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1.
The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca2+ (Cai2+) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Cai2+ (to micromolar concentrations) observed after the addition of ionophore plus Ca2+ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Cai2+, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Cai2+ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Cai2+ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Cai2+. calcium; connexin43; lens gap junctions 相似文献
2.
The subcellular spatial and temporal organization ofagonist-induced Ca2+ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca2+ concentration([Ca2+]i)in peripheral cytoplasmic processes from where activation propagated asa[Ca2+]iwave toward the central regions of the cell. The average propagation velocity of the[Ca2+]iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca2+]idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca2+]ideclined monotonically, whereas in the central region[Ca2+]idecreased in an oscillatory fashion. Propagating[Ca2+]iwaves were preceded by small, highly localized[Ca2+]itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa2+ release was 23 nM, and theunderlying flux of Ca2+ amountedto ~1-2 × 1018mol/s or ~0.3 pA, consistent with aCa2+ flux through a single orsmall number of endoplasmic reticulum Ca2+-release channels. 相似文献
3.
Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway 总被引:1,自引:0,他引:1
In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa2+ concentration([Ca2+]i)and also stimulated inositol 1,4,5-trisphosphate(IP3) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP3 generation and[Ca2+]iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa2+-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca2+. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca2+]iincrease, IP3 generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca2+]iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca2+]i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca2+]iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa2+ in pertussis toxin-sensitiveG protein-mediated MAPK activation. 相似文献
4.
Aromolaran AS Zima AV Blatter LA 《American journal of physiology. Cell physiology》2007,293(1):C106-C118
The role of glycolytically generated ATP in Ca2+/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca2+ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca2+ concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + -hydroxybutyrate (-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca2+ concentration ([Ca2+]i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca2+]i. The rise of [Ca2+]i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca2+]i. In the absence of extracellular Ca2+ 2-DG caused an increase in [Ca2+]i, suggesting that inhibition of glycolysis directly triggered release of Ca2+ from intracellular endoplasmic reticulum (ER) Ca2+ stores. The inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca2+ response. Ca2+ release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca2+]i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca2+]i. Propagating [Ca2+]i waves were preceded by [Ca2+]i oscillations and small, highly localized elevations of [Ca2+]i (Ca2+ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca2+ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca2+ release was attenuated. Similar results were obtained with pyr + -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg2+ concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca2+ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP3R. Ca2+/calmodulin-dependent kinase II; glycolysis; calcium regulation 相似文献
5.
Humez S Monet M van Coppenolle F Delcourt P Prevarskaya N 《American journal of physiology. Cell physiology》2004,287(6):C1733-C1746
In this study, we investigated ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained intracellular pH (pHi) decrease from 7.41 to 7.11. Inhibition of Ca2+ influx, lowering extracellular Ca2+, and buffering cytoplasmic Ca2+ inhibited ATP-induced acidification, thereby demonstrating that acidification is a consequence of Ca2+ entry. We show that ATP induced reuptake of Ca2+ by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced after the dissipation of the mitochondrial proton gradient by rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, after inhibition of Ca2+ uptake into the mitochondria by ruthenium red, and after inhibition of the F0F1-ATPase with oligomycin. ATP-induced acidification was not induced by either stimulation of the Cl/HCO3 exchanger or inhibition of the Na+/H+ exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable Ca2+ from the endoplasmic reticulum, assessed by measuring change in cytosolic Ca2+ induced by thapsigargin or ATP in a Ca2+-free medium. This latter finding reveals cross talk between pHi and Ca2+ homeostasis in which the Ca2+-induced intracellular acidification can in turn regulate the amount of Ca2+ that can be released from the endoplasmic reticulum. Furthermore, pHi decrease was capable of reducing cell growth. Taken together, our results suggest that ATP-induced acidification in DU-145 cells results from specific effect of mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP. prostate; cancer; acidification 相似文献
6.
Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels 总被引:7,自引:0,他引:7
He ML Zemkova H Koshimizu TA Tomić M Stojilkovic SS 《American journal of physiology. Cell physiology》2003,285(2):C467-C479
Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals 相似文献
7.
A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line 总被引:1,自引:0,他引:1
Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn2+, and 50 µM Cu2+. Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC50 for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca2+ and 2 mM Mg2+, and 33 µM in low-Mg2+, nominally Ca2+-free saline. Overall, the results are most consistent with activation of a P2X7 receptor by ATP4. However, low permeability to N-methyl-D-glucamine+ and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X7 receptor activation. ATP stimulation of internalization occurs in Na+-free, Ca2+-free, or low-Mg2+ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X7 receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp 相似文献
8.
Effects of cytoplasmic Ca2+ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca2+ up to 2.5?105M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca2+]i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca2+]i from1.4?106 to 2.5?105 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca2+]i from zero to 1.38?106M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca2+with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca2+ on the ATP-dependent H+ effluxwas measured. No marked difference in H+ effluxes was detectedbetween zero and 2.5?105 M [Ca2+]i; but, at 104M the ATP-dependent H+ efflux was almost zero. Ca2+ efflux experimentswere done to investigate dependencies on [Ca2+]i and [ATP]i.The efflux was about 1 pmol cm2 s1 at all [Ca2+]iconcentrations tested (1.38?106, 2.5?105, 104M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]i. Thepossibility of a Ca2+-extruding pump is discussed.
1 Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985) 相似文献
9.
Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception 总被引:1,自引:0,他引:1
Vicario I Obeso A Rocher A López-Lopez JR González C 《American journal of physiology. Cell physiology》2000,279(1):C51-C61
Thenotion that intracellular Ca2+ (Cai2+)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa2+ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Cai2+ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca2+ concentration([Ca2+]i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca2+. We found thatthreshold [Ca2+]i for high extracellularK+ (Ke+) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high Ke+)-inducedrelease of CA. The same agents produced Cai2+transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Cai2+responses elicited by high Ke+. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca2+]i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Cai2+ stores do not playa significant role in the instant-to-instant chemoreception process. 相似文献
10.
Mohanty Madhumita Jena; Ye Maian; Li Xingli; Rossi Noreen F. 《American journal of physiology. Cell physiology》2001,281(2):C555
Hypotonicswelling increases the intracellular Ca2+ concentration([Ca2+]i) in vascular smooth muscle cells(VSMC). The source of this Ca2+ is not clear. To study thesource of increase in [Ca2+]i in response tohypotonic swelling, we measured [Ca2+]i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca2+]i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores with vasopressin did not inhibit the increasein [Ca2+]i in response to hypotonic swelling.Exposure of 45Ca2+-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in45Ca2+ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa2+ from the intracellular stores from a novel sitedistinct from the IP3-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores. 相似文献
11.
Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells 总被引:2,自引:0,他引:2
Jung SR Kim MH Hille B Nguyen TD Koh DS 《American journal of physiology. Cell physiology》2004,286(3):C573-C579
In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca2+ concentration ([Ca2+]i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca2+]i. Low concentrations of ATP (<10 µM) induced intracellular Ca2+ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca2+]i rise and increased the exocytosis rate sevenfold. The contribution of Ca2+ or cAMP pathways to exocytosis was tested by using the Ca2+ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca2+]i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca2+ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate 相似文献
12.
Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells 总被引:1,自引:0,他引:1
Liu Xibao; Rojas Eduardo; Ambudkar Indu S. 《American journal of physiology. Cell physiology》1998,275(2):C571
This study examines theCa2+ influx-dependent regulationof the Ca2+-activatedK+ channel(KCa) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theKCa current and cytosolic Ca2+ concentration([Ca2+]i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa2+ and addition ofLa3+ orGd3+, but notZn2+, inhibited the increases inKCa current and[Ca2+]i.Ca2+ influx during refill (i.e.,addition of Ca2+ to cells treatedwith CCh and then atropine inCa2+-free medium) failed to evokeincreases in the KCa current but achieved internal Ca2+ storerefill. When refill was prevented by thapsigargin,Ca2+ readdition induced rapidactivation of KCa. These dataprovide further evidence that intracellularCa2+ accumulation provides tightbuffering of[Ca2+]iat the site of Ca2+ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca2+ influx-dependentregulation of the sustained KCacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa2+ into the internalCa2+ store and release via theinositol 1,4,5-trisphosphate-sensitive channel. 相似文献
13.
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
14.
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
15.
Intravascular pressure regulates local and global Ca2+ signaling in cerebral artery smooth muscle cells 总被引:1,自引:0,他引:1
The regulationof intracellular Ca2+ signals in smooth muscle cells andarterial diameter by intravascular pressure was investigated in ratcerebral arteries (~150 µm) using a laser scanning confocal microscope and the fluorescent Ca2+ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca2+spark frequency 2.6-fold, Ca2+ wave frequency 1.9-fold, andglobal intracellular Ca2+ concentration([Ca2+]i) 1.4-fold in smooth muscle cells,and constricted arteries. Ryanodine (10 µM), an inhibitor ofryanodine-sensitive Ca2+ release channels, or thapsigargin(100 nM), an inhibitor of the sarcoplasmic reticulumCa2+-ATPase, abolished sparks and waves, elevated global[Ca2+]i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 µM), a voltage-dependentCa2+ channel (VDCC) blocker, significantly reduced sparks,waves, and global [Ca2+]i, and dilatedpressurized (60 mmHg) arteries. Steady membrane depolarization elevatedCa2+ signaling similar to pressure and increased transientCa2+-sensitive K+ channel current frequencye-fold for ~7 mV, and these effects were prevented by VDCCblockers. Data are consistent with the hypothesis that pressure inducesa steady membrane depolarization that activates VDCCs, leading to anelevation of spark frequency, wave frequency, and global[Ca2+]i. In addition, pressure inducescontraction via an elevation of global[Ca2+]i, whereas the net effect of sparks andwaves, which do not significantly contribute to global[Ca2+]i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction. 相似文献
16.
Buckley Barbara J.; Whorton A. R. 《American journal of physiology. Cell physiology》1997,273(4):C1298
Tunicamycin is anucleoside antibiotic that inhibits protein glycosylation andpalmitoylation. The therapeutic use of tunicamycin is limited inanimals because of its toxic effects, particularly in cerebralvasculature. Tunicamycin decreases palmitoylation of the endothelialisoform of nitric oxide synthase, stimulates nitric oxide synthesis,and increases the concentration of intracellular calcium([Ca2+]i)in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton.FASEB J. 11: A110, 1997). In the present study,we investigated the mechanism by which tunicamycin alters[Ca2+]iusing the Ca2+-sensitive dye fura2. We found that tunicamycin increased[Ca2+]iwithout increasing levels of inositol phosphates. When cells wereincubated in the absence of extracellularCa2+,[Ca2+]irapidly rose in response to tunicamycin, although a full response wