Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry     

[Ca2+]i-reducing action of cAMP in rat pancreatic beta -cells: involvement of thapsigargin-sensitive stores

In the presentstudy, we examined the ability of adenosine 3',5'-cyclicmonophosphate (cAMP) to reduce elevated levels of cytosolicCa²⁺ concentration([Ca²⁺]_i)in pancreatic -cells.[Ca²⁺]_iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single-cells by fura 2 and autofluorescence microfluorometry. Sustained[Ca²⁺]_ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activatorforskolin (10 µM), and an incretin glucagon-likepeptide-1-(7-36) amide (10⁹ M), as well as byglucose (16.7 mM). The[Ca²⁺]_i-reducingeffects of cAMP were greater at elevated glucose (8.3-16.7 mM)than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA),H-89, counteracted[Ca²⁺]_i-reducingeffects of cAMP but not those of glucose. Okadaic acid, a phosphataseinhibitor, at 10-100 nM also reduced sustained [Ca²⁺]_ielevation in a concentration-dependent manner. Glucose, but not DBcAMP,increased NAD(P)H in -cells.[Ca²⁺]_i-reducingeffects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitorof the endoplasmic reticulum (ER)Ca²⁺ pump. In contrast,[Ca²⁺]_i-reducingeffects of cAMP were not altered by ryanodine, an ERCa²⁺-release inhibitor,Na⁺-free conditions, or diazoxide,an ATP-sensitive K⁺ channelopener. In conclusion, the cAMP-PKA pathway reduces[Ca²⁺]_ielevation by sequestering Ca²⁺ inthapsigargin-sensitive stores. This process does not involve, but ispotentiated by, activation of -cell metabolism. Together with theknown[Ca²⁺]_i-increasingaction of cAMP, our results reveal dual regulation of -cell[Ca²⁺]_iby the cAMP-signaling pathway and by a physiological incretin.

     

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     

Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the -subunits of the G protein gustducin (G_gust) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of Ca²⁺ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca²⁺] ([Ca²⁺]_i) in a dose- and time-dependent manner. Chelating extracellular Ca²⁺ with EGTA blocked the increase in [Ca²⁺]_i induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [Ca²⁺]_i induced by bombesin, but did not attenuate the [Ca²⁺]_i increase elicited by DB or PTC. These results indicate that Ca²⁺ influx mediates the increase in [Ca²⁺]_i induced by DB and PTC in STC-1 cells. Preincubation with the L-type voltage-sensitive Ca²⁺ channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 min inhibited the increase in [Ca²⁺]_i elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca²⁺]_i induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [Ca²⁺]_i and cholecystokinin release through Ca²⁺ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells. type 2 family taste receptors; gastrointestinal peptides; phospholipase C ₂; Ca²⁺ fluxes; enteroendocrine cells; cholecystokinin secretion     

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.

     

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and proliferation in pancreatic -cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca²⁺]_i involves Ca²⁺-induced Ca²⁺ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca²⁺]_i and insulin release in BRIN-BD11 -cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca²⁺]_i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K⁺ concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca²⁺]_i elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K⁺. Ovine prolactin increased [Ca²⁺]_i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca²⁺]_i but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca²⁺]_i. Our study indicates that hGH stimulates rise in [Ca²⁺]_i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells. c-Src; growth hormone receptor; prolactin receptor; Ca²⁺-induced Ca²⁺ release     

Effect of beta -adrenoceptor activation on [Ca2+]i regulation in murine skeletal myotubes

The presentstudy used real-time confocal microscopy to examine the effects of the₂-adrenoceptor agonistsalbutamol on regulation of intracellularCa²⁺ concentration([Ca²⁺]_i)in myotubes derived from neonatal mouse limb muscles.Immunocytochemical staining for ryanodine receptors and skeletal musclemyosin confirmed the presence of sarcomeres. The myotubes displayedboth spontaneous and ACh-induced rapid (<2-ms rise time)[Ca²⁺]_itransients. The[Ca²⁺]_itransients were frequency modulated by both low and high concentrations of salbutamol. Exposure to -bungarotoxin and tetrodotoxin inhibited ACh-induced[Ca²⁺]_itransients and the response to low concentrations of salbutamol but notthe response to higher concentrations. Preexposure to caffeineinhibited the subsequent[Ca²⁺]_iresponse to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the[Ca²⁺]_iresponse to caffeine. Inhibition of dihydropyridine-sensitive Ca²⁺ channels with nifedipine orPN-200-110 did not prevent[Ca²⁺]_ielevations induced by higher concentrations of salbutamol. The effectsof salbutamol were mimicked by the membrane-permeant analog dibutyryladenosine 3',5'-cyclic monophosphate. Thesedata indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulumCa²⁺ release.     

