Ca2+]i regulates trafficking of Cav1.3 (alpha1D Ca2+ channel) in insulin-secreting cells

Chronic exposure of pancreatic -cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of -cell basal intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We have investigated the relationship between the basal intracellular free Ca²⁺ and the L-type (Ca_v1.3) Ca²⁺ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca²⁺ influx, which was attenuated when the basal [Ca²⁺]_i was elevated. Using voltage-clamp techniques, we found that changing [Ca²⁺]_i could regulate the amplitude of the Ca²⁺ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Ca_v1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca²⁺]_i conditions by deconvolution microscopy. The [Ca²⁺]_i-dependent translocation of Ca_v1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca²⁺]_i. These results suggest that Ca_v1.3 channel trafficking is involved in glucose desensitization of pancreatic -cells. internalization; intracellular free calcium; glucose desensitization     

Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

To study the effects of -opioid receptor stimulation onintracellular Ca²⁺ concentration([Ca²⁺]_i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca²⁺]_iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca²⁺]_itransient, which results from the influx ofCa²⁺ and the subsequentmobilization of Ca²⁺ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca²⁺]_iand inhibited the[Ca²⁺]_itransient induced by caffeine, which mobilizesCa²⁺ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa²⁺ from its intracellular poolinto the cytoplasm. The Ca²⁺responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient were significantly attenuated when the extracellular pH(pH_e) was loweredto 6.8, which itself reduced intracellular pH(pH_i) and increased[Ca²⁺]_i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa⁺/H⁺exchange blocker, or 0.2 mM Ni²⁺,a putativeNa⁺/Ca²⁺exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa⁺/H⁺andNa⁺/Ca²⁺exchanges. When glucose at 50 mM, known to activate theNa⁺/H⁺exchange, was added, both the resting[Ca²⁺]_iand pH_i increased. Interestingly,the effects of U-50488H on [Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solutionwas superfused, the resting[Ca²⁺]_iincreased; the increase was abolished by 0.2 mMNi²⁺, but thepH_i remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca²⁺]_iand electrically induced[Ca²⁺]_itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa²⁺ from SR. Activation of bothNa⁺/H⁺andNa⁺/Ca²⁺exchanges, leading to an elevation of[Ca²⁺]_i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

     

Two-step Ca2+ intracellular release underlies excitation-contraction coupling in mouse urinary bladder myocytes

The relative contributions of Ca²⁺-induced Ca²⁺ release (CICR) versus Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) to excitation-contraction coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder (UB) smooth muscle cells (UBSMCs). Confocal Ca²⁺ images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30-ms depolarization to 0 mV, intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased in several small, discrete areas just beneath the cell membrane. These Ca²⁺ "hot spots" then spread slowly through the myoplasm as Ca²⁺ waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few Ca²⁺ sparks, which declined quickly. The number of Ca²⁺ sparks, or hot spots, was closely related to the depolarization duration in the range of 5–20 ms. There was an apparent threshold depolarization duration of 10 ms within which to induce enough Ca²⁺ transients to spread globally and then induce a contraction. Application of 100 µM ryanodine to the pipette solution did not change the resting [Ca²⁺]_i or the VDCC current, but it did abolish Ca²⁺ hot spots elicited by depolarization. Application of 3 µM xestospongin C reduced ACh-induced Ca²⁺ release but did not affect depolarization-induced Ca²⁺ events. The addition of 100 µM ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [Ca²⁺]_i rise triggered by a single action potential is not due mainly to Ca²⁺ influx through VDCCs but is attributable to the subsequent two-step CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺-activated K⁺ current; voltage-dependent Ca²⁺ channel     

Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0

During maturation of oocytes,Cl conductance (G_Cl) oscillatesand intracellular pH (pH_i) increases. ElevatingpH_i permits the protein synthesis essential to maturation.To examine whether changes in G_Cl andpH_i are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespH_i, decreases intracellular Cl concentration([Cl]_i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pH_ichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pH_i. In contrast, hyperpolarization elevates pH_i. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pH_iand decreases [Cl]_i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpH_i elevation, although the decrease in[Cl]_i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pH_i and decreases[Cl]_i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte G_Cl may exert an effect onpH_i via shrinkage, thus inducing meiotic maturation.

