Glucocorticoids prolong Ca(2+) transients in hippocampal-derived H19-7 neurons by repressing the plasma membrane Ca(2+)-ATPase-1

Calcium ions (Ca(2+)) play an important role in mediating an array of structural and functional responses in cells. In hippocampal neurons, elevated glucocorticoid (GC) levels, as seen during stress, perturb calcium homeostasis and result in altered neuronal excitability and viability. Ligand- and voltage-gated calcium channels have been the presumed targets of hormonal regulation; however, circumstantial evidence has suggested the possibility that calcium extrusion might be an important target of GC regulation. Here we demonstrate that GC-induced repression of the plasma membrane Ca(2+)-ATPase-1 (PMCA1) is an essential determinant of intracellular Ca(2+) levels ([Ca(2+)](i)) in cultured hippocampal H19-7 cells. In particular, GC treatment caused a prolongation of agonist-evoked elevation of [Ca(2+)](i) that was prevented by the expression of exogenous PMCA1. Furthermore, selective inhibition of PMCA1 using the RNA interference technique caused prolongation of Ca(2+) transients in the absence of GC treatment. Taken together, these observations suggest that GC-mediated repression of PMCA1 is both necessary and sufficient to increase agonist-evoked Ca(2+) transients by down-regulating Ca(2+) extrusion mechanisms in the absence of effects on calcium channels. Prolonged exposure to GCs, resulting in concomitant accumulation of [Ca(2+)](i), is likely to compromise neuronal function and viability.     

Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.     

The effect of calcium pump inhibitors on the response of intracellular calcium to caffeine in snail neurones

We have measured intracellular free calcium ([Ca(2+)]i) using Fura-2 or Ca(2+)-sensitive microelectrodes in voltage-clamped neurones of the snail, Helix aspersa. Caffeine-induced transient increases in [Ca(2+)]i were normally followed by a brief fall of [Ca(2+)]i below its pre-caffeine level. We investigated the cause of this undershoot by raising [Ca(2+)]i; and by inhibiting the plasma membrane or endoplasmic reticulum Ca ATPases (PMCA or SERCA respectively). When the cell membrane potential was decreased from -60 to -25mV, steady-state [Ca(2+)]i increased. The caffeine-induced transients were smaller while the undershoots were larger than in control conditions. When the PMCA was inhibited by high pH the steady-state [Ca(2+)]i increased by 100-400nM. The caffeine-induced [Ca(2+)]i increase and the subsequent undershoot both became larger. Injection of orthovanadate, which inhibits the PMCA and increases [Ca(2+)]i, did not block either effect of caffeine. But when the SERCA was inhibited by cyclopiazonic acid the undershoot disappeared. The phosphodiesterase inhibitor IBMX did not influence the undershoot. These results suggest that the undershoot is generated by the Ca(2+)] ATPase of the stores rather than that of the plasma membrane. Since the undershoot increased as [Ca(2+)]i increased, we conclude that at higher levels of [Ca(2+)]i the stores refill more rapidly.     

Ca(2+)-dependent K(+) current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells

We characterized changes in membrane currents and the cytosolic Ca(2+) concentration, [Ca(2+)](i), in response to caffeine, and compared them with those in response to muscarine using the perforated patch-clamp technique and fura-2 microfluorimetry in guinea-pig adrenal chromaffin cells. Catecholamine release from single voltage-clamped cells was monitored with amperometry using carbon microelectrodes. Caffeine produced a transient outward current (I(out)) at holding potentials over - 60 mV, increasing in amplitude with increasing the potentials. It also evoked a rapid increase of [Ca(2+)](i) at all potentials examined. The current-voltage relation revealed that the activation of K(+) channels was responsible for the I(out) evoked by caffeine. Both current and [Ca(2+)](i) responses were reversibly abolished by cyclopiazonic acid, an inhibitor of Ca(2+)-pump ATPase. At - 30 mV, the caffeine-induced I(out), but not [Ca(2+)](i), was partly inhibited by either charybdotoxin or apamin. In the majority of cells tested, caffeine induced a larger I(out) but a smaller [Ca(2+)](i) increase than muscarine. Caffeine and muscarine increased catecholamine release from voltage-clamped single cells concomitant with the transient increase of [Ca(2+)](i), and there was a positive correlation between them. These results indicate that caffeine activates Ca(2+)-dependent K(+) channels and catecholamine secretion due to the release of Ca(2+) from internal stores in voltage-clamped adrenal chromaffin cells of the guinea-pig. There seems to be a spatial difference between [Ca(2+)](i) increased by Ca(2+) release from caffeine-sensitive stores and that released from muscarine (inositol 1,4,5-trisphosphate)-sensitive ones.     

