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1.
Intracellular signal transduction during gastrin-induced histamine secretion in rat gastric ECL cells 总被引:8,自引:0,他引:8
Zanner Robert; Hapfelmeier Gerhard; Gratzl Manfred; Prinz Christian 《American journal of physiology. Cell physiology》2002,282(2):C374
Activation of Gqprotein-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. 相似文献
2.
Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C 总被引:1,自引:0,他引:1
Bayguinov O Hagen B Kenyon JL Sanders KM 《American journal of physiology. Cell physiology》2001,281(5):C1512-C1523
Localized Ca2+ transients resulting from inositoltrisphosphate (IP3)-dependent Ca2+ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa2+ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca2+([Ca2+]o) reduced localized Ca2+transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca2+ transients and STOCs showed increasedcoupling strength between Ca2+ transients and STOCs when[Ca2+]o was reduced. Gd3+ (10 µM) did not affect Ca2+ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca2+influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca2+ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca2+ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca2+ transients. Single channelstudies indicated that large-conductance Ca2+-activatedK+ (BK) channels were inhibited by aCa2+-dependent PKC. In summary 1)Ca2+ release from IP3 receptor-operated storesactivates Ca2+-activated K+ channels;2) Ca2+ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca2+ sensitivity of BK channels, reducing the couplingstrength between localized Ca2+ transients and BK channels. 相似文献
3.
Muscarinic stimulation increases basal Ca(2+) and inhibits spontaneous Ca(2+) transients in murine colonic myocytes 总被引:1,自引:0,他引:1
Bayguinov O Hagen B Sanders KM 《American journal of physiology. Cell physiology》2001,280(3):C689-C700
Localized Ca2+ transients inisolated murine colonic myocytes depend on Ca2+ releasefrom inositol 1,4,5-trisphosphate (IP3) receptors.Localized Ca2+ transients couple to spontaneous transientoutward currents (STOCs) and mediate hyperpolarization responses inthese cells. We used confocal microscopy and whole cell patch-clamprecording to investigate how muscarinic stimulation, which causesformation of IP3, can suppress Ca2+ transientsand STOCs that might override the excitatory nature of cholinergicresponses. ACh (10 µM) reduced localized Ca2+ transientsand STOCs, and these effects were associated with a rise in basalcytosolic Ca2+. These effects of ACh were mimicked bygeneralized rises in basal Ca2+ caused by ionomycin(250-500 nM) or elevated external Ca2+ (6 mM).Atropine (10 µM) abolished the effects of ACh. Pretreatment of cellswith nicardipine (1 µM), or Cd2+ (200 µM) had no effecton responses to ACh. An inhibitor of phospholipase C, U-73122, blockedCa2+ transients and STOCs but did not affect the increasein basal Ca2+ after ACh stimulation. Xestospongin C (Xe-C;5 µM), a membrane-permeable antagonist of IP3 receptors,blocked spontaneous Ca2+ transients but did not prevent theincrease of basal Ca2+ in response to ACh. Gd3+(10 µM), a nonselective cation channel inhibitor, prevented the increase in basal Ca2+ after ACh and increased thefrequency and amplitude of Ca2+ transients and waves.Another inhibitor of receptor-mediated Ca2+ influxchannels, SKF-96365, also prevented the rise in basal Ca2+after ACh and increased Ca2+ transients and development ofCa2+ waves. FK-506, an inhibitor ofFKBP12/IP3 receptor interactions, had no effect onthe rise in basal Ca2+ but blocked the inhibitory effectsof increased basal Ca2+ and ACh on Ca2+transients. These results suggest that the rise in basalCa2+ that accompanies muscarinic stimulation of colonicmuscles inhibits localized Ca2+ transients that couldcouple to activation of Ca2+-activated K+channels and reduce the excitatory effects of ACh. 相似文献
4.
Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells 总被引:8,自引:0,他引:8
Janiak R Wilson SM Montague S Hume JR 《American journal of physiology. Cell physiology》2001,280(1):C22-C33
To examine the natureof inositol 1,4,5-trisphosphate (IP3)-sensitive andryanodine (Ryn)-sensitive Ca2+ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca2+ concentration([Ca2+]i) were measured using fura2-AM fluorescence. Properties of elementary local Ca2+release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca2+ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca2+ transients but had little or no effecton the initial IP3-mediated intracellular Ca2+transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP3-induced intracellularCa2+ transients but failed to attenuate the initialCaf-induced intracellular Ca2+ transient. These resultssuggest that in canine PASMC, IP3-, and Ryn-sensitiveCa2+ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca2+ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca2+ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca2+ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa2+ stores, ANG II (8 nM) induced slow, sustained[Ca2+]i increases originating at sites nearthe cell surface, which were abolished by depleting IP3stores. Discrete quantal-like events expected due to the coordinatedopening of IP3 receptor clusters ("Ca2+puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca2+ stores and elementary Ca2+ release eventsin isolated PASMC. 相似文献
5.
Kapur N Mignery GA Banach K 《American journal of physiology. Cell physiology》2007,292(4):C1510-C1518
During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca2+ signals during the G0 phase, the transition from G1 to S, and from G2 to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G1 and G2, we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca2+ concentration during cell cycle progression. With confocal imaging of the Ca2+-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca2+ oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca2+ influx but depended on inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release and the refilling of intracellular stores by a store-operated Ca2+ influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca2+ oscillations were confined to the G1/S phase (70% oscillating cells vs. G2/M with 15% oscillating cells) of the cell cycle. ATP induced Ca2+ oscillations, and activation of SOC could be induced in G1/S and G2/M synchronized cells. Intracellular Ca2+ stores were not depleted, and all three IP3 receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP3-mediated Ca2+ release is necessary for cell cycle progression through G1/S. Because the IP3 receptor sensitizer thimerosal induced Ca2+ oscillations only in G1/S, we propose that changes in IP3 receptor sensitivity or basal levels of IP3 could be the basis for the G1/S-confined Ca2+ oscillations. pluripotent; IP3; store operated Ca entry; IP3 receptor 相似文献
6.
Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes 总被引:1,自引:0,他引:1
Kim Sung Joon; Ahn Seung Cheol; Kim Jin Kyung; Kim Young Chul; So Insuk; Kim Ki Whan 《American journal of physiology. Cell physiology》1997,273(6):C1947
We investigatedthe relationship between voltage-operatedCa2+ channel current and thecorresponding intracellular Ca2+concentration([Ca2+]i)change (Ca2+ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca2+ current(ICa) andconcomitantly raised[Ca2+]i.Both responses were suppressed by nicardipine, an L-typeCa2+ channel blocker, and thevoltage dependence of Ca2+transient was similar to the current-voltage relation ofICa. When pulseduration was increased by up to 900 ms, peakCa2+ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca2+ buffering capacity(B value), determined as the ratio ofthe time integral ofICa divided bythe amplitude of Ca2+ transient,was not significantly increased after depletion of Ca2+ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca2+-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca2+-activatedK+ current[IK(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of IK(Ca) withirregular effects on Ca2+transients. The above results suggest that, in guinea pig gastric myocyte, Ca2+ transient is tightlycoupled to ICaduring depolarization, and global[Ca2+]iis not significantly affected byCa2+-inducedCa2+ release from sarcoplasmicreticulum during depolarization. 相似文献
7.
