitors. Imidazole antimycotics, especially econazole and miconazole, were the most potent inhibitors (IC50 approximately equal to 10(-6) M). The inhibitor sensitivity profile was similar to IA-type cytochrome P450. Calmodulin antagonists increased the plasma membrane permeability to Mn2+ in cells with filled Ca2+ stores, and this effect was also blocked by imidazole antimycotics. On this basis, we propose a model in which activation of a cytochrome P450, situated at the Ca2+ stores, opens a plasma membrane Ca2+ pathway. This activity would be inhibited by Ca2+ inside the stores by a calmodulin-dependent mechanism.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Modeling of Ca2+ flux in pancreatic beta-cells: role of the plasma membrane and intracellular stores

We have developed a detailed mathematical model of ionic flux in beta-cells that includes the most essential channels and pumps in the plasma membrane. This model is coupled to equations describing Ca2+, inositol 1,4,5-trisphosphate (IP3), ATP, and Na+ homeostasis, including the uptake and release of Ca2+ by the endoplasmic reticulum (ER). In our model, metabolically derived ATP activates inward Ca2+ flux by regulation of ATP-sensitive K+ channels and depolarization of the plasma membrane. Results from the simulations support the hypothesis that intracellular Na+ and Ca2+ in the ER can be the main variables driving both fast (2-7 osc/min) and slow intracellular Ca2+ concentration oscillations (0.3-0.9 osc/min) and that the effect of IP3 on Ca2+ leak from the ER contributes to the pattern of slow calcium oscillations. Simulations also show that filling the ER Ca2+ stores leads to faster electrical bursting and Ca2+ oscillations. Specific Ca2+ oscillations in isolated beta-cell lines can also be simulated.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.     

Increased plasma membrane permeability to Ca2+ in anti-Ig-stimulated B lymphocytes is dependent on activation of phosphoinositide hydrolysis

The biochemical basis of Ca2+ mobilization after anti-Ig binding to B cell Ag-R has been further characterized by flow cytometric analysis of indo-1-loaded B cells. The ability to distinguish intracellular Ca2+ release from extracellular Ca2+ influx by using an extracellular calcium depletion-repletion approach has allowed us to study the relationship between the mobilization of Ca2+ from these sources. Studies involving manipulation of the Ca2+ gradient across the plasma membrane indicate that a significant portion of the Ca2+ mobilization response is preserved even when the normal inwardly directed Ca2+ gradient is reversed. In the presence of an extracellular calcium concentration ([Ca2+]o) of 10 microM, the response to anti-Ig is not blocked by the organic Ca2+ channel blockers. This response is not reduced by further depletion of [Ca2+]o by EGTA Ca2+-binding buffers. Thus, the Ca2+ response that occurs when [Ca2+]o less than or equal to 10 microM represents intracellular calcium release. Analysis of B cells stimulated with anti-Ig in low Ca2+ medium ([Ca2+]o = less than 10 microM) followed by repletion of [Ca2+]o to 1 to 5 mM reveals that a significant increase in permeability of the plasma membrane to Ca2+ develops in the stimulated cells. The resultant Ca2+ influx is nimodipine (20 microM) sensitive. Both intracellular Ca2+ release and Ca2+ influx are reduced in parallel as the concentration of anti-Ig stimulus is decreased, suggesting that Ca2+ influx may be coupled to the release of intracellular stores. Neomycin blocks anti-Ig-stimulated formation of inositol trisphosphate, which mediates release of Ca2+ from the endoplasmic reticulum. It also blocks the anti-Ig-induced release of intracellular Ca2+ stores as well as Ca2+ influx, indicating that both responses may be dependent upon phosphatidylinositol 4,5-bisphosphate hydrolysis.     

Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes.     

