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1.
Several regulated Ca2+ entry pathways have been identified, with capacitative Ca2+ entry (CCE) being the most characterized. In the present study, we examined Ca2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca2+ release from intracellular stores, and increased Ca2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca2+ entry when CCE was blocked by gadolinium (Gd3+). AA-induced Ca2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca2+ release and Ca2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca2+, and to activate a second Ca2+ entry pathway that is dependent on depletion of Ca2+ from ryanodine-sensitive stores.  相似文献   

2.
Activation of Ca2+ entry upon cell stimulation by agonists can be accomplished by different mechanisms, including store-operated calcium entry (SOCE) and the action of phospholipase A2 (PLA2) products. In adipocytes there are two relatively independent pathways for Ca2+ mobilization from intracellular stores. In the present work we studied, whether these pathways are coupled with particular mechanisms of Ca2+ entry into the cell. It is shown that acetylcholine (ACh) induces oscillatory responses in cytosolic Ca2+ concentration independently of SOCE inhibition by YM-58483 or 2-APB. These oscillations were abolished by the addition of La3+, which inhibits both store-operated calcium (SOC) and ARC channels (regulated by arachidonic acid, AA). The responses to ACh were suppressed by AACOCF3 inhibiting PLA2 of type IV and VIA (iPLA2). Oscillations evoked by fetal bovine serum (FBS) were distinguished by the baseline spiking and, in contrast, were terminated by YM-58483 and La3+ but were not dependent on AACOCF3. The same cell could respond to ACh and FBS at their sequential addition in any order with the intermediate wash. Oscillatory responses of a similar (base or elevated line) form to phenylephrine decayed only gradually after the inhibition of phospholipase C or inositol 1,4,5-trisphosphate receptor, and were partially attenuated by the inhibitors YM-58483 and La3+ without appreciable influence of AACOCF3. AA at concentrations 1–10 μM caused oscillations when added after spontaneous cessation of ACh-induced oscillations or itself, with a discernible effect produced at lower concentrations after ACh. Calmodulin inhibitor R24571 caused oscillations, which could be suppressed by YM-58483 or AACOCF3 suggesting activation of SOCE and iPLA2, respectively. Taken together, these results indicate that the mechanism of Ca2+ entry activation depends on the signaling pathway involved by an agonist. ACh does not employ SOCE but activates PLA2 with probable participation of the form VIA, which entails the action of its product(s) on ARC channels and likely on lysophospholipid-activated channels. FBS acts through SOCE without participation of PLA2. These two versions can coexist in the case of phenylephrine.  相似文献   

3.
Store-operated Ca2+ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca2+ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca2+ release from intracellular Ca2+ stores and Ca2+ entry from the extracellular space. Ca2+ release was not affected by treatment with ST, but Ca2+ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca2+ sequestration into Ca2+ stores. The Ca2+ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca2+ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca2+ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La3+ and 2-APB. The large increase in Ca2+ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca2+ stores. These results show that SOCE remains activated even after the refilling of Ca2+ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.  相似文献   

4.
The cytoplasmic Ca2+ concentration ([Ca2+]cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca2+ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca2+]cyt. We measured changes of [Ca2+]cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca2+] from 1.3 mM to 5 mM induced an incrase in [Ca2+]cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd3+, 10 μM La3+ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca2+ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca2+) resulted in a decrease of [Ca2+]cyt from 105±10 nM to 88.5±10 nM with La3+, from 103±12 to 89±12 nM with Gd3+ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca2+ entry beside its function in Ca2+ signaling contributes to the regulation of resting [Ca2+]cyt.  相似文献   

5.
Ca2+ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca2+ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca2+ permeable channels, and limited electrophysiological analyses of Ca2+ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca2+-release-activated Ca2+ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca2+ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca2+-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca2+ current with the same properties, including high selectivity for Ca2+ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca2+-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.  相似文献   

6.
Depletion of Ca2+ stores inthe sarcoplasmic reticulum (SR) activates extracellularCa2+ influx via capacitativeCa2+ entry (CCE). Here, CCE levelsin proliferating and growth-arrested human pulmonary artery smoothmuscle cells (PASMCs) were compared by digital imaging fluorescencemicroscopy. Resting cytosolic freeCa2+ concentration([Ca2+]cyt)in proliferating PASMCs was twofold higher than that in growth-arrestedcells. Cyclopiazonic acid (CPA; 10 µM), which inhibits SRCa2+-ATPase and depletes inositol1,4,5-trisphosphate-sensitiveCa2+ stores, transiently increased[Ca2+]cytin the absence of extracellularCa2+. The addition of 1.8 mMCa2+ to the extracellular solutionin the presence of CPA induced large increases in[Ca2+]cyt,indicative of CCE. The CPA-induced SRCa2+ release in proliferatingPASMCs was twofold higher than that in growth-arrested cells, whereasthe transient rise of[Ca2+]cytdue to CCE was fivefold greater in proliferating cells. CCE wasinsensitive to nifedipine but was significantly inhibited by 50 mMK+, which reduces the drivingforce for Ca2+ influx, and by 0.5 mM Ni2+, a putative blocker ofstore-operated Ca2+ channels.These data show that augmented CCE is associated with proliferation ofhuman PASMCs and may be involved in stimulating and maintaining cell growth.

