Inhibition Of Capacitative Calcium Entry Research Articles

Cyclic AMP relaxation of rat aortic smooth muscle is mediated in part by decrease of depletion of intracellular Ca2 + stores and inhibition of capacitative calcium entry

Despite a large number of studies, the mechanism by which 3′,5′-cyclic monophosphate (cAMP) induces vasorelaxation is not fully understood. The comparison between results obtained in different vessels or species has often been the source of conflicting reports. In order to shed more light onto this mechanism, we studied the effects of forskolin in phenylephrine-pre-contracted endothelium-denuded rat aorta and measured cAMP levels in rat aortic myocytes by enzyme-immunoassay. Nanomolar forskolin relaxed phenylephrine-induced contractions. This effect was mimicked by dibutyryl-cAMP and was potentiated by rolipram or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB-203580). Nifedipine and verapamil partially relaxed phenylephrine-induced contractions, while further application of cAMP-elevating agents fully relaxed these contractions. In Ca2+-free extracellular solution, forskolin reduced phenylephrine-induced transient contractions and reduced the Ca2+-induced contraction after depletion of intracellular stores. Nanomolar concentrations of forskolin increased basal cAMP levels only in the presence of rolipram or phenylephrine, which did not modify intracellular levels of cAMP by themselves. In conclusion, relaxation by cAMP is mediated in part by decrease of depletion of intracellular Ca2+ stores and inhibition of capacitative calcium entry. This study provides the first evidence that inhibition of PDE4 or p38-MAPK potentiates the vasodilator effect of cAMP-elevating agents in rat aortic myocytes.

Glucose deprivation induced increase in O‐GlcNAc levels in cardiomyocytes is not mediated via increased OGT expression

O‐Linked N‐acetylglucosamine (O‐GlcNAc) is a post‐translational modification of proteins that plays an important role in mediating the response of cells to stress. Previous studies in human hepatocellular carcinoma and Neuro‐2a neuroblastoma cells demonstrated that glucose deprivation increased O‐GlcNAc levels by up‐regulation of O‐GlcNAc transferase (OGT) in an AMP‐activated protein kinase‐dependent manner. The goal of this study was to examine the effect of glucose deprivation (GD) on O‐GlcNAc levels in neonatal rat ventricular cardiomyocytes (NRVM). GD significantly increased NRVM O‐GlcNAc levels within 1‐2 hours and O‐GlcNAc levels continued to increase over the next 6 hours. GD increased p38 and Akt phosphorylation, but had no effect OGT protein levels. Increasing hexosamine biosynthesis with glucosamine (5mM) significantly attenuated the effects of GD; however, inhibition of OGT (Alloxan, 5mM or TT04, 5µM) or O‐GlcNAcase (PUGNAc or NAG‐Bt, 100µM) had no effect. Surprisingly, inhibition of capacitative calcium entry (CCE) with SKF 96365 (5μM) markedly inhibited GD‐induced increase in O‐GlcNAc. These results demonstrate that GD rapidly increases O‐GlcNAc levels in NRVM independent of changes in OGT expression. The mechanism underlying the GD‐induced increase in O‐GlcNAcylation in NRVMs remains to be determined; however, it appears to be mediated at least in part via hexosamine biosynthesis and CCE. (Supported by NIH grants: HL067464 and HL079364)

Inhibition of capacitative calcium entry suppresses ouabain‐induced stimulation of sodium‐hydrogen exchange in rat optic nerve astrocytes