Gender-specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle cells of female rat

The hypothesisthat vascular protection in females and its absence in males reflectsgender differences in [Ca²⁺]_i andCa²⁺ mobilization mechanisms of vascular smooth musclecontraction was tested in fura 2-loaded aortic smooth muscle cellsisolated from intact and gonadectomized male and female Wistar-Kyoto(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca²⁺), the resting length and[Ca²⁺]_i were significantlydifferent in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than inintact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,phenylephrine (Phe, 10⁵ M) caused transient increasein [Ca²⁺]_i to 428 ± 13 nMfollowed by maintained increase to 201 ± 8 nM and 32% cellcontraction. In intact female WKY, the Phe-induced [Ca²⁺]_i transient was notsignificantly different, but the maintained [Ca²⁺]_i (159 ± 7 nM) and cellcontraction (26%) were significantly less than in intact male WKY. InCa²⁺-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)caused transient increases in[Ca²⁺]_i and contraction that werenot significantly different between males and females. Membranedepolarization by 51 mM KCl caused 31% cell contraction and increased[Ca²⁺]_i to 259 ± 9 nM in intactmale WKY, which were significantly greater than a 24% contraction and214 ± 8 nM [Ca²⁺]_i in intactfemale WKY. Maintained Phe- and KCl-stimulated cell contraction and[Ca²⁺]_i were significantly greaterin SHR than WKY in all groups of rats. Reduction in cell contractionand [Ca²⁺]_i in intact femalescompared with intact males was significantly greater in SHR (~30%)than WKY (~20%). No significant differences in cell contraction or[Ca²⁺]_i were observed betweencastrated males, ovariectomized (OVX) females, and intact males, orbetween OVX females with 17-estradiol implants and intact females.Exogenous application of 17-estradiol (10⁸ M) tocells from OVX females caused greater reduction in Phe- and KCl-inducedcontraction and [Ca²⁺]_i in SHR thanWKY. Thus the basal, maintained Phe- and depolarization-induced [Ca²⁺]_i and contraction of vascularsmooth muscle triggered by Ca²⁺ entry from theextracellular space exhibit differences depending on gender and thepresence or absence of female gonads. Cell contraction and[Ca²⁺]_i due to Ca²⁺release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca²⁺]_i in vascular smoothmuscle of female rats is greater in SHR than WKY rats.

     

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     

Glucose-induced mixed [Ca2+]c oscillations in mouse beta-cells are controlled by the membrane potential and the SERCA3 Ca2+-ATPase of the endoplasmic reticulum

Stimulatory concentrations of glucose induce two patterns of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) oscillations in mouse islets: simple or mixed. In the mixed pattern, rapid oscillations are superimposed on slow ones. In the present study, we examined the role of the membrane potential in the mixed pattern and the impact of this pattern on insulin release. Simultaneous measurement of [Ca²⁺]_c and insulin release from single islets revealed that mixed [Ca²⁺]_c oscillations triggered synchronous oscillations of insulin secretion. Simultaneous recordings of membrane potential in a single -cell within an islet and of [Ca²⁺]_c in the whole islet demonstrated that the mixed pattern resulted from compound bursting (i.e., clusters of membrane potential oscillations separated by prolonged silent intervals) that was synchronized in most -cells of the islet. Each slow [Ca²⁺]_c increase during mixed oscillations was due to a progressive summation of rapid oscillations. Digital image analysis confirmed the good synchrony between subregions of an islet. By contrast, islets from sarco(endo)plasmic reticulum Ca²⁺-ATPase isoform 3 (SERCA3)-knockout mice did not display typical mixed [Ca²⁺]_c oscillations in response to glucose. This results from a lack of progressive summation of rapid oscillations and from altered spontaneous electrical activity, i.e., lack of compound bursting, and membrane potential oscillations characterized by lower-frequency but larger-depolarization phases than observed in SERCA3^+/+ -cells. We conclude that glucose-induced mixed [Ca²⁺]_c oscillations result from compound bursting in all -cells of the islet. Disruption of SERCA3 abolishes mixed [Ca²⁺]_c oscillations and augments -cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the -cell membrane potential during glucose stimulation. electrical activity; insulin-secreting cell; thapsigargin     

Sphingosine-1-phosphate activates BKCa channels independently of G protein-coupled receptor in human endothelial cells