Metabolic inhibition with cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes 总被引:5,自引:0,他引:5
Wang YX Zheng YM Abdullaev I Kotlikoff MI 《American journal of physiology. Cell physiology》2003,284(2):C378-C388
We examined the effectsof metabolic inhibition on intracellular Ca2+ release insingle pulmonary arterial smooth muscle cells (PASMCs). Severemetabolic inhibition with cyanide (CN, 10 mM) increased intracellularcalcium concentration ([Ca2+]i) and activatedCa2+-activated Cl currents[ICl(Ca)] in PASMCs, responses that were greatlyinhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents andCa2+ sparks in PASMCs. In Xenopus oocytes, CNalso induced Ca2+ release and activatedICl(Ca), and these responses were inhibited by thapsigarginand cyclopiazonic acid to deplete sarcoplasmic reticulum (SR)Ca2+, whereas neither heparin nor anti-inositol1,4,5-trisphosphate receptor (IP3R) antibodies affected CNresponses. In both PASMCs and oocytes, CN-evoked Ca2+release was inhibited by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and oligomycin or CCCP andthapsigargin. Whereas hypoxic stimuli resulted in Ca2+release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition withCN increases [Ca2+]i in both pulmonary andsystemic artery myocytes by stimulating Ca2+ release fromthe SR and mitochondria. 相似文献
8.
Differential regulation of Ca(2+) sparks and Ca(2+) waves by UTP in rat cerebral artery smooth muscle cells 总被引:6,自引:0,他引:6
Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activatesphospholipase C, shifted Ca2+ signaling from sparks towaves in the smooth muscle cells of rat cerebral arteries. UTPdecreased the frequency of Ca2+ sparks and transientCa2+-activated K+ (KCa) currentsand increased the frequency of Ca2+ waves. The UTP-inducedreduction in Ca2+ spark frequency did not reflect adecrease in global cytoplasmic Ca2+, Ca2+influx through voltage-dependent Ca2+ channels (VDCC), orCa2+ load of the sarcoplasmic reticulum (SR), since globalCa2+ was elevated, blocking VDCC did not prevent theeffect, and SR Ca2+ load did not decrease. However,blocking protein kinase C (PKC) with bisindolylmaleimide I did preventUTP reduction of Ca2+ sparks and transient KCacurrents. UTP decreased the effectiveness of caffeine, which increasesthe Ca2+ sensitivity of ryanodine-sensitiveCa2+ release (RyR) channels, to activate transientKCa currents. This work supports the concept thatvasoconstrictors shift Ca2+ signaling modalities fromCa2+ sparks to Ca2+ waves through the concertedactions of PKC on the Ca2+ sensitivity of RyR channels,which cause Ca2+ sparks, and of inositol trisphosphate(IP3) on IP3 receptors to generateCa2+ waves. 相似文献
9.
Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells 总被引:2,自引:0,他引:2
Kim YV Di Cello F Hillaire CS Kim KS 《American journal of physiology. Cell physiology》2004,286(1):C31-C42
Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR14) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining, we showed for the first time that PAR14 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura 2 were used to monitor intracellular Ca2+ concentration ([Ca2+]i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca2+]i rise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca2+]i increase, whereas PAR1-AP exhibited sustained [Ca2+]i rise. The PAR1-AP-induced sustained [Ca2+]i rise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF-96365. Restoration of extracellular Ca2+ to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca2+ resulted in significant [Ca2+]i rise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca2+]i rise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca2+]i rise to high PAR1-AP concentration. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC, whereas PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated Ca2+ influx without significantly affecting brain endothelial barrier functions. store-operated calcium influx; desensitization; transendothelial electrical resistance; digital imaging 相似文献
10.
To study the effects of flow on in situendothelial intracellular calcium concentration([Ca2+]i) signaling, rat aortic rings wereloaded with fura 2, mounted on a tissue flow chamber, and divided intocontrol and flow-pretreated groups. The latter was perfused with bufferat a shear stress of 50 dyns/cm2 for 1 h. Endothelial[Ca2+]i responses to ACh or shear stresseswere determined by ratio image analysis. Moreover, ACh-induced[Ca2+]i elevation responses were measured ina calcium-free buffer, or in the presence of SKF-96365, to elucidatethe role of calcium influx in the flow effects. Our results showed that1) ACh increased endothelial[Ca2+]i in a dose-dependent manner, and theseresponses were incremented by flow-pretreatment; 2) thedifferences in ACh-induced [Ca2+]i elevationbetween control and flow-pretreated groups were abolished by SKF-96365or by Ca2+-free buffer; and 3) in the presenceof 105 M ATP, shear stress induced dose-dependent[Ca2+]i elevation responses that were notaltered by flow-pretreatment. In conclusion, flow-pretreatment augmentsthe ACh-induced endothelial calcium influx in rat aortas ex vivo. 相似文献
11.
Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry 总被引:6,自引:0,他引:6
Bradley E Hollywood MA McHale NG Thornbury KD Sergeant GP 《American journal of physiology. Cell physiology》2005,289(3):C625-C632
The aim of the present study was to investigate the properties and role of capacitative Ca2+ entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca2+ entry in IC was larger in cells with depleted intracellular Ca2+ stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca2+ entry blockers Gd3+ (10 µM), La3+ (10 µM), and Ni2+ (100 µM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca2+ entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 µM), wortmannin (10 µM), and nifedipine (1 µM). Spontaneous transient inward currents were recorded from IC voltage-clamped at 60 mV. These events were not significantly affected by Gd3+ (10 µM) or La3+ (10 µM) and were only slightly decreased in amplitude by 100 µM Ni2+. The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells. smooth muscle; patch clamp; spontaneous transient inward currents 相似文献
12.
IP(3) receptors, IP(3) transients, and nucleus-associated Ca(2+) signals in cultured skeletal muscle 总被引:2,自引:0,他引:2
Jaimovich E Reyes R Liberona JL Powell JA 《American journal of physiology. Cell physiology》2000,278(5):C998-C1010
Inositol1,4,5-trisphosphate (IP3) receptors (IP3R) andryanodine receptors (RyR) were localized in cultured rodent muscle fractions by binding of radiolabeled ligands (IP3 andryanodine), and IP3R were visualized in situ byfluorescence immunocytological techniques. Also explored was the effectof K+ depolarization on IP3 mass andCa2+ transients studied using a radio-receptor displacementassay and fluorescence imaging of intracellular fluo 3. RyR werelocated in a microsomal fraction; IP3R were preferentiallyfound in the nuclear fraction. Fluorescence associated withanti-IP3R antibody was found in the region of the nuclearenvelope and in a striated pattern in the sarcoplasmic areas. Anincrease in external K+ affected membrane potential andproduced an IP3 transient. Rat myotubes displayed afast-propagating Ca2+ signal, corresponding to theexcitation-contraction coupling transient and a much slowerCa2+ wave. Both signals were triggered by high externalK+ and were independent of external Ca2+. Slowwaves were associated with cell nuclei and were propagated leaving"glowing" nuclei behind. Different roles are proposed for atleast two types of Ca2+ release channels, each mediating anintracellular signal in cultured skeletal muscle. 相似文献
13.
Barrera NP Morales B Villalón M 《American journal of physiology. Cell physiology》2004,287(4):C1114-C1124
An increase in intracellular free Ca2+ concentration ([Ca2+]i) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP3) receptor-dependent Ca2+ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP3 peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca2+ from internal stores and an influx of extracellular Ca2+, respectively. In addition, ATP-dependent Ca2+ mobilization required protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP3 receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca2+]i component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca2+ influx is also dependent on IP3 receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP3 receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP3 receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP3 receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP3 receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP3 receptor can directly trigger Ca2+ influx in response to ATP. transduction mechanisms; P2Y receptor; calcium influx 相似文献
14.
L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis 总被引:2,自引:0,他引:2
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. 相似文献
15.
Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells 总被引:2,自引:0,他引:2
The effects of inhibitors of CaMKII on intracellular Ca2+ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca2+]i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca2+ resulted in a dose-dependent increase of [Ca2+]i consisting of a rapid and transient Ca2+ spike followed by a small sustained plateau phase of elevated [Ca2+]i. Exposure to KN-93 in the absence of extracellular Ca2+ caused a transient rise of [Ca2+]i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca2+ from intracellular endoplasmic reticulum (ER) Ca2+ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca2+ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP3) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca2+ response, suggesting that exposure to KN-93 affects Ca2+ release from an IP3-sensitive store. Depletion of Ca2+ stores by exposure to ATP or to the ER Ca2+ pump inhibitor thapsigargin triggered robust capacitative Ca2+ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca2+ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca2+ from intracellular stores and activation of CCE. Ca2+/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry 相似文献
16.
Spontaneous transient currents, due to activation of Ca2+-dependent K+ and Cl– channels, occur in corpus cavernosum smooth muscle cells (CCSMC) of the penis. The Ca2+ events responsible for triggering Ca2+-dependent Cl– channels have never been identified in vascular muscle. We used high-speed fluorescence imaging combined with patch-clamp electrophysiology to provide the first characterization of Ca2+ events underlying these currents. Freshly isolated rat CCSMC loaded with fluo-4 exhibited localized, spontaneous elevations of intracellular Ca2+ (Ca2+ sparks) in 57% of cells. There was an average of 6.4 ± 0.5 release sites/cell with a frequency of 0.9 ± 1 Hz/cell and peak amplitude F/Fo of 67 ± 10%. We addressed the controversy of whether these events are mediated by ryanodine or inositol 1,4,5 trisphosphate (IP3) receptors. Caffeine caused either a global Ca2+ rise at high concentrations or an increase in spark frequency at lower concentrations, whereas ryanodine dramatically reduced the amplitude and frequency of sparks. 2-Aminoethoxydiphenyl borate, an inhibitor of IP3 receptors, had no effect on spark frequency. Combined imaging and electrophysiological recording revealed strong coupling between Ca2+ sparks and biphasic transient currents, a relationship never before shown in vascular muscle. Moreover, spark frequency increased on depolarization, an effect abolished with the blockade of Ca2+ channels, consistent with Ca2+ influx regulating Ca2+ release from stores. We establish for the first time that Ca2+ sparks occur in CCSMC and arise from Ca2+ release through ryanodine receptors. Moreover, the voltage dependence of spark frequency demonstrated here provides novel functional evidence for voltage-dependent Ca2+ influx in CCSMC. calcium signaling; potassium and chloride channels; ryanodine receptors 相似文献
17.
Stimulus-induced currents in isolated taste receptor cells of the larval tiger salamander 总被引:3,自引:1,他引:2
Gustatory receptor cells, isolated from the lingual epitheliumof larval tiger salamanders (Ambystoma tigrinum), possess avariety of voltage- and ion-dependent conductances, includinga transient Na+ -current (INa), a voltage-gated Ca2+ -current(IA). a transient K+ -current (IA), a delayed rectifier K+ -current(IK), and a Ca2+ -activated K+ -current (IK(Ca))- By use ofwhole-cell and excised-patch tight-seal recording techniques,we examined the effects of taste stimuli on the conductancesof taste cells from the tiger salamander. Depolarizing receptorpotentials elicited by NaCl were associated with slow, gradedinward currents which were composed of amiloride-sensitive andtetrodoxin-(TTX)-sensitive components. Potassium chloride producedmaintained inward currents, which usually showed both phasicand tonic components and were only partially blocked by tetraethylammoniumchloride (TEA). Citric and acetic acids elicited slow depolarizationsin taste cells. Under voltage-clamp, acids produced graded inwardcurrents which were composed of two components: one attributableto a transient block of voltage-dependent K+ -channels and asmaller component which may have resulted from an increasedconductance to cations. Quinine hydrochloride elicited slowdepolarization of taste cells which was associated with a slowlydeveloping maintained inward current under voltage-clamp. Quininesuppressed both voltage-dependent inward and outward currents.In some taste cells, L-arginine elicited small outward currentswhich were attributable to an increase in K+ conductance. Inother cells, L-arginine produced a decrease in voltage-dependentoutward currents and generated depolarizations associated withinward currents. These results indicate that several independentmechanisms, including amiloride-sensitive Na+ -channels, andstimulus modulation of voltage-dependent K+ -channels, are involvedin taste cell responses to chemical stimuli. More than one mechanismis typically present in a single cell.
