Coupling of L-type voltage-sensitive calcium channels to P2X(2) purinoceptors in PC-12 cells

Extracellular ATPelevates cytosolic Ca²⁺ by activating P2X and P2Ypurinoceptors and voltage-sensitive Ca²⁺ channels (VCCCs)in PC-12 cells, thereby facilitating catecholamine secretion. Weinvestigated the mechanism by which ATP activates VSCCs.2-Methylthioadenosine 5'-triphosphate (2-MeS-ATP) and UTP were used aspreferential activators of P2X and P2Y, respectively. Nifedipineinhibited the ATP- and 2-MeS-ATP-evoked cytosolic Ca²⁺concentration increase and [³H]norepinephrine secretion,but not the UTP-evoked responses. Studies with Ca²⁺ channelblockers indicated that L-type VSCCs were activated after the P2Xactivation. Mn²⁺ entry profiles and studies withthapsigargin revealed that Ca²⁺ entry, rather thanCa²⁺ release, was sensitive to nifedipine. AlthoughP2X₂ and P2X₄ receptor mRNAs were detected,studies with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acidrevealed that P2X₂ was mainly coupled to the L-type VSCCs. The inhibitory effect of nifedipine did not occur in the absence ofextracellular Na⁺, suggesting that Na⁺ influx,which induces depolarization, was essential for theP2X₂-mediated activation of VSCCs. We report thatdepolarization induced by Na⁺ entry through theP2X₂ purinoceptors effectively activates L-type VSCCs inPC-12 cells.

     

Mechanism of Catecholamine Synthesis Inhibition by Neuropeptide Y: Role of Ca2+ Channels and Protein Kinases

Abstract: We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective Ca²⁺ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The L-type Ca²⁺ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by ～90% and attenuated the inhibitory effect of NPY. In contrast, the N-type Ca²⁺ channel blocker ω-conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP-dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stimulation of Ca²⁺/phospholipid-dependent protein kinase (PKC) with phorbol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concentration-dependent inhibition of depolarization-stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels, possibly through a PKC-mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization-stimulated catecholamine synthesis.     

Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms

Published data suggest that the neuropeptide calcitonin gene-related peptide (CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca²⁺ concentration ([Ca²⁺]_int) and membrane potential (E_m) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC₄(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca²⁺]_int: a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC₅₀ of 0.30 nM, and the maximal effect (initially 3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca²⁺ mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca²⁺ influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca²⁺ discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular E_m, with maximal effect at 20 nM and an EC₅₀ of 0.30 nM. This effect was attenuated with charybdotoxin (–20%) or glyburide (glibenclamide; –80%), suggesting that E_m hyperpolarization is induced by both Ca²⁺-activated and ATP-sensitive K⁺ channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells. osteoblastic cells; calcium; membrane potential; potassium channels; adenosine 3',5'-cyclic monophosphate     

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     

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

Spontaneous transient outward currents(STOCs) were recorded from smooth muscle cells of theguinea pig taenia coli using the whole cell patch-clamp technique.STOCs were resolved at potentials positive to 50 mV. Treatingcells with caffeine (1 mM) caused a burst of outward currentsfollowed by inhibition of STOCs. Replacing extracellularCa²⁺ with equimolarMn²⁺ caused STOCs to "rundown." Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM)inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs"remained in the presence of these drugs. Mini-STOCs were reduced byapamin (500 nM), an inhibitor of small-conductance Ca²⁺-activatedK⁺ channels (SK channels).Application of ATP or 2-methylthioadenosine 5'-triphosphate(2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATPpersisted in the presence of charybdotoxin but were blocked bycombination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did notincrease STOCs in the presence of pyridoxal phosphate6-azophenyl-2',4'-disulfonic acid, aP₂ receptor blocker. Similarly,pretreatment of cells with U-73122 (1 µM), an inhibitor ofphospholipase C (PLC), abolished the effects of 2-MeS-ATP. XestosponginC, an inositol 1,4,5-trisphosphate(IP₃) receptor blocker,attenuated STOCs, but these events were not affected by ryanodine. Thedata suggest that purinergic activation through P_2Y receptors results in localizedCa²⁺ release via PLC- andIP₃-dependent mechanisms. Releaseof Ca²⁺ is coupled to STOCs, whichare composed of currents mediated by large-conductanceCa²⁺-activatedK⁺ channels and SK channels. Thelatter are thought to mediate hyperpolarization and relaxationresponses of gastrointestinal muscles to inhibitory purinergic stimulation.