     

Impaired [Ca(2+)](i) and pH(i) responses to kappa-opioid receptor stimulation in the heart of chronically hypoxic rats

-Opioid receptor (-OR)stimulation with U50,488H, a selective -OR agonist, or activation ofprotein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), anactivator of PKC, decreased the electrically induced intracellularCa²⁺ ([Ca²⁺]_i) transient andincreased the intracellular pH (pH_i) in single ventricularmyocytes of rats subjected to 10% oxygen for 4 wk. The effects ofU50,488H were abolished by nor-binaltorphimine, a selective -ORantagonist, and calphostin C, a specific inhibitor of PKC, while theeffects of PMA were abolished by calphostin C andethylisopropylamiloride (EIPA), a potent Na⁺/H⁺exchange blocker. In both right hypertrophied and leftnonhypertrophied ventricles of chronically hypoxic rats, the effects ofU50,488H or PMA on [Ca²⁺]_i transient andpH_i were significantly attenuated and completely abolished,respectively. Results are first evidence that the[Ca²⁺]_i and pH_i responses to-OR stimulation are attenuated in the chronically hypoxic rat heart,which may be due to reduced responses to PKC activation. Responses toall treatments were the same for right and left ventricles, indicatingthat the functional impairment is independent of hypertrophy. -ORmRNA expression was the same in right and left ventricles of bothnormoxic and hypoxic rats, indicating no regional specificity.

     

Mechanical stress-induced Ca2+ entry and Cl- current in cultured human aortic endothelial cells

A fluid streamthrough a microtube was applied to cultured human aortic endothelialcells to investigate the endothelial responses of both the ioniccurrents and intracellular Ca²⁺concentration([Ca²⁺]_i)to mechanical stimulation. The fluid stream induced an increase in[Ca²⁺]_ithat was dependent on both the flow rate and the extracellular Ca²⁺ concentration.Gd³⁺ and niflumic acid inhibitedthe fluid stream-induced increase in[Ca²⁺]_i,whereas Ba²⁺ andtetraethylammonium ion exhibited no effect. The fluid stream-induced [Ca²⁺]_iincrease was accompanied by the activation of an inward current at52.8 mV. The reversal potential of the fluid stream-induced current shifted to positive potentials when the externalCl concentration wasreduced but was not affected by variation of the externalNa⁺ concentration. During theexposure to the fluid stream,[Ca²⁺]_iwas voltage dependent, i.e., depolarization decreased[Ca²⁺]_i.We therefore conclude that the fluid stream-induced current is largelycarried by Cl and that theCl current may thus play arole in modulating the Ca²⁺ influxby altering the membrane potential of endothelial cells.

     

Involvement of stretch-activated cation channels in hypotonically induced insulin secretion in rat pancreatic beta-cells

In isolated rat pancreatic -cells, hypotonic stimulation elicited an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) at 2.8 mM glucose. The hypotonically induced [Ca²⁺]_c elevation was significantly suppressed by nicardipine, a voltage-dependent Ca²⁺ channel blocker, and by Gd³⁺, amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca²⁺]_c elevation was not inhibited by suramin, a P₂ purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of -cells and produced outwardly rectifying cation currents; Gd³⁺ inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd³⁺ significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic -cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca²⁺ channels and thus elevating insulin secretion. calcium ion; swelling; patch-clamp; gadolinium     

Ciglitizone inhibits cell proliferation in human uterine leiomyoma via activation of store-operated Ca2+ channels