3Present address: Department of Physiology, Tokyo Medical andDental University, 5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113,Japan 相似文献
18.
Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes 总被引:4,自引:0,他引:4
Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 µM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 µM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 µM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 µM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 µM), which sensitizes the IP3R to IP3. In cells voltage clamped at 40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity. calcium ion release; calcium ion transients; smooth muscle 相似文献
19.
Hypoxia reduces KCa channel activity by inducing Ca2+ spark uncoupling in cerebral artery smooth muscle cells 总被引:1,自引:0,他引:1
Zhao G Adebiyi A Xi Q Jaggar JH 《American journal of physiology. Cell physiology》2007,292(6):C2122-C2128
Arterial smooth muscle cell large-conductance Ca2+-activated potassium (KCa) channels have been implicated in modulating hypoxic dilation of systemic arteries, although this is controversial. KCa channel activity in arterial smooth muscle cells is controlled by localized intracellular Ca2+ transients, termed Ca2+ sparks, but hypoxic regulation of Ca2+ sparks and KCa channel activation by Ca2+ sparks has not been investigated. We report here that in voltage-clamped (–40 mV) cerebral artery smooth muscle cells, a reduction in dissolved O2 partial pressure from 150 to 15 mmHg reversibly decreased Ca2+ spark-induced transient KCa current frequency and amplitude to 61% and 76% of control, respectively. In contrast, hypoxia did not alter Ca2+ spark frequency, amplitude, global intracellular Ca2+ concentration, or sarcoplasmic reticulum Ca2+ load. Hypoxia reduced transient KCa current frequency by decreasing the percentage of Ca2+ sparks that activated a transient KCa current from 89% to 63%. Hypoxia reduced transient KCa current amplitude by attenuating the amplitude relationship between Ca2+ sparks that remained coupled and the evoked transient KCa currents. Consistent with these data, in inside-out patches at –40 mV hypoxia reduced KCa channel apparent Ca2+ sensitivity and increased the Kd for Ca2+ from 17 to 32 µM, but did not alter single-channel amplitude. In summary, data indicate that hypoxia reduces KCa channel apparent Ca2+ sensitivity via a mechanism that is independent of cytosolic signaling messengers, and this leads to uncoupling of KCa channels from Ca2+ sparks. Transient KCa current inhibition due to uncoupling would oppose hypoxic cerebrovascular dilation. transient calcium-activated potassium current 相似文献
20.
Kim MY Liang GH Kim JA Kim YJ Oh S Suh SH 《American journal of physiology. Cell physiology》2006,290(4):C1000-C1008
The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca2+-activated K+ (BKCa) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca2+ concentration ([Ca2+]i), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca2+]i. [Ca2+]i and membrane potentials were strongly correlated. In whole cell clamped cells, BKCa currents were activated by increasing [Ca2+]i via cell dialysis with pipette solution, and the activated BKCa currents were further enhanced by S1P. When [Ca2+]i was buffered at 1 µM, the S1P concentration required to evoke half-maximal activation was 403 ± 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BKCa channel activity in a reversible manner and shifted the relationship between Ca2+ concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5'-O-(2-thiodiphosphate) (GDPS; 1 mM) using a patch pipette, GDPS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BKCa current and channel activation. These results suggest that S1P enhances BKCa channel activity by increasing Ca2+ sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca2+ influx through Ca2+ entry channels. Inasmuch as S1P activates BKCa channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca2+ mobilization in human endothelial cells. sphingolipid metabolites; intracellular second messenger; Ca2+ mobilization 相似文献