Objective: Earlier we showed that 1 μM ouabain increased capacitative calcium entry (CCE) but did not increase cell sodium1. Here we studied cytoplasmic pH recovery after an acid load in ouabain‐treated cells and tested whether pH response were linked to altered calcium entry.Methods: Ammonium chloride (20mM) was used to acid load the cells. BCECF and FURA‐2 were used to measure intracellular pH and calcium.Results: Ammonium chloride removal causes the pHi to fall abruptly, followed by gradual recovery. lμM ouabain increased the rate of pHi recovery both in the presence and in the absence of bicarbonate in the bathing medium. NHE inhibitor DMA reduced the rate of pHi recovery. Ouabain did not detectably alter cytoplasmic Ca2+ (Ca2+i) but CCE inhibitors SKF96365 and 2APB reduced both the basal and ouabain‐stimulated rates of pHi recovery. Calcium chelator BAPTA‐AM also inhibited basal and ouabain‐stimulated pHi recovery. Western blot showed presence of NHE1 but not NHE2, NHE3 or NHE4.Conclusion: Ouabain‐induced stimulation of pHi recovery was due, at least in part, to stimulation of NHE1. Although lμM ouabain failed to alter Ca2+i, the findings suggest the NHE response may be dependent on CCE.Funding: NIH Grant EY014069

Capacitative Calcium Entry Contributes to the Differential Transactivation of the Epidermal Growth Factor Receptor in Response to Thiazolidinediones

Thiazolidinediones (TZDs) are synthetic ligands for the peroxisome proliferator-activated receptor gamma (PPARgamma) but also elicit PPARgamma-independent effects, most notably activation of mitogen-activated protein kinases (MAPKs). Ciglitazone rapidly activates extracellular signal-regulated kinase (Erk) MAPK, an event requiring c-Src kinase-dependent epidermal growth factor receptor (EGFR) transactivation, whereas troglitazone only weakly activates Erk and does not induce EGFR transactivation; the mechanism underlying this difference remains unclear. In this study, both ciglitazone and troglitazone increased Src activation. Similar effects were observed with Delta2-derivatives of each TZD, compounds that bind PPARgamma but do not lead to its activation, further indicating a PPARgamma-independent mechanism. Neither EGFR kinase nor Pyk2 inhibition prevented Src activation; however, inhibition of Src kinase activity prevented Pyk2 activation. Intracellular calcium chelation blocks TZD-induced Pyk2 activation; here, Src activation by both TZDs and ciglitazone-induced EGFR transactivation were prevented by calcium chelation. Accordingly, both TZDs increased calcium concentrations from intracellular stores; however, only ciglitazone produced a secondary calcium influx in the presence of extracellular calcium. Removal of extracellular calcium or inhibition of capacitative calcium entry by 2-APB prevented ciglitazone-induced EGFR transactivation and Erk activation but did not affect upstream kinase signaling pathways. These results demonstrate that upstream kinases (i.e., Src and Pyk2) are required but not sufficient for EGFR transactivation by TZDs. Moreover, influx of extracellular calcium through capacitative calcium entry may be an unrecognized component that provides a mechanism for the differential induction of EGFR transactivation by these compounds.

EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells.

In rabbit corneal epithelial cells (RCEC), we determined whether capacitative calcium entry (CCE) mediates the mitogenic response to epidermal growth factor, EGF. [Ca2+]i was measured with single-cell fluorescence imaging of fura2-loaded RCEC. EGF (5 ng/ml) maximally increased [Ca2+]i 4.4-fold. Following intracellular store (ICS) calcium depletion in calcium-free medium with 10 microM cyclopiazonic acid (CPA) (endoplasmic reticulum calcium ATPase inhibitor), calcium addback elicited plasma membrane Ca2+ influx as a result of activation of plasma membrane store operated channel (SOC) activity. Based on Mn2+ quench measurements of fura2 fluorescence, 5 ng/ml EGF enhanced such influx 2.3-fold, whereas with Rp-cAMPS (protein kinase A inhibitor) plus EGF it increased by 5.3-fold. In contrast, SOC activation was blocked with 100 microM 2-aminoethyldiphenylborate (2-APB, store-operated channel inhibitor). During exposure to either 50 microM UO126 (MEK-1/2 inhibitor) or 10 microM forskolin (adenylate cyclase activator), 5 ng/ml EGF failed to affect [Ca2+]i. RT-PCR detected gene expression of: 1) transient receptor potential (TRP) protein isoforms 1, 3, 4, 6 and 7; 2) IP3R isoforms 1-3. Immunocytochemistry, in conjunction with confocal and immunogold electron microscopy, detected plasma membrane localization of TRP4 expression. Inhibition of CCE with 2-APB and/or CPA, eliminated the 2.5-fold increase in intracellular [3H]-thymidine incorporation induced by EGF. Taken together, CCE in RCEC mediates the mitogenic response to EGF. EGF induces CCE through its stimulation of Erkl/2 activity, whereas PKA stimulation suppresses these effects of EGF. TRP4 may be a component of plasma membrane SOC activity, which is stimulated by ICS calcium depletion.