The role of the Ca2+ binding ligand Asn879 in the function of the plasma membrane Ca2+ pump

Asn879 in the transmembrane segment M6 of the plasma membrane Ca²⁺ pump (PMCA human isoform 4xb) has been proposed to coordinate Ca²⁺ at the transport site through its carboxylate. This idea agrees with the fact that this Asn is conserved in other Ca²⁺-ATPases but is replaced by Asp, Glu, and other residues in closely related 2P-type ATPases of different ionic specificity. Previous mutagenesis studies have shown that the substitution of Ala for Asn abolishes the activity of the enzyme (Adebayo et al., 1995; Guerini et al., 1996). We have constructed a mutant PMCA in which the Asn879 was substituted by Asp. The mutant protein was expressed in Saccharomyces cerevisiae, solubilized and purified by calmodulin affinity chromatography. The Asn879Asp PMCA mutant exhibited about 30% of the wild type Ca²⁺-dependent ATPase activity and only a minor reduction of the apparent affinity for Ca²⁺. The decrease in the Ca²⁺-ATPase of the mutant enzyme was in parallel with the reduction in the amount of phosphoenzyme formed from Ca²⁺ plus ATP. Noteworthy, the mutation nearly eliminated the ability of the enzyme to hydrolyze pNPP which is maximal in the absence of Ca²⁺ revealing a major effect of the mutation on the Ca²⁺-independent reactions of the transport cycle. At a pH low enough to protonate the Asp carboxylate the pNPPase activity of Asn879Asp increased, suggesting that the binding of protons to Asn879 is essential for the activities catalyzed by E₂-like forms of the enzyme.     

Oxidative-induced membrane damage in diabetes lymphocytes: Effects on intracellular Ca2 + homeostasis

Oxidative stress is linked to several human diseases, including diabetes. However, the intracellular signal transduction pathways regulated by reactive oxygen species (ROS) remain to be established. Deleterious effects of ROS stem from interactions with various ion transport proteins such as ion channels and pumps, primarily altering Ca^{2 +} homeostasis and inducing cell dysfunction. This study characterized the Ca^{2 +} transport system in lymphocytes of patients with type-2 diabetes, evaluating the possible correlation between cell modifications and the existence of specific oxidative stress damage. Lymphocytes from type-2 diabetes patients displayed oxidative stress features (accumulation of some ROS species, membrane peroxidation, increase in protein carbonyls, increase in SOD and Catalase activity) and Ca^{2 +} dyshomeostasis (modified voltage-dependent and inositol 1,4,5-triphosphate-mediated Ca^{2 +} channel activities, decrease in Ca^{2 +} pumps activity). The data support a correlation between oxidative damage and alterations in intracellular Ca^{2 +} homeostasis, possibly due to modification of the ionic control in lymphocytes of type-2 diabetes patients.     

Spatio-temporal modelling explains the effect of reduced plasma membrane Ca2+ efflux on intracellular Ca2+ oscillations in hepatocytes

In many non-excitable eukaryotic cells, including hepatocytes, Ca²⁺ oscillations play a key role in intra- and intercellular signalling, thus regulating many cellular processes from fertilisation to death. Therefore, understanding the mechanisms underlying these oscillations, and consequently understanding how they may be regulated, is of great interest. In this paper, we study the influence of reduced Ca²⁺ plasma membrane efflux on Ca²⁺ oscillations in hepatocytes. Our previous experiments with carboxyeosin show that a reduced plasma membrane Ca²⁺ efflux increases the frequency of Ca²⁺ oscillations, but does not affect the duration of individual transients. This phenomenon can be best explained by taking into account not only the temporal, but also the spatial dynamics underlying the generation of Ca²⁺ oscillations in the cell. Here we divide the cell into a grid of elements and treat the Ca²⁺ dynamics as a spatio-temporal phenomenon. By converting an existing temporal model into a spatio-temporal one, we obtain theoretical predictions that are in much better agreement with the experimental observations.     

Coupling between intracellular Ca2+ stores and the Ca2+ permeability of the plasma membrane. Comparison of the effects of thapsigargin, 2,5-di-(tert-butyl)-1,4-hydroquinone, and cyclopiazonic acid in rat thymic lymphocytes.

The regulation of Ca2+ uptake by receptors is incompletely understood. It has been proposed that the Ca2+ permeability of the plasma membrane increases in response to depletion of a critical intracellular Ca2+ storage compartment (Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W. (1989) J. Biol. Chem. 264, 12266-12271). This hypothesis is based largely on the effect of thapsigargin, an inhibitor of endomembrane CA(2+)-ATPases. Due to the existence of an endogenous leak, inhibition of Ca2+ uptake by thapsigargin induces depletion of the stores. This is accompanied by increased plasmalemmal Ca2+ permeability, without change in the level of inositol phosphates. On the other hand, depletion of the intracellular stores by 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ), a chemically unrelated inhibitor of the Ca(2+)-ATPases, fails to induce Ca2+ influx (Kass, G. E., Duddy, S. K., Moore, G. A., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). In an attempt to reconcile these observations, we analyzed in lymphocytes the mode of action of thapsigargin and BHQ. In addition, we tested the effects of cyclopiazonic acid (CPA), a blocker of the skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase. All three compounds released Ca2+ from a common intracellular compartment. Thapsigargin and low concentrations of BHQ and CPA concomitantly elevated the plasmalemmal Ca2+ permeability. Higher concentrations of BHQ and CPA produced a secondary inhibition of the Ca2+ entry pathway, by a mechanism seemingly unrelated to their effects on the internal stores. This inhibitory side effect can account for the reported discrepancies between the effects of thapsigargin and BHQ. The data provide further support for the notion that endomembrane Ca2+ stores are functionally coupled to the plasma membrane Ca2+ permeability pathway.     

The Neurospora plasma membrane Ca2+ pump

Plasma membrane vesicles isolated from the eukaryotic microorganism Neurospora crassa by the concanavalin A method catalyze Mg2+-ATP dependent 45Ca2+ accumulation. Since the ATP-responsive vesicles are functionally inverted, the Ca2+ transport system presumably operates as a Ca2+ exit pump in the intact cell. The mechanism of the Ca2+ pump system involves two components: 1) an electrogenic, proton-translocating ATPase (EC 3.6.1.3), which utilizes the chemical energy of ATP hydrolysis to generate a transmembrane electrical potential and pH gradient, and 2) a Ca2+/H+ antiporter, which utilizes the transmembrane pH gradient to energize the active transport of Ca2+. Evidence for this mechanism is presented and the possible implications of these findings for the mechanisms of Ca2+ pumps in other cells are discussed.     

Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.     

Ca2+ homeostasis in unstimulated platelets

Unstimulated platelets maintain a low cytosolic free Ca2+ concentration and a steep plasma membrane Ca2+ gradient. The mechanisms that are required have not been completely defined. In the present studies, 45Ca2+ was used to examine the kinetics of Ca2+ exchange in intact unstimulated platelets. Quin2 was used to measure the cytosolic free Ca2+ concentration. Under steady-state conditions, the maximum rate of Ca2+ exchange across the platelet plasma membrane, 2 pmol/10(8) platelets/min, was observed at extracellular free Ca2+ concentrations 20-fold less than in plasma. Two intracellular exchangeable Ca2+ pools were identified. The size of the more rapidly exchanging pool (t 1/2, 17 min) and the cytosolic free Ca2+ concentration were relatively unaffected by large changes in the extracellular Ca2+ concentration. In contrast, the size of the more slowly exchanging Ca2+ pool (t 1/2, 300 min) varied with the extracellular Ca2+ concentration, which suggests that it is physically as well as kinetically distinct from the rapidly exchangeable Ca2+ pool. The locations of the Ca2+ pools were determined by differential permeabilization of 45Ca2+-loaded platelets with digitonin. 45Ca2+ in the rapidly exchanging pool was released with lactate dehydrogenase, which suggests that it is located in the cytosol. 45Ca2+ in the slowly exchanging pool was released with markers for both the dense tubular system and mitochondria, but inhibition of mitochondrial Ca2+ uptake with carbonyl cyanide m-chlorophenylhydrazone had no effect on the size of the slowly exchangeable Ca2+ pool or the cytosolic free Ca2+ concentration. In contrast, addition of metabolic inhibitors (KCN plus carbonyl cyanide m-chlorophenylhydrazone plus deoxyglucose) or trifluoperazine caused a decrease in the size of the slowly exchangeable Ca2+ pool and an increase in the cytosolic free Ca2+ concentration. These observations suggest that Ca2+ homeostasis in unstimulated platelets is maintained by limiting Ca2+ influx from plasma, actively promoting Ca2+ efflux, and sequestering Ca2+ within an internal site, which is most likely the dense tubular system and not mitochondria.     

