Cadmium uptake and toxicity via voltage-sensitive calcium channels

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.     

Diverse effects of sphingosine on calcium mobilization and influx in differentiated HL-60 cells

Sphingosine induces a biphasic increase in cytosolic-free Ca(2+)([Ca(2+)](i)) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (beta-aminoethyl ether) N, N, N', N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca(2+)influx does not appear due to Ca(2+)influx through store-operated Ca(2+)(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca(2+)influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca(2+)](i)elicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca(2+)from intracellular Ca(2+)stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca(2+)influx through an undefined Ca(2+)channel and cause a blockade of SOC channels.     

Effect of the antidepressant sertraline on Ca2+fluxes in Madin-Darby canine renal tubular cells

The effect of the antidepressant sertraline on cytosolic-free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether sertraline changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Sertraline at concentrations between 1and 100 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ implicating Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholiapase A2 but not by store-operated Ca2+ channel blockers and protein kinase C/A modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline partly reduced inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from endoplasmic reticulum. Inhibition of phospholipase C did not much alter sertraline-induced [Ca2+]i rise. Collectively, in MDCK cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets.

Progesterone rapidly increased intracellular free calcium ([Ca2+]i) in human sperm, removal of extracellular Ca2+ prevented the increase in [Ca2+]i. The Ca2+ influx was not blocked by the T-type Ca2+ channel blocker mibefradil. However T-type calcium channels do appear to be present in human sperm because the neoglycoprotein mannose-albumin, an inducer of the acrosome reaction, was able to promote Ca2+ influx, which was blocked by mibefradil and more potently inhibited by Ni2+ than Cd2+. The receptor for progesterone that promotes the Ca2+ influx was located on the plasma membrane using FITC-progesterone-albumin. It is concluded that progesterone stimulates Ca2+ influx in human sperm via a unique Ca2+ channel possibly similar to a store-operated channel (SOC) or a receptor-operated channel (ROC). We have found that progesterone metabolites, such as pregnanolone and pregnanediol, promote a rapid rise in [Ca2+]i and aggregation in human platelets, similar to that observed with thrombin. The increase in [Ca2+]i was prevented when extracellular Ca2+ was removed or by the SOC inhibitor SKF-96365. The phospholipase C inhibitor U-73122 also prevented the increase in [Ca2+]i, suggesting that these metabolites interact with a cell surface receptor on the platelet to activate phospholipase C to produce inositol-P3, which mobilizes intracellular Ca2+, thereby activating the SOC in the plasma membrane. Progesterone and estradiol conjugated to albumin, also produced a rapid increase in [Ca2+]i, which was prevented by Ca2+ removal from the medium or when SKF-96365 or U-73122 were added. It is proposed that human platelets possess cell surface receptors for steroids.     

Glucocorticoids increase Ca2+ influx through dihydropyridine-sensitive channels linked to activation of protein kinase C in vascular smooth muscle cells.

To clarify whether protein kinase is associated with glucocorticoid-induced Ca2+ influx into vascular smooth muscle cells, we investigated the effects of protein kinase inhibitors on dexamethasone-induced 45Ca2+ uptake and dihydropyridine binding in A7r5 cells. Protein kinase C inhibitors (staurosporine and UCN-01) abolished the dexamethasone-induced 45Ca2+ uptake and [methyl-3H]PN 200-110 binding. In contrast, KT5720 and KT5823, which are more specific inhibitors of cAMP-dependent protein kinase and cGMP-dependent protein kinase, respectively, did not affect the effects of dexamethasone. Treatment with 100 nM dexamethasone for 48 hours increased protein kinase C activity in A7r5 cells. These results suggest that glucocorticoids increase Ca2+ influx through dihydropyridine-sensitive channels, linked to activation of protein kinase C in vascular smooth muscle cells.     

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.     

A spirochete surface protein uncouples store-operated calcium channels in fibroblasts: a novel cytotoxic mechanism

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca(2+) release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca(2+) transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn(2+) after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca(2+) with EGTA. Msp pretreatment reduced the amplitude of [Ca(2+)](i) transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca(2+), Msp reduced Ca(2+) efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca(2+) release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca(2+) depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca(2+) entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca(2+) release from endoplasmic reticulum stores, and it inhibits subsequent Ca(2+) influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.     