Regulation of growth factor induced gene expression by calcium signalling: integrated mRNA and protein expression analysis.

There is considerable indirect evidence that growth factor induced changes in the intracellular concentration of calcium play an important role in the regulation of the mammalian cell cycle. However, the precise mechanism by which this may be achieved remains unclear. Here we show that SKF-96365, an inhibitor of growth factor induced capacitative calcium entry (CCE), inhibits cell cycle progression by preventing entry into S phase. SKF-96365 changes the temporal profile of growth factor induced calcium signalling and recent studies have shown that alterations in the temporal and spatial patterns of calcium signalling can differentially regulate gene expression. We have therefore sought to examine the effect of inhibition of CCE on growth factor induced gene expression during G1. To achieve this we have initiated a combined transcriptomic and proteomic approach to measure CCE regulated gene expression using cDNA arrays and two-dimensional polyacrylamide gel electrophoresis, respectively. The initial results of this on-going analysis are reported here. They reveal that inhibition of CCE influences the expression of 29 genes at the mRNA level and 22 genes at the protein level. We report the identification of the mRNAs whose expression is altered by inhibition of CCE and describe the potential functional significance of some of these changes. The value of integrating a transcriptomic and two-dimensional gel electrophoresis based proteomic approach to studies of gene expression is discussed.

Nifedipine block of capacitative calcium entry in cultured human uterine smooth-muscle cells.

To determine whether nifedipine inhibits capacitative calcium entry at clinically relevant concentrations using cultured human myocytes as a model for human myometrium. Myocyte cultures were initiated from the myometrium of term pregnant women who underwent cesarean delivery. Paired cells were chosen for study. The cell of interest was stimulated by an intercellular calcium wave from the adjacent cell. In this fashion, release of sarcoplasmic reticulum (SR) calcium was accomplished with minimal disturbance of the plasma membrane and the subplasmalemmal space (SPS) of the cell studied. Depletion of the SR calcium stores by the calcium wave activated the capacitative calcium current, elevated calcium in the SPS, and activated calcium-activated potassium channels. A cell-attached patch clamp was used to monitor the outward current resulting from the calcium activation of these potassium channels. Calcium green-1 fluorescence was used to simultaneously monitor changes of the deep cytosolic calcium concentrations. Experiments were performed at varying concentrations of nifedipine (0-10 micromol/L). Nifedipine reduced outward potassium currents in a dose-dependent manner. Nifedipine at 100 nmol/L resulted in greater than a 50% reduction of outward current, indicating a significant inhibition of capacitative calcium entry at that concentration. Higher concentrations of nifedipine abolished outward current. Experiments designed to detect indirect effects of nifedipine on capacitative calcium entry were negative. Nifedipine block of capacitative calcium entry occurred at concentrations similar to those required to block L-type voltage-activated calcium channels. These data suggest that block of capacitative calcium entry may be an important mechanism of action when nifedipine is clinically used for tocolysis of preterm labor.

Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin☆

Yessotoxin (YTX) is a polyether toxin of marine origin that has been classified among the diarrheic shellfish poisoning (DSP) toxins group due to its lipophilic nature. However, unlike other DSP toxins, YTX does not produce diarrhea and its mechanisms of action are unknown. We studied the effect of YTX on the cytosolic calcium levels of freshly isolated human lymphocytes by means of fluorescence imaging microscopy. We showed that YTX produced a calcium influx through nifedipine and SKF 96365 (1-[β-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1 H-imidazole hydrochloride)-sensitive channels. This Ca 2+ entry was not affected by the DSP toxin okadaic acid, which inhibits protein phosphatases. In addition, YTX also produced an inhibition of capacitative calcium entry activated by thapsigargin or by preincubation in a Ca 2+-free medium. This capacitative calcium entry was not sensitive to nifedipine. Furthermore, the inhibitory effect of YTX was dependent on the time of addition of the toxin. We suggest that YTX may interact with calcium channels in a way similar to that described for other polyether marine compounds such as brevetoxins and maitotoxin, although an involvement of other second messengers is also likely.

Inhibition of capacitative calcium entry is not obligatory for relaxation of the mouse anococcygeus by the NO/cyclic GMP signalling pathway.

1. The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. 2. In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic field stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 microM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microM). 3. In single smooth muscle cells, loaded with Fura-2, both carbachol and Tg produced sustained elevations in cytoplasmic calcium levels ([Ca2+]i). SNP inhibited the rise in [Ca2+]i produced by carbachol, an effect attenuated by ODQ. In contrast, neither SNP nor 8-Br-cyclic GMP reduced the elevated [Ca2+]i associated with Tg. 4. In beta-escin skinned preparations, NO had no effect on tone induced by calcium (1 microM in the presence of 100 microM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. 5. The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by refilling the internal stores, since reduction in [Ca2+]i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation.

The Inhibition of Capacitative Calcium Entry Due to ATP Depletion but Not Due to Glucosamine Is Reversed by Staurosporine

The capacitative Ca2+ entry pathway in J774 macrophages is rapidly inhibited by the amino sugar glucosamine. This pathway is also inhibited by treatments such as 2-deoxy-D-glucose (2dGlc) or glucose deprivation that inhibit glycolysis and lead to significant decreases in cellular ATP and other trinucleotides. We sought to determine whether glucosamine's effect on capacitative Ca2+ entry was also due to ATP depletion, as has been suggested recently for its link to insulin resistance. In contrast to brief treatments with 2dGlc, there was no significant decrease in ATP following exposure to glucosamine. In addition, the 2dGlc-mediated inhibition of capacitative Ca2+ influx was reversed by staurosporine, a microbial alkaloid that inhibits a broad range of protein kinases. Staurosporine was also able to reverse the inhibition of capacitative Ca2+ entry seen following other treatments that decreased cellular ATP levels, including cytochalasin B and iodoacetic acid. Other inhibitors of protein kinase C, including bisindolylmaleimide, K252a, H-7, and calphostin C, were unable to mimic this effect of staurosporine. However, the inhibition of capacitative Ca2+ influx in the presence of glucosamine was not reversed by staurosporine. These data indicate that the inhibitory action on capacitative Ca2+ entry of glucosamine is distinct from that caused by ATP depletion.

Reduced Capacitative Calcium Entry Correlates with Vesicle Accumulation and Apoptosis

A preneoplastic variant of Syrian hamster embryo cells, sup(+), exhibits decreased endoplasmic reticulum calcium levels and subsequently undergoes apoptosis in low serum conditions (Preston, G. A., Barrett, J. C., Biermann, J. A., and Murphy, E. (1997) Cancer Res. 57, 537-542). This decrease in endoplasmic reticulum calcium appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane. Thus we investigated whether inhibition of capacitative calcium entry per se could reduce endoplasmic reticulum calcium and induce apoptosis of cells. We find that treatment with either SKF96365 (30-100 microM) or cell-impermeant 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N', N'-tetraacetic acid (5-10 mM) is able to induce apoptosis of cells in conditions where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis of cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of cells with bafilomycin (30-100 nM), an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles, decreased capacitative entry, and induced apoptosis. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis.