     

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]_e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca²⁺ influx may mediate this effect. We show that increase in [Os]_e (by 18–125%) differentially suppresses L-type and T-type Ca²⁺ channel currents (I_L and I_T, respectively); I_L was more sensitive than I_T. Hyperosmotic suppression of I_L depended on the magnitude of increase in [Os]_e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of I_L and I_T persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca²⁺ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca²⁺ influx involves Ca²⁺ channel proteins. We therefore recorded the activity of L-type Ca²⁺ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]_e reduced the activity of Ca²⁺ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca²⁺ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca²⁺ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Effects of PKCalpha activation on Ca2+ pump and KCa channel in deoxygenated sickle cells

We have previously shown that a pretreatment with phorbol12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC),reduced deoxygenation-induced K⁺loss and Ca²⁺ uptake and preventedcell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007,1995). The present study explores the detailed mechanism of thisPMA-induced inhibition. The main findings are, first, the detection ofPKC and PKC in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The -isoform only is translocated to the membrane and activated by PMAand by elevation of cytosolicCa²⁺. Second, PMA is demonstratedto activate Ca²⁺ efflux indeoxygenated SS cells by a direct stimulation of the Ca²⁺ pump. PMA, moreover, inhibitsdeoxygenation-induced, charybdotoxin-sensitive K⁺ efflux in SS cells. Thisinhibition is partly indirect and explained by the reduceddeoxygenation-induced rise in cytosolicCa²⁺ resulting fromCa²⁺ pump stimulation. However, asignificant inhibition of theCa²⁺-activatedK⁺ channels(K_Ca channels) by PMA can also bedemonstrated when the channels are activated byCa²⁺ plus ionophore, underconditions in which the Ca²⁺ pumpis operating near its maximal extrusion rate, but swamped byCa²⁺ plus ionophore. The data thussuggest a PKC-mediated phosphorylation both of theCa²⁺ pump and of theK_Ca channel or an auxiliaryprotein.

     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

Intracellular signal transduction during gastrin-induced histamine secretion in rat gastric ECL cells

Activation of G_qprotein-coupled receptors usually causes a biphasic increase inintracellular calcium concentration ([Ca²⁺]_i)that is crucial for secretion in nonexcitable cells. In gastric enterochromaffin-like (ECL) cells, stimulation with gastrin leads to aprompt biphasic calcium response followed by histamine secretion. Thisstudy investigates the underlying signaling events in this neuroendocrine cell type. In ECL cells, RT-PCR suggested the presence of inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes1-3. The IP₃R antagonist 2-aminoethoxydiphenyl borateabolished both gastrin-induced elevation of[Ca²⁺]_i and histamine release. Thapsigarginincreased [Ca²⁺]_i, however, without inducinghistamine secretion. In thapsigargin-pretreated cells, gastrinincreased [Ca²⁺]_i through calcium influxacross the plasma membrane. Both nimodipine and SKF-96365 inhibitedgastrin-induced histamine release. The protein kinase C (PKC) activatorphorbol 12-myristate 13-acetate induced histamine secretion, an effectthat was prevented by nimodipine. In summary, gastrin-stimulatedhistamine release depends on IP₃R activation andplasmalemmal calcium entry. Gastrin-induced calcium influx wasmediated by dihydropyridine-sensitive calcium channels that appear tobe L-type channels activated through a pathway involving activation of PKC.

     

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.