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μM) and capsaicin (1 μM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 μM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 μM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.     

Phosphatidylinositol 3-kinase facilitates bile acid-induced Ca(2+) responses in pancreatic acinar cells

Bile acids are known to induce Ca(2+) signals in pancreatic acinar cells. We have recently shown that phosphatidylinositol 3-kinase (PI3K) regulates changes in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) elicited by CCK by inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). The present study sought to determine whether PI3K regulates bile acid-induced [Ca(2+)](i) responses. In pancreatic acinar cells, pharmacological inhibition of PI3K with LY-294002 or wortmannin inhibited [Ca(2+)](i) responses to taurolithocholic acid 3-sulfate (TLC-S) and taurochenodeoxycholate (TCDC). Furthermore, genetic deletion of the PI3K gamma-isoform also decreased [Ca(2+)](i) responses to bile acids. Depletion of CCK-sensitive intracellular Ca(2+) pools or application of caffeine inhibited bile acid-induced [Ca(2+)](i) signals, indicating that bile acids release Ca(2+) from agonist-sensitive endoplasmic reticulum (ER) stores via an inositol (1,4,5)-trisphosphate-dependent mechanism. PI3K inhibitors increased the amount of Ca(2+) in intracellular stores during the exposure of acinar cells to bile acids, suggesting that PI3K negatively regulates SERCA-dependent Ca(2+) reloading into the ER. Bile acids inhibited Ca(2+) reloading into ER in permeabilized acinar cells. This effect was augmented by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), suggesting that both bile acids and PI3K act synergistically to inhibit SERCA. Furthermore, inhibition of PI3K by LY-294002 completely inhibited trypsinogen activation caused by the bile acid TLC-S. Our results indicate that PI3K and its product, PIP(3), facilitate bile acid-induced [Ca(2+)](i) responses in pancreatic acinar cells through inhibition of SERCA-dependent Ca(2+) reloading into the ER and that bile acid-induced trypsinogen activation is mediated by PI3K. The findings have important implications for the mechanism of acute pancreatitis since [Ca(2+)](i) increases and trypsinogen activation mediate key pathological processes in this disorder.     

Increased Ca(2+) sparks and sarcoplasmic reticulum Ca(2+) stores potentially determine the spontaneous activity of pulmonary vein cardiomyocytes

Pulmonary veins (PVs) contain cardiomyocytes with spontaneous activity that may be responsible for PV arrhythmia. Abnormal Ca(2+) regulation is known to contribute to PV arrhythmogenesis. The purpose of this study was to investigate whether PV cardiomyocytes with spontaneous activity have different intracellular Ca(2+) ([Ca(2+)](i)) transients, Ca(2+) sparks and responses to isoproterenol and ryanodine receptor modulators (magnesium and FK506) than do PV cardiomyocytes without spontaneous activity and left atrial (LA) cardiomyocytes. Through fluorescence and confocal microscopy, we evaluated the [Ca(2+)](i) transients and Ca(2+) sparks in isolated rabbit PV and LA cardiomyocytes. PV cardiomyocytes with spontaneous activity had larger [Ca(2+)](i) transients and sarcoplasmic reticulum (SR) Ca(2+) stores than PV cardiomyocytes without spontaneous activity or LA cardiomyocytes. PV cardiomyocytes with spontaneous activity also had a higher incidence and frequency of Ca(2+) sparks, and had Ca(2+) sparks with larger amplitudes than other cardiomyocytes. Magnesium (5.4 mM) reduced the [Ca(2+)](i) transient amplitude and beating rate in PV cardiomyocytes with spontaneous activity. However, in contrast with other cardiomyocytes, low doses (1.8 mM) of magnesium did not reduce the [Ca(2+)](i) transients amplitude in PV cardiomyocytes with spontaneous activity. FK506 (1 muM) diminished the SR Ca(2+) stores in PV cardiomyocytes with spontaneous activity to a lesser extent than that in other cardiomyocytes. Isoproterenol (10 nM) increased the [Ca(2+)](i) transient amplitude to a lesser extent in LA cardiomyocytes than in PV cardiomyocytes with or without spontaneous activity. In conclusion, our results suggest that enhanced [Ca(2+)](i) transients, increased Ca(2+) sparks and SR Ca(2+) stores may contribute to the spontaneous activity of PV cardiomyocytes.     

Release of Ca(2+) from intracellular stores and entry of extracellular Ca(2+) are involved in sea squirt sperm activation.

A rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is required to activate sperm of all organisms studied. Such elevation of [Ca(2+)](i) can occur either by influx of extracellular Ca(2+) or by release of Ca(2+) from intracellular stores. We have examined these sources of Ca(2+) in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca(2+)-sensitive dye fura-2 to monitor [Ca(2+)](i) by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca(2+)], as well as by the presence of high [K(+)](o) or the Ca channel blockers pimozide, penfluridol, or Ni(2+), but not nifedipine or Co(2+). The accompanying rise in [Ca(2+)](i) was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca(2+) entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca(2+)](i) increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca(2+)](i) could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca(2+) from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP(3)) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca(2+) and that thimerosal stimulates release of Ca(2+) from IP(3)-sensitive intracellular stores.     

Ca(2+) homeostasis, Ca(2+) signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.     

Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)](i) transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH.     

Resistance to Tumor Necrosis Factor-Induced Cell Death Mediated by PMCA4 Deficiency

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor (TNF)-resistant lines from L929 cells. Using this approach, we discovered that the plasma membrane calcium ATPase 4 (PMCA4) is required for TNF-induced cell death in L929 cells. Under basal conditions, PMCA4-deficient (PMCA(mut)) cells have a normal phenotype. However, stimulation with TNF induces an abnormal increase in the intracellular calcium concentration ([Ca(2+)](i)). The substantially elevated [Ca(2+)](i) caused resistance to TNF-induced cell death. We found that an increase in the total volume of acidic compartments (VAC), mainly constituted by lysosomes, is a common event in cell death caused by a variety of agonists. The increased [Ca(2+)](i) in PMCA(mut) cells promoted lysosome exocytosis, which, at least in part, accounted for the inhibition of TNF-induced increase in VAC and cell death. Promoting lysosome exocytosis by calcium inhibited TNF-induced cell death in wild-type L929 cells, while inhibition of lysosome exocytosis or increase of VAC by sucrose restored the sensitivity of PMCA(mut) cells to TNF-induced cell death. Thus, increase of the volume of acidic compartment is a part of the cell death process, and the antideath effect of calcium is mediated, at least in part, by inhibition of the TNF-induced increase in VAC.     

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.     

VEGF and ATP act by different mechanisms to increase microvascular permeability and endothelial [Ca(2+)](i)

Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) by stimulating Ca(2+) influx into endothelial cells. To determine whether VEGF-mediated Ca(2+) influx is stimulated by release of Ca(2+) from intracellular stores, we measured the effect of Ca(2+) store depletion on VEGF-mediated increased L(p) and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) of frog mesenteric microvessels. Inhibition of Ca(2+) influx by perfusion with NiCl(2) significantly attenuated VEGF-mediated increased [Ca(2+)](i). Depletion of Ca(2+) stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca(2+)](i) or the increase in L(p). In contrast, ATP-mediated increases in both [Ca(2+)](i) and L(p) were inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca(2+) influx. VEGF also increased Mn(2+) influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca(2+)](i) and L(p) even when Ca(2+) stores were depleted and under conditions that prevented ATP-mediated increases in [Ca(2+)](i) and L(p). This suggests that VEGF acts through a Ca(2+) store-independent mechanism, whereas ATP acts through Ca(2+) store-mediated Ca(2+) influx.     

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.     

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).     

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).     

Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.     

cADP ribose and [Ca(2+)](i) regulation in rat cardiac myocytes

cADP ribose (cADPR)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1-10 microM) induced sustained, concentration-dependent [Ca(2+)](i) responses ranging from 50 to 500 nM, which were completely inhibited by 20 microM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca(2+)](i) waves, increasing concentrations of cADPR increased wave frequency up to approximately 250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca(2+)](i) transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca(2+)](i) responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca(2+)](i) responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca(2+)](i) response. These results indicate that in cardiac myocytes, cADPR induces Ca(2+) release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.     

Diabetes alters intracellular calcium transients in cardiac endothelial cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+)](i)) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+)](i) homeostasis due to altered sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+) regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+)](i) homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+)](i) transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+) ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+)](i) sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.