The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca²⁺]_i. [Ca²⁺]_i and membrane potentials were strongly correlated. In whole cell clamped cells, BK_Ca currents were activated by increasing [Ca²⁺]_i via cell dialysis with pipette solution, and the activated BK_Ca currents were further enhanced by S1P. When [Ca²⁺]_i was buffered at 1 µM, the S1P concentration required to evoke half-maximal activation was 403 ± 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BK_Ca channel activity in a reversible manner and shifted the relationship between Ca²⁺ concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5'-O-(2-thiodiphosphate) (GDPS; 1 mM) using a patch pipette, GDPS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BK_Ca current and channel activation. These results suggest that S1P enhances BK_Ca channel activity by increasing Ca²⁺ sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca²⁺ influx through Ca²⁺ entry channels. Inasmuch as S1P activates BK_Ca channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca²⁺ mobilization in human endothelial cells. sphingolipid metabolites; intracellular second messenger; Ca²⁺ mobilization     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

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.

     

Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of epsilon-protein kinase C-dependent pathway

Certain angina and coronary artery disease forms do not respond to Ca²⁺ channel blockers, and a role for vasoactive eicosanoids such as PGF₂ in Ca²⁺ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF₂-induced coronary smooth muscle contraction is Ca²⁺ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca²⁺), PGF₂ (10^-5 M) caused transient [Ca²⁺]_i increase followed by maintained [Ca²⁺]_i increase and 34% cell contraction. Ca²⁺ channel blockers verapamil and diltiazem (10^-6 M) abolished maintained PGF₂-induced [Ca²⁺]_i increase but only partially inhibited PGF₂-induced cell contraction to 17%. Verapamil-insensitive PGF₂ contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and -PKC V1-2. PGF₂ caused Ca²⁺-dependent -PKC and Ca²⁺-independent -PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF₂-induced -but not -PKC translocation. PMA (10^-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca²⁺]_i increase and -PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca²⁺ influx, caused 36% cell contraction and [Ca²⁺]_i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause - or -PKC translocation. Thus a significant component of PGF₂-induced contraction of coronary smooth muscle is Ca²⁺ antagonist insensitive, involves Ca²⁺-independent -PKC activation and translocation, and may represent a signaling mechanism of Ca²⁺ antagonist-resistant coronary vasospasm. eicosanoids; calcium; vascular smooth muscle     

P2 purinoceptor of the globular substance in the otoconial membrane of the guinea pig inner ear

The biological characteristics of the globular substance, aprecursor of otoconia, are unclear. In the present study, the ATP-induced internal free Ca²⁺ concentration([Ca²⁺]_i) changes of the globular substanceand the ATP distribution in the vestibular organ were investigatedusing a Ca²⁺ indicator, fluo 3, and an adeninenucleotide-specific fluorochrome, quinacrine, by means of confocallaser scanning microscopy. [Ca²⁺]_i showed arapid and dose-dependent increase in response to ATP with a 50%effective concentration (EC₅₀) of 16.7 µM. Thisreaction was independent of external Ca²⁺, indicating thepresence of an internal Ca²⁺ reservoir. Neither adenosine,,-methylene-ATP, 3'-O-(4-benzoylbenzoyl)-ATP, ADP, norUTP evoked this reaction, whereas 2-methylthio-ATP induced an increaseof [Ca²⁺]_i with an EC₅₀ of 14.4 µM. Moreover, P2 antagonists, reactive blue 2 and suramin, and aphospholipase C inhibitor, U-73122, inhibited the ATP-induced[Ca²⁺]_i increase. These findings indicate thepresence of a P2Y purinoceptor on the globular substance. In addition,granular fluorescence was observed in the quinacrine-stained macularsensory epithelium, indicating the presence of ATP-containing granulesin this tissue. These results suggest that a paracrine mechanisminvolving ATP may exist in the macula and that this mechanism regulatesthe biological behavior of the globular substance.

     

Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor

We determined the effect of aromatic aminoacid stimulation of the human extracellular Ca²⁺-sensingreceptor (CaR) on intracellular Ca²⁺ concentration([Ca²⁺]_i) in single HEK-293 cells. Additionof L-phenylalanine or L-tryptophan (at 5 mM)induced [Ca²⁺]_i oscillations from a restingstate that was quiescent at 1.8 mM extracellular Ca²⁺concentration ([Ca²⁺]_e). Each[Ca²⁺]_i peak returned to baseline values, andthe average oscillation frequency was ~1 min¹ at37°C. Oscillations were not induced or sustained if the[Ca²⁺]_e was reduced to 0.5 mM, even in thecontinued presence of amino acid. Average oscillation frequency inresponse to an increase in [Ca²⁺]_e (from 1.8 to 2.5-5 mM) was much higher (~4 min¹) than thatinduced by aromatic amino acids. Oscillations in response to[Ca²⁺]_e were sinusoidal whereas those inducedby amino acids were transient. Thus both amino acids andCa²⁺, acting through the same CaR, produce oscillatoryincreases in [Ca²⁺]_i, but the resultantoscillation pattern and frequency allow the cell to discriminate whichagonist is bound to the receptor.