Potentiation of Fcepsilon receptor I-activated Ca(2+) current (I(CRAC)) by cholera toxin: possible mediation by ADP ribosylation factor

Antigen-evoked influx of extracellular Ca(2+) into mast cells may occur via store-operated Ca(2+) channels called calcium release-activated calcium (CRAC) channels. In mast cells of the rat basophilic leukemia cell line (RBL-2H3), cholera toxin (CT) potentiates antigen-driven uptake of (45)Ca(2+) through cAMP-independent means. Here, we have used perforated patch clamp recording at physiological temperature to test whether cholera toxin or its substrate, Gs, directly modulates the activity of CRAC channels. Cholera toxin dramatically amplified (two- to fourfold) the Ca(2)+ release-activated Ca(2+) current (I(CRAC)) elicited by suboptimal concentrations of antigen, without itself inducing I(CRAC), and this enhancement was not mimicked by cAMP elevation. In contrast, cholera toxin did not affect the induction of I(CRAC) by thapsigargin, an inhibitor of organelle Ca(2+) pumps, or by intracellular dialysis with low Ca(2+) pipette solutions. Thus, the activity of CRAC channels is not directly controlled by cholera toxin or Gsalpha. Nor was the potentiation of I(CRAC) due to enhancement of phosphoinositide hydrolysis or calcium release. Because Gs and the A subunit of cholera toxin bind to ADP ribosylation factor (ARF) and could modulate its activity, we tested the sensitivity of antigen-evoked I(CRAC) to brefeldin A, an inhibitor of ARF-dependent functions, including vesicle transport. Brefeldin A blocked the enhancement of antigen-evoked I(CRAC) without inhibiting ADP ribosylation of Gsalpha, but it did not affect I(CRAC) induced by suboptimal antigen or by thapsigargin. These data provide new evidence that CRAC channels are a major route for Fcin receptor I-triggered Ca(2+) influx, and they suggest that ARF may modulate the induction of I(CRAC) by antigen.     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.     

Muscarinic acetylcholine receptors activate TRPC6 channels in PC12D cells via Ca2+ store-independent mechanisms

In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6.     

Maitotoxin potently promotes Ca2+ influx in mouse spermatogenic cells and sperm, and induces the acrosome reaction

Maitotoxin (MTX), a potent marine toxin, activates Ca2+ entry via nonselective cation channels in a wide variety of cells. The identity of the channels involved in MTX action remains unknown. In mammalian sperm, Ca2+ entry through store-operated channels regulates a number of physiological events including the acrosome reaction (AR). Here we report that MTX produced an increase in the intracellular concentration of Ca2+ ([Ca2+]i) in spermatogenic cells that depended on extracellular Ca2+. Ni2+ and SKF96365 diminished the MTX-activated Ca2+ uptake, at concentrations they inhibit store-operated channels, and in a similar manner as they inhibit the Ca2+ influx activated following depletion of intracellular stores by thapsigargin (Tpg). In addition, MTX significantly increased [Ca2+]i in single mature sperm and effectively induced the AR with a half-maximal concentration (ED50) of approximately 1.1 nM. Notably, SKF96365 similarly inhibited the MTX-induced increase in sperm [Ca2+]i and the AR triggered by the toxin, Tpg and zona pellucida. These results suggest that putative MTX-activated channels may be involved in the Ca2+ influx required for mouse sperm AR.     

Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle

The acrosome reaction (AR) of sperm is a prerequisite for fusion with the egg. In sea urchins, the complete AR (CAR) consists of exocytosis of the acrosomal vesicle (AV) and polymerization of acrosomal actin to form the approximately 1 micro m long acrosomal process. The fucose sulfate polymer (FSP) of egg jelly stimulates Ca(2+) entry through two distinct Ca(2+) channels and induces the CAR. Here we report that the second channel is blocked by SKF96365 (SKF), an inhibitor of store-operated channels. SKF also blocks the thapsigargin (TG), trifluoperazine (TFP), and calmidizolium (CMZ) stimulated Ca(2+) entry into sperm. These data indicate that the second Ca(2+) channel is a store-operated channel (SOC) that may be regulated by calmodulin. The TG, TFP, and CMZ-induced intracellular Ca(2+) elevations are similar to those induced by FSP, but the sperm acrosomal process does not polymerize. An antibody to bindin, the major protein of the AV, showed that in a significant percentage of these drug-treated sperm, the AV had undergone exocytosis. When NH(4)Cl was added to increase intracellular pH, the TG-treated sperm polymerized actin to form the acrosomal process. We conclude that the second Ca(2+) channel of sea urchin sperm is a SOC that triggers AV exocytosis.     