This study investigated the acute effects of a peroxisome proliferator-activated receptor (PPAR)- ligand, ciglitizone, on cell proliferation and intracellular Ca²⁺ signaling in human normal myometrium and uterine leiomyoma. Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured with fura-2 AM, and cellular viabilities were determined by viable cell count and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay. Ciglitizone (100 µM) induced greater inhibition of cell proliferation in uterine leiomyoma than in myometrium. Ciglitizone also dose-dependently increased [Ca²⁺]_i in both myometrium and uterine leiomyoma; these [Ca²⁺]_i increases were inhibited by PPAR- antagonists and raloxifene. Ciglitizone-induced [Ca²⁺]_i increase showed only an initial peak in normal myometrial cells, whereas in uterine leiomyoma there was a second sustained [Ca²⁺]_i increase as well. The initial [Ca²⁺]_i increase in both myometrium and uterine leiomyoma resulted from the release of Ca²⁺ by the sarcoplasmic reticulum via activation of ryanodine receptors. The second [Ca²⁺]_i increase was observed only in uterine leiomyoma because of a Ca²⁺ influx via an activation of store-operated Ca²⁺ channels (SOCCs). Cell proliferation was inhibited and secondary [Ca²⁺]_i increase in uterine leiomyoma was attenuated by cotreatment of ciglitizone with a SOCC blocker, lanthanum. The results suggest that ciglitizone inhibits cell proliferation and increases [Ca²⁺]_i through the activation of SOCCs, especially in human uterine leiomyoma. peroxisome proliferator-activated receptor-; intracellular calcium; uterine cells     

Erythropoietin-modulated calcium influx through TRPC2 is mediated by phospholipase Cgamma and IP3R

In the present study, we examined the mechanisms through which erythropoietin (Epo) activates the calcium-permeable transient receptor potential protein channel (TRPC)2. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in intracellular calcium concentration ([Ca²⁺]_i). This increase in [Ca²⁺]_i was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U-73122 but not by the inactive analog U-73343, demonstrating the requirement for PLC activity in Epo-modulated Ca²⁺ influx in primary erythroid cells. To determine whether PLC is involved in the activation of TRPC2 by Epo, cell models were used to examine this interaction. Single CHO-S cells that expressed transfected Epo receptor (Epo-R) and TRPC2 were identified, and [Ca²⁺]_i was quantitated. Epo-induced Ca²⁺ influx through TRPC2 was inhibited by pretreatment with U-73122 or by downregulation of PLC1 by RNA interference. PLC activation results in the production of inositol 1,4,5-trisphosphate (IP₃), and TRPC2 has IP₃ receptor (IP₃R) binding sites. To determine whether IP₃R is involved in Epo-R signaling, TRPC2 mutants were prepared with partial or complete deletions of the COOH-terminal IP₃R binding domains. In cells expressing TRPC2 IP₃R binding mutants and Epo-R, no significant increase in [Ca²⁺]_i was observed after Epo stimulation. TRPC2 coassociated with Epo-R, PLC, and IP₃R, and the association between TRPC2 and IP₃R was disrupted in these mutants. Our data demonstrate that Epo-R modulates TRPC2 activation through PLC; that interaction of IP₃R with TRPC2 is required; and that Epo-R, TRPC2, PLC, and IP₃R interact to form a signaling complex. transient receptor potential protein channels; erythropoietin receptor; calcium channels     

Role of calcium channels in carboxyl-terminal parathyroid hormone receptor signaling

Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(1–84) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([Ca²⁺]_i) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca²⁺]_i was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca²⁺]_i was observed in OC59 cells in response to the CPTH fragment hPTH-(53–84) (250 nM). No [Ca²⁺]_i signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(1–34) nor a mutant CPTH analog, [Ala^55–57]hPTH-(53–84), that does not to bind to CPTHRs, increased [Ca²⁺]_i in OC59 cells. The [Ca²⁺]_i response to hPTH-(53–84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), -agatoxin IVA (10 nM), and -conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs. calcium influx; osteocytes