     

Suppression of cAMP by phosphoinositol/Ca2+ pathway in the cardiac kappa -opioid receptor

To determine whetherthe phosphoinositol/Ca²⁺ pathwayinteracts with the adenylate cyclase/adenosine 3',5'-cyclicmonophosphate (cAMP) pathway in the cardiac -receptor, the effectsof U-50488, a specific -receptor agonist, on the intracellularCa²⁺ concentration([Ca²⁺]_i)and forskolin-induced accumulation of cAMP in rat ventricular myocyteswere determined after interference of thephosphoinositol/Ca²⁺ pathway.U-50488 suppressed the forskolin-induced accumulation of cAMP andelevated[Ca²⁺]_i,which were blocked by norbinaltorphimine, a specific -receptor antagonist, and pertussis toxin. The effects of U-50488 werequalitatively similar to those of A-23187, aCa²⁺ ionophore, but opposite tothose of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM), a[Ca²⁺]_ichelator. Abolition of U-50488-induced elevation of[Ca²⁺]_iby BAPTA-AM also abolished the effect of U-50488 on forskolin-induced accumulation of cAMP. Inhibition of the phospholipase C by specific inhibitors, U-73122 and neomycin, abolished the effects of U-50488 onboth[Ca²⁺]_iand forskolin-induced accumulation of cAMP. The results showed for thefirst time that -receptor stimulation may suppress cAMP accumulationvia activation of thephosphoinositol/Ca²⁺ pathway inthe rat heart.

     

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     

Impaired G(s)alpha and adenylyl cyclase cause beta-adrenoceptor desensitization in chronically hypoxic rat hearts

The effectsof -adrenoceptor stimulation with isoproterenol on electricallyinduced contraction and intracellular calcium ([Ca²⁺]_i) transient, and cAMP inmyocytes from both hypertrophied right and nonhypertrophied leftventricles of rats exposed to 10% oxygen for 4 wk, were significantlyattenuated. The increased [Ca²⁺]_i transientin response to cholera toxin was abolished, whereas increased cAMPafter NaF significantly attenuated. The biologically activeisoform, G_s-small (45 kDa), was reduced while thebiologically inactive isoform, G_s-large (52 kDa),increased. The increased electrically induced[Ca²⁺]_i transient and cAMP with 10-100µM forskolin were significantly attenuated in chronically hypoxicrats. The content of G_i2, the predominantisoform of G_i protein in the heart, was unchanged. Resultsindicate that impaired functions of G_s protein and adenylyl cyclase cause -adrenoceptor desensitization. The impaired function of the G_s protein may be due to reducedG_s-small and/or increased G_s-large, whichdoes not result from changes in G_i protein. Responses toall treatments were the same for right and left ventricles, indicatingthat the impaired cardiac functions are not secondary to cardiac hypertrophy.

     

Na(+)/Ca(2+) exchange and its role in intracellular Ca(2+) regulation in guinea pig detrusor smooth muscle

The role of Na⁺/Ca²⁺ exchange inregulating intracellular Ca²⁺ concentration([Ca²⁺]_i) in isolated smooth muscle cellsfrom the guinea pig urinary bladder was investigated. Incrementalreduction of extracellular Na⁺ concentration resulted in agraded rise of [Ca²⁺]_i; 50-100 µMstrophanthidin also increased [Ca²⁺]_i. Asmall outward current accompanied the rise of[Ca²⁺]_i in low-Na⁺ solutions(17.1 ± 1.8 pA in 29.4 mM Na⁺). The quantity ofCa²⁺ influx through the exchanger was estimated from thecharge carried by the outward current and was ~30 times that which isnecessary to account for the rise of [Ca²⁺]_i,after correction was made for intracellular Ca²⁺ buffering.Ca²⁺ influx through the exchanger was able to loadintracellular Ca²⁺ stores. It is concluded that the levelof resting [Ca²⁺]_i is not determined by theexchanger, and under resting conditions (membrane potential 50 to60 mV), there is little net flux through the exchanger. However, asmall rise of intracellular Na⁺ concentration would besufficient to generate significant net Ca²⁺ influx.