Opposite regulation of KCNQ5 and TRPC6 channels contributes to vasopressin-stimulated calcium spiking responses in A7r5 vascular smooth muscle cells

Physiologically relevant concentrations of [Arg⁸]-vasopressin (AVP) induce repetitive action potential firing and Ca²⁺ spiking responses in the A7r5 rat aortic smooth muscle cell line. These responses may be triggered by suppression of KCNQ potassium currents and/or activation of non-selective cation currents. Here we examine the relative contributions of KCNQ5 channels and TRPC6 non-selective cation channels to AVP-stimulated Ca²⁺ spiking using patch clamp electrophysiology and fura-2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 channel expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown resulted in more positive resting membrane potentials and induced spontaneous action potential firing and Ca²⁺ spiking. However physiological concentrations of AVP induced additional depolarization and increased Ca²⁺ spike frequency in KCNQ5 knockdown cells. AVP activated a non-selective cation current that was reduced by TRPC shRNA treatment or removal of external Na⁺. Neither resting membrane potential nor the AVP-induced depolarization was altered by knockdown of TRPC6 channel expression. However, both TRPC6 shRNA and removal of external Na⁺ delayed the onset of Ca²⁺ spiking induced by 25 pM AVP. These results suggest that suppression of KCNQ5 currents alone is sufficient to excite A7r5 cells, but AVP-induced activation of TRPC6 contributes to the stimulation of Ca²⁺ spiking.     

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     

Signal Transduction in Smooth Muscle: Selected Contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca2+ sensitivity

To clarify the contribution of intracellularCa²⁺ concentration([Ca²⁺]_i)-dependent and -independentsignaling mechanisms in arteriolar smooth muscle (aSM) to modulation ofarteriolar myogenic tone by nitric oxide (NO), released in response toincreases in intraluminal flow from the endothelium, changes in aSM[Ca²⁺]_i and diameter of isolated rat gracilismuscle arterioles (pretreated with indomethacin) were studied byfluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca²⁺]_i significantly increased andmyogenic tone developed in response to elevations of extracellularCa²⁺ concentration. The Ca²⁺ channelinhibitor nimodipine substantially decreased[Ca²⁺]_i and completely inhibited myogenictone. Dilations to intraluminal flow (that were inhibited byN-nitro-L-arginine methyl ester)or dilations to the NO donorS-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were notaccompanied by substantial decreases in aSM[Ca²⁺]_i. 8-Bromoguanosine cGMP and thecGMP-specific phosphodiesterase inhibitor zaprinast significantlydilated arterioles yet elicited only minimal decreases in[Ca²⁺]_i. Thus flow-induced endothelialrelease of NO elicits relaxation of arteriolar smooth muscle by acGMP-dependent decrease of the Ca²⁺ sensitivity of thecontractile apparatus without substantial changes in thepressure-induced level of [Ca²⁺]_i.

     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

     

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) using double-barreled, Ca²⁺-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca²⁺]_i was approximately twofold in MHS compared with MHN quiescent myoballs (232 ± 35 vs. 112 ± 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca²⁺]_i in both groups; however, the concentration required to cause a rise in [Ca²⁺]_i elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca²⁺ buffer BAPTA reduced [Ca²⁺]_i, raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca²⁺]_i, and reduced the amount of Ca²⁺ release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca²⁺]_i levels in these cells. calcium homeostasis; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid     

Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

     

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

A sustained increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca²⁺ stores by thapsigargin (TG), induced an increase in the [Ca²⁺]_i and cell death. However, the TG-induced death was not related to the [Ca²⁺]_i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca²⁺-dependent anti-apoptotic mechanism. After TG treatment, Ca²⁺-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca²⁺-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca²⁺]_i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca²⁺-dependent MAPK activation counteracts death signaling; removal of Ca²⁺ activated a second caspase-independent death pathway. sarco(endo)plasmic reticulum calcium ion adenosine triphosphatase; cytosolic calcium ion concentration; mitogen-activated protein kinase     