Octanol blocks fluid secretion by inhibition of capacitative calcium entry in rat mandibular salivary acinar cells

The aliphatic alcohol octanol is thought to modulate enzyme secretion from the exocrine pancreas by the inhibition of gap junction permeability. We have now investigated the effects of octanol on salivary secretion and intracellular calcium concentration ([Ca2+]i), measured in isolated perfused rat mandibular glands and in isolated mandibular acinar cells respectively. Stimulation of perfused glands with 10 μM carbachol (CCh) evoked a rapid increase in fluid secretion followed by a decrease to a sustained elevated level. Application of 1 mM octanol during CCh stimulation inhibited fluid secretion reversibly. In isolated acini, the CCh-induced [Ca2+]iincrease was reversibly inhibited by the same concentration of octanol. However, octanol also inhibited the increase in [Ca2+]iin single acinar cells where gap junctions were no longer functional, indicating that octanol directly affected the intracellular Ca2+signalling pathway. The initial increase in [Ca2+]iinduced by 0.5–10 μM CCh, which is due to Ca2+release from IP3-sensitive Ca2+stores, was not affected by pretreatment with octanol. In contrast, CCh-, phenylephrine- or thapsigargin-induced Ca2+entry was almost completely and reversibly inhibited by octanol. Octanol also blocked agonist-evoked Ca2+entry in pancreatic acinar cells, and thapsigargin-evoked Ca2+entry in fibroblasts. These data strongly suggest that octanol blocks salivary secretion from mandibular gland by the inhibition of capacitative Ca2+entry, and raise the possibility that octanol may be a useful tool for inhibiting agonist-evoked Ca2+entry pathways.

Multiple effects of econazole on calcium signaling: depletion of thapsigargin-sensitive calcium store, activation of extracellular calcium influx, and inhibition of capacitative calcium entry

The effect of econazole on intracellular calcium levels ([Ca 2+] i) in Madin Darby canine kidney cells was investigated using fura-2 fluorimetry. Econazole increased [Ca 2+] i dose-dependently at 5–50 μM. The Ca 2+ signal consisted of an initial rise, a gradual decay and a sustained plateau. Extracellular Ca 2+ removal partially reduced the econazole response. Mn 2+ quench of fura-2 fluorescence confirmed econazole-induced Ca 2+ influx. The econazole-sensitive intracellular Ca 2+ store overlaps with that sensitive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+ pump, because 25 μM econazole depleted the thapsigargin-sensitive store, and conversely, thapsigargin abolished the econazole response. Econazole (25–50 μM) partially inhibited capacitative Ca 2+ entry induced by cyclopiazonic acid, another endoplasmic reticulum Ca 2+ pump inhibitor, measured by depleting internal Ca 2+ store in Ca 2+-free medium followed by adding 10 mM CaCl 2. Econazole induced capacitative Ca 2+ entry itself. Pretreatment with La 3+ (100 μM) partially inhibited 25 μM econazole-induced Mn 2+ quench of fura-2 fluorescence, and La 3+ immediately reduced 20 μM econazole-induced Ca 2+ signal when added at the peak of the signal, suggesting that econazole induced Ca 2+ influx via two separate pathways: one is sensitive to La 3+, the other is not. La 3+ enlarged 25 μM econazole-induced [Ca 2+] i transient during the decay phase. The econazole response was not altered when the cytosolic level of inositol 1,4,5-trisphosphate was inhibited by the phospholipase C inhibitor U73122.

2049 Effect of BCL-2 on apoptosis associated with the inhibition of capacitative calcium entry

2049 Effect of BCL-2 on apoptosis associated with the inhibition of capacitative calcium entry