     

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.

     

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.

     

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     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

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     

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     

Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells

The L-type Ca²⁺ channel is the primary voltage-dependent Ca²⁺-influx pathway in many excitable and secretory cells, and direct phosphorylation by different kinases is one of the mechanisms involved in the regulation of its activity. The aim of this study was to evaluate the participation of Ser/Thr kinases and tyrosine kinases (TKs) in depolarization-induced Ca²⁺ influx in the endocrine somatomammotrope cell line GH3. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using a spectrofluorometric method with fura 2-AM, and 12.5 mM KCl (K⁺) was used as a depolarization stimulus. K⁺ induced an abrupt spike (peak) in [Ca²⁺]_i that was abolished in the presence of nifedipine, showing that K⁺ enhances [Ca²⁺]_i, preferably activating L-type Ca²⁺ channels. H89, a selective PKA inhibitor, significantly reduced depolarization-induced Ca²⁺ mobilization in a concentration-related manner when it was applied before or after K⁺, and okadaic acid, an inhibitor of Ser/Thr phosphatases, which has been shown to regulate PKA-stimulated L-type Ca²⁺ channels, increased K⁺-induced Ca²⁺ entry. When PKC was activated by PMA, the K⁺-evoked peak in [Ca²⁺]_i, as well as the plateau phase, was significantly reduced, and chelerythrine (a PKC inhibitor) potentiated the K⁺-induced increase in [Ca²⁺]_i, indicating an inhibitory role of PKC in voltage-dependent Ca²⁺ channel (VDCC) activity. Genistein, a TK inhibitor, reduced the K⁺-evoked increase in [Ca²⁺]_i, but, unexpectedly, the tyrosine phosphatase inhibitor orthovanadate reduced not only basal Ca²⁺ levels but, also, Ca²⁺ influx during the plateau phase. Both results suggest that different TKs may act differentially on VDCC activation. Activation of receptor TKs with epidermal growth factor (EGF) or vascular endothelial growth factor potentiated K⁺-induced Ca²⁺ influx, and AG-1478 (an EGF receptor inhibitor) decreased it. However, inhibition of the non-receptor TK pp60 c-Src enhanced K⁺-induced Ca²⁺ influx. The present study strongly demonstrates that a complex equilibrium among different kinases and phosphatases regulates VDCC activity in the pituitary cell line GH3: PKA and receptor TKs, such as vascular endothelial growth factor receptor and EGF receptor, enhance depolarization-induced Ca²⁺ influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and may therefore participate in the regulation of a plethora of intracellular processes, such as hormone secretion, gene expression, protein synthesis, and cell proliferation, in pituitary cells. phosphatases; protein kinase A; protein kinase C; epidermal growth factor     

Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells

To clarifyinteractions between the cytoskeleton and activity of L-typeCa²⁺ (Ca_L) channels in vascular smooth muscle(VSM) cells, we investigated the effect of disruption of actinfilaments and microtubules on the L-type Ca²⁺ current[I_Ba(L)] of cultured VSM cells (A7r5 cellline) using whole cell voltage clamp. The cells were exposed to eachdisrupter for 1 h and then examined electrophysiologically andmorphologically. Results of immunostaining using anti--actin andanti--tubulin antibodies showed that colchicine disrupted both actinfilaments and microtubules, cytochalasin D disrupted only actinfilaments, and nocodazole disrupted only microtubules.I_Ba(L) was greatly reduced in cells that wereexposed to colchicine or cytochalasin D but not to nocodazole.Colchicine even inhibited I_Ba(L) by about 40%when the actin filaments were stabilized by phalloidin or when thecells were treated with phalloidin plus taxol to stabilize bothcytoskeletal components. These results suggest that colchicine mustalso cause some inhibition of I_Ba(L) due toanother unknown mechanism, e.g., a direct block of Ca_Lchannels. In summary, actin filament disruption of VSM cells inhibitsCa_L channel activity, whereas disrupting the microtubulesdoes not.