Intravascular pressure regulates local and global Ca2+ signaling in cerebral artery smooth muscle cells

The regulationof intracellular Ca²⁺ signals in smooth muscle cells andarterial diameter by intravascular pressure was investigated in ratcerebral arteries (~150 µm) using a laser scanning confocal microscope and the fluorescent Ca²⁺ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca²⁺spark frequency 2.6-fold, Ca²⁺ wave frequency 1.9-fold, andglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) 1.4-fold in smooth muscle cells,and constricted arteries. Ryanodine (10 µM), an inhibitor ofryanodine-sensitive Ca²⁺ release channels, or thapsigargin(100 nM), an inhibitor of the sarcoplasmic reticulumCa²⁺-ATPase, abolished sparks and waves, elevated global[Ca²⁺]_i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 µM), a voltage-dependentCa²⁺ channel (VDCC) blocker, significantly reduced sparks,waves, and global [Ca²⁺]_i, and dilatedpressurized (60 mmHg) arteries. Steady membrane depolarization elevatedCa²⁺ signaling similar to pressure and increased transientCa²⁺-sensitive K⁺ channel current frequencye-fold for ~7 mV, and these effects were prevented by VDCCblockers. Data are consistent with the hypothesis that pressure inducesa steady membrane depolarization that activates VDCCs, leading to anelevation of spark frequency, wave frequency, and global[Ca²⁺]_i. In addition, pressure inducescontraction via an elevation of global[Ca²⁺]_i, whereas the net effect of sparks andwaves, which do not significantly contribute to global[Ca²⁺]_i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.

     

Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle

Prakash, Y. S., H. F. M. van der Heijden, M. S. Kannan, andG. C. Sieck. Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle. J. Appl.Physiol. 82(6): 1836-1843, 1997.Relaxation ofairway smooth muscle (ASM) by -adrenoceptor agonists involvesreduction of intracellular Ca²⁺concentration([Ca²⁺]_i).In porcine ASM cells, acetylcholine induces[Ca²⁺]_ioscillations that display frequency modulation by agonist concentration and basal[Ca²⁺]_i.We used real-time confocal microscopy to examine the effect ofsalbutamol (1 nM to 1 µM), a₂-adrenoceptor agonist, on[Ca²⁺]_ioscillations in freshly dissociated porcine ASM cells. Salbutamol decreased the frequency of[Ca²⁺]_ioscillations in a concentration-dependent fashion, completely inhibiting the oscillations at 1 µM. These effects were mimicked by acell-permeant analog of adenosine 3,5-cyclicmonophosphate. The inhibitory effect of salbutamol was partiallyreversed by BAY K 8644. Salbutamol reduced[Ca²⁺]_ieven when sarcoplasmic reticulum (SR)Ca²⁺ reuptake andCa²⁺ influx were blocked.Lanthanum blockade of Ca²⁺ effluxattenuated the inhibitory effect of salbutamol on[Ca²⁺]_i.The[Ca²⁺]_iresponse to caffeine was unaffected by salbutamol. On the basis ofthese results, we conclude that₂-adrenoceptor agonists have little effect on SR Ca²⁺ releasein ASM cells but reduce[Ca²⁺]_iby inhibiting Ca²⁺ influx throughvoltage-gated channels and by enhancingCa²⁺ efflux.

     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

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     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Polyunsaturated fatty acids mobilize intracellular Ca2+ in NT2 human teratocarcinoma cells by causing release of Ca2+ from mitochondria

In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca²⁺ ([Ca²⁺]_m), which fluctuates in coordination with intracellular Ca²⁺ ([Ca²⁺]_i). Polyunsaturated FA (PUFA) have been shown to cause [Ca²⁺]_i mobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca²⁺]_i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca²⁺]_i response to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca²⁺]_i mobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP₃) receptor activation, as well as IP₃-sensitive internal Ca²⁺ stores. Furthermore, PUFA-mediated [Ca²⁺]_i mobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca²⁺]_m with X-rhod-1 and ⁴⁵Ca²⁺ indicate that PUFA induce Ca²⁺ efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, blocks PUFA-induced Ca²⁺ efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca²⁺ release from mitochondria, possibly via the Ca²⁺ uniporter, appears to be the underlying mechanism for PUFA-induced [Ca²⁺]_i mobilization in NT2 cells. arachidonic acid; mitochondrial Ca²⁺ uniporter; G protein-coupled receptor; IP₃ receptor     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells

Studies were performed to identify the molecular component responsible for store-operated Ca²⁺ entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca²⁺-selective and -nonselective cation channels in a variety of cells, we screened TRPC1–TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated Ca²⁺ (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). SOC was measured as the increase in [Ca²⁺]_i after extracellular Ca²⁺ was increased from <10 nM to 1 mM in the continued presence of thapsigargin. We found that TRPC4 antisense, which reduced plasmalemmal expression of TRPC4, inhibited SOC by 83%. Incubation with scrambled TRPC4 oligonucleotides did not affect SOC. Immunohistochemical staining identified expressed TRPC4 in the glomeruli of mouse renal sections. The results of RT-PCR performed to distinguish between TRPC4- and TRPC4- were consistent with expression of both isoforms in brain but with only TRPC4- expression in MMC. These studies show that TRPC4- may form the homotetrameric SOC in mouse mesangial cells. canonical transient receptor potential; TRPC4-; TRPC4-; TRPC1; fura 2; glomerulus     

Mechanical signals and mechanosensitive modulation of intracellular [Ca(2+)] in smooth muscle

We testedthe hypothesis that strain is the primary mechanical signal in themechanosensitive modulation of intracellular Ca²⁺concentration ([Ca²⁺]_i) in airway smoothmuscle. We found that [Ca²⁺]_i wassignificantly correlated with muscle length during isotonic shorteningagainst 20% isometric force (F_iso). When the isotonic loadwas changed to 50% F_iso, data points from the 20 and 50% F_iso experiments overlapped in thelength-[Ca²⁺]_i relationship. Similarly, datapoints from the 80% F_iso experiments clustered near thosefrom the 50% F_iso experiments. Therefore, despite 2.5- and4-fold differences in external load, [Ca²⁺]_idid not deviate much from the length-[Ca²⁺]_irelation that fitted the 20% F_iso data. Maximal inhibition of sarcoplasmic reticular (SR) Ca²⁺ uptake by 10 µMcyclopiazonic acid (CPA) did not significantly change[Ca²⁺]_i in carbachol-induced isometriccontractions and isotonic shortening. CPA also did not significantlychange myosin light-chain phosphorylation or force redevelopment whencarbachol-activated muscle strips were quickly released from optimallength (L_o) to 0.5 L_o. These results are consistent with thehypothesis and suggest that SR Ca²⁺ uptake is not theunderlying mechanism.

     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Metabolic inhibition with cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes

We examined the effectsof metabolic inhibition on intracellular Ca²⁺ release insingle pulmonary arterial smooth muscle cells (PASMCs). Severemetabolic inhibition with cyanide (CN, 10 mM) increased intracellularcalcium concentration ([Ca²⁺]_i) and activatedCa²⁺-activated Cl currents[I_Cl(Ca)] in PASMCs, responses that were greatlyinhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents andCa²⁺ sparks in PASMCs. In Xenopus oocytes, CNalso induced Ca²⁺ release and activatedI_Cl(Ca), and these responses were inhibited by thapsigarginand cyclopiazonic acid to deplete sarcoplasmic reticulum (SR)Ca²⁺, whereas neither heparin nor anti-inositol1,4,5-trisphosphate receptor (IP₃R) antibodies affected CNresponses. In both PASMCs and oocytes, CN-evoked Ca²⁺release was inhibited by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and oligomycin or CCCP andthapsigargin. Whereas hypoxic stimuli resulted in Ca²⁺release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition withCN increases [Ca²⁺]_i in both pulmonary andsystemic artery myocytes by stimulating Ca²⁺ release fromthe SR and mitochondria.