Participation of phospholipase A2 isoforms in the control of calcium influx into electrically non-excitable cells

The participation of phospholipase A2 isoforms in capacitative store-operated Ca2+ influx into Jurkat leukemic T and MDCK cells was investigated. Preincubation of Jurkat cells with either bromophenacyl bromide (an inhibitor of secreted phospholipase A2, sPLA2) or Helss (an inhibitor of calcium independent phospholipase A2--iPLA2) resulted in a significant inhibition of the calcium influx. The extent of this inhibition depended on the pH of the extracellular millieu; it increased with alkalisation. The rate of Ca2+ influx into MDCK cells was reduced by bromophenacyl bromide. Preincubation of these cells with Helss resulted in the stimulation of the influx. These observations suggest the participation of different PLA2 isoforms in the regulation of Ca2+ influx. They also show that the extent that PLA2 isoforms control the influx depends on the pH of the medium. Finally, these data indicate that various phospholipase A2 isoforms may play a role in the control of Ca2+ influx in different cell lines.     

Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone.

The hypothalamic decapeptide gonadotropin-releasing hormone stimulates mobilization of two discrete pools of calcium in clonal (alphaT3-1) and primary pituitary gonadotropes. A multidisciplinary approach was implemented to investigate the effects of discrete calcium fluctuations on the signaling pathways linking the gonadotropin-releasing hormone receptor to activation of mitogen-activated protein kinases and immediate early genes. Blockade of calcium influx through nifedipine-sensitive voltage-gated calcium channels reduced buserelin-induced activation of extracellular signal-regulated kinase (ERK) and c-Fos while activation of c-Jun N-terminal kinase and c-Jun was unaffected. Inhibition of buserelin-stimulated ERK activity by nifedipine was also observed in rat pituitary cells in primary culture. Direct activation of alphaT3-1 cell L-type calcium channels with the agonist Bay-K 8644 resulted in phosphorylation of ERK and induction of c-Fos. However, simple voltage-induced channel activation did not produce a sufficient calcium signal, since depolarization with 35 mM KCl failed to induce activation of ERK. Depletion of intracellular calcium stores with thapsigargin did not affect buserelin-induced ERK activation. An inhibitor of protein kinase C decreased calcium influx through nifedipine-sensitive calcium channels and phosphorylation of ERK induced by buserelin. Pharmacological inhibition of protein kinase C did not block Bay-K 8644-induced ERK activation. These observations suggest that calcium influx through L-type channels is required for GnRH-induced activation of ERK and c-Fos and that the influence of calcium lies downstream of protein kinase C.     

Properties of a native cation channel activated by Ca2+ store depletion in vascular smooth muscle cells

Depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) influx in smooth muscle cells, but the native store-operated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca(2+) stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of store-operated channels, which are activated upon passive depletion of Ca(2+) stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+) ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Loading SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which slowly depletes Ca(2+) stores without a rise in intracellular Ca(2+), activated the same 3-pS channels in cell-attached membrane patches as well as whole cell nonselective cation currents in SMC. TG- and BAPTA-activated 3-pS channels were cation-selective but poorly discriminated among Ca(2+), Sr(2+), Ba(2+), Na(+), K(+), and Cs(+). Open channel probability did not change at negative membrane potentials but increased significantly at high positive potentials. Activation of 3-pS channels did not depend on intracellular Ca(2+) concentration. Neither TG nor a variety of second messengers (including Ca(2+), InsP3, InsP4, GTPgammaS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels in inside-out membrane patches. Thus, 3-pS nonselective cation channels are present and activated by TG or BAPTA-induced depletion of intracellular Ca(2+) stores in intact SMC. These native store-operated cation channels can account for capacitative Ca(2+) influx in SMC and can play an important role in regulation of vascular tone.     

Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.