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.     

Ca2+ control of electrolyte permeability in plasma membrane vesicles from cat pancreas

Summary The influence of Ca²⁺ and other cations on electrolyte permeability has been studied in isolated membrane vesicles from cat pancreas.Ca²⁺ in the micromolar to millimolar concentration range, as well as Mg²⁺, Sr²⁺, Mn²⁺ and La³⁺ at a tested concentration of 10^–4 m, increased Na⁺ permeability when applied at the vesicle inside. When added to the vesicle outside, however, they decreased Na⁺ permeability. Ba²⁺ was effective from the outside but not from the vesicle inside.When Ca²⁺ was present at both sides of the membrane, Na⁺ efflux was not affected as compared to that in the absence of Ca²⁺. Monovalent cations such as Rb⁺, Cs⁺, K⁺, Tris⁺ and choline⁺ decreased Na⁺ permeability when present at the vesicle outside at a concentration range of 10 to 100mm. Increasing Na⁺ concentrations from 10 to 100mm at the vesicle inside increased Na⁺ permeability.The temperature dependence of Na⁺ efflux revealed that the activation energy increased in the lower temperature range (0 to 10°C) when Ca²⁺ was present at the outside or at both sides, but not when present at the vesicle inside only or in the absence of Ca²⁺.The results suggest that the Ca²⁺ outside effect is due to binding of calcium to negatively charged phospholipids with a consequent reduction of both fluidity and Na⁺ permeability of the membrane. The Ca²⁺-inside effect most likely involves interaction with proteins with consequent increase in Na⁺ permeability.The data are consistent with current hypotheses on secretagogue-induced fluid secretion in acinar cells of the pancreas according to which secretagogues elicit NaCl and fluid secretion by liberating Ca²⁺ from cellular membranes and by stimulating Ca²⁺ influx into the cell. The increased intracellular Ca²⁺ concentration in turn increases the contraluminal Na⁺ permeability which leads to NaCl influx. The luminal sodium pump finally transports Na⁺ ions into the lumen.     

Positive regulation of capacitative Ca2+ entry by intracellular Ca2+ in Xenopus oocytes expressing rat TRP4

We have investigated the role of intracellular Ca2+ in the opening of capacitative Ca2+ entry (CCE) channels formed with rat TRP4 (rTRP4) using Xenopus oocytes. In rTRP4-expressing oocytes pretreated with thapsigargin, perfusion with A23187, a Ca2+ ionophore, significantly potentiated the delayed phase of the CCE-mediated Cl- current response evoked by extracellular perfusion with Ca2+, without affecting the transient phase of CCE response. In control oocytes, the potentiation of delayed CCE response by A23187 was not significant. Using cut-open recording in combination with artificial intracellular perfusion of oocytes, CCE-mediated Cl- response was recorded at controlled cytosolic Ca2+ concentrations. Intracellular perfusion with a Ca2+ free solution containing 10 mM EGTA abolished most of the CCE responses of both non-injected and rTRP4-expressing oocytes. The native CCE response was not fully recovered by subsequent increases in the intracellular Ca2+ concentration up to 300 nM. However, CCE response of the rTRP4-expressing oocytes was restored at an internal Ca2+ concentration of 110 nM. Blockade of endogenous Cl- channels with anion channel blocker isolated Ca2+ current flowing through CCE channels and clarified the difference in the sensitivity to an internal Ca2+ concentration. These findings indicate that recombinant CCE channels formed with rTRP4 are positively regulated by cytosolic Ca2+ at higher sensitivity compared to oocyte-endogenous CCE channels.     

Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.