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7.
In vascular endothelial cells, depletion of intracellularCa2+ stores elicited capacitativeCa2+ entry (CCE) that resulted inbiphasic changes of intracellular Ca2+ concentration([Ca2+]i)with a rapid initial peak of[Ca2+]ifollowed by a gradual decrease to a sustained plateau level. Weinvestigated the rates of Ca2+entry, removal, and sequestration during activation of CCE and theirrespective contributions to the biphasic changes of[Ca2+]i.Ca2+ buffering by mitochondria,removal byNa+/Ca2+exchange, and a fixed electrical driving force forCa2+ (voltage-clamp experiments)had little effect on the CCE signal. The rates of entry ofMn2+ andBa2+, used as unidirectionalsubstitutes for Ca2+ entry throughthe CCE pathway, were constant and did not follow the concomitantchanges of[Ca2+]i.Pharmacological inhibition of the plasma membraneCa2+ pump, however, abolished thesecondary decay phase of the CCE transient. The disparity between thebiphasic changes of[Ca2+]iand the constant rate of Ca2+entry during CCE was the result of a delayed,Ca2+-dependent activation of thepump. These results suggest an important modulatory role of the plasmamembrane Ca2+ pump in the netcellular gain of Ca2+ during CCE.

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8.
Inositol lipid signaling relies on an InsP3-induced Ca2+ release from intracellular stores and on extracellular Ca2+ entry, which takes place when the Ca2+ stores become depleted of Ca2+. This interplay between Ca2+ release and Ca2+ entry has been termed capacitative Ca2+ entry and the inward current calcium release activated current (CRAC) to indicate gating of Ca2+ entry by Ca2+-store depletion. The signaling pathway and the gating mechanism of capacitative Ca2+ entry, however, are largely unknown and the molecular participants in this process have not been identified. In this article we review genetic, molecular, and functional studies of wild-type and mutantDrosophila photoreceptors, suggesting that thetransient receptor potential mutant (trp) is the first putative capacitative Ca2+ entry mutant. Furthermore, several lines of evidence suggest that thetrp gene product TRP is a candidate subunit of the plasma membrane channel that is activated by Ca2+ store depletion.  相似文献   

9.
10.
2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca2+ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca2+]i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca2+ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca2+]i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca2+]i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca2+]i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca2+ and SOC channel blockers such as SK&F96365, La3+ and Gd3+. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca2+]i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca2+]i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.  相似文献   

11.
Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.  相似文献   

12.
The term excitation-coupled Ca2+ entry (ECCE) designates the entry of extracellular Ca2+ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca2+ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn2+. Here, we have examined the effects of these agents on the L-type Ca2+ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La3+, and Gd3+) and dantrolene all inhibited the L-type Ca2+ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca2+ channel displays permeability to Mn2+ in the absence of external Ca2+ and produces a Ca2+ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca2+ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca2+; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca2+-free solution and were unaffected by Ca2+ removal; and (3) after block of SR Ca2+ release by 200 μM ryanodine, normal myotubes still displayed a large Ca2+ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca2+ entry attributed to ECCE.  相似文献   

13.
Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca2+ influx is triggered by NE, the Ca2+ entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca2+ influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca2+ response after repetitive stimulation. Ca2+ entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca2+ entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca2+ entry. Moreover, NE failed to induce further Ca2+ entry after the development of Ca2+ entry induced by thapsigargin (Tg), suggesting that the pathway of Ca2+ entry induced by NE might be identical to that of Tg. Interestingly, Ca2+ entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca2+-activated K+ (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca2+ entry coupled to BK channels and that NE-induced Ca2+ entry is important in the regulation of AANAT. serotonin N-acetyltransferase; pineal gland  相似文献   

14.
Cannabinoid CB1-receptor stimulation in DDT1 MF-2 smooth muscle cells induces a rise in [Ca2+]i, which is dependent on extracellular Ca2+ and modulated by thapsigargin-sensitive stores, suggesting capacitative Ca2+ entry (CCE), and by MAP kinase. Non-capacitative Ca2+ entry (NCCE) stimulated by arachidonic acid (AA) partly mediates histamine H1-receptor-evoked increases in [Ca2+]i in DDT1 MF-2 cells. In the current study, both Ca2+ entry mechanisms and a possible link between MAP kinase activation and increasing [Ca2+]i were investigated. In the whole-cell patch clamp configuration, the CB-receptor agonist CP 55, 940 evoked a transient, Ca2+-dependent K+ current, which was not blocked by the inhibitors of CCE, 2-APB, and SKF 96365. AA, but not its metabolites, evoked a transient outward current and inhibited the response to CP 55,940 in a concentration-dependent manner. CP 55,940 induced a concentration-dependent release of AA, which was inhibited by the CB1 antagonist SR 141716. The non-selective Ca2+ channel blockers La3+ and Gd3+ inhibited the CP 55,940-induced current at concentrations that had no effect on thapsigargin-evoked CCE. La3+ also inhibited the AA-induced current. CP 55,940-induced AA release was abolished by Gd3+ and by phospholipase A2 inhibition using quinacrine; this compound also inhibited the outward current. The CP 55,940-induced AA release was strongly reduced by the MAP kinase inhibitor PD 98059. The data suggest that in DDT1 MF-2 cells, AA is an integral component of the CB1 receptor signaling pathway, upstream of NCCE and, via PLA2, downstream of MAP kinase.  相似文献   

15.
Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca2+ signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca2+ oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs.LTD4-induced Ca2+ oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca2+ entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca2+ oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP3R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca2+ oscillations. Ca2+ oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP3R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3.We conclude that IL-13 enhances frequency of LTD4-induced Ca2+ oscillations in human ASMCs, which may be cooperatively modulated by IP3R, RyR systems and possibly by SOCE.  相似文献   

16.
In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca2+ release from the intracellular stores followed by a sustained Ca2+ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca2+ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca2+ -ATPase inhibitor, stimulated Ca2+ entry into the cells by a mechanism other than emptying Ca2+ stores. In addition, Ca2+ entry into the Ca2+ -depleted cells was stimulated by low basal level of cytosolic Ca2+, not by the emptying of intracellular Ca2+ stores. Both the Ca2+ release and influx were dependent on high and low concentrations of extracellular Ca2+. At low concentrations, Mn2+ entered the cell through the Ca2+ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn2+ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca2+ release was inhibited by low concentrations of Ni2+, La3+, and EGTA, while Ca2+ influx was inhibited by high concentrations. Thus, in T cells Ca2+ influx occurs independently of IP3-dependent Ca2+ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca2+ influx. Extracellular Ca2+ influenced Ca2+ release and influx through the action of two plasma membrane Ca2+ entry pathways with different pharmacological and biochemical properties.  相似文献   

17.
Summary The mechanisms of Cl-channel activation in the plasmalemma ofNitellopsis obtusa was studied by measuring both the transient inward current under voltage clamp and Cl efflux during the action potential. 9-anthracenecarboxylic acid (A-9-C) at 1.0mm inhibited both the transient inward current and the Cl efflux, but did not uncouple the sudden cessation of the cytoplasmic streaming. Since this excitation-cessation coupling is caused by a transient increase in the cytoplasmic Ca2+ concentration, these results suggest that A-9-C inhibited not the Ca2+ channel but specifically the Cl channel. The following results were found between the Ca2+-channel activation and the Cl-channel activation: (1) The Ca2+-channel blocker La3+ uncoupled the excitation-cessation coupling and inhibited both the transient inward current and the Cl efflux, although the Cl-channel blocker A-9-C did not affect the excitation-cessation coupling. (2) The Cl efflux was greatly reduced by depletion of Ca2+ from the external solution and restored by an increase in the external Ca2+ concentration. (3) An increase in the external ionic, strength which increases Ca2+ entry (T. Shiina & M. Tazawa,J. Membrane Biol. 96:263–276, 1987) enhanced the Cl efflux. (4) Mg2+, which cannot pass through the Ca2+ channel, reduced both the transient inward current and the Cl efflux. (5) Although Sr2+ can pass through the plasmalemma Ca2+ channel, Cl-channel activation by Sr2+ was only partial. These findings support the hypothesis that voltage-dependent Ca2+-channel activation, which increases the free Ca2+ concentration in the cytoplasm, is necessary for the subsequent Cl-channel activation.  相似文献   

18.
《Cell calcium》2008,43(6):606-617
We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP3 receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.  相似文献   

19.
Previous results with potato tuber discs showed that a treatment with abscisic acid stimulated K+ uptake. In this investigation, we determine the relationship between increased K' uptake and H+extrusion, and Ca2+ fluxes by treating tissues with specific Ca2+ channel blocker (La3+), calmodulin (CaM) inhibitors (chlorpromazine and W7), and with Ca2+ ionophore (A23187). K+ uptake increased with increasing external pH whether tissues were treated with ABA or not. Treatment of tissues with La3+ inhibited K+ uptake, whereas CaM inhibitors have no effect. By contrast ABA and A23187 produced a synergistic effect, suggesting that ABA may act in part, on K+ uptake, like a Ca2+ agonist, in accord with Huddart's hypothesis. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

20.
Summary We have investigated muscarinic receptor-operated Ca2+ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca2+ release and extracellular Ca2+ entry. The carbachol (Cch) dose response of intracellular Ca2+ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K 0.510 or 30 m, depending on the method for [Ca2+] i determination). However, similar data for Ca2+ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca2+ release and a second higher affinity site withK 0.52.5 m. In addition, the Ca2+ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K+ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca2+ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca2+] i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca2+ channel blocker La3+ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca2+ in these cells by increasing Ca2+ entry. Organic Ca2+ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca2+ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K+ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP3) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP3 formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca2+ entry in HSG-PA cells. One is associated with IP3 formation and intracellular Ca2+ release and is independent of membrane potential; the other is less dependent on IP3 formation and intracellular Ca2+ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.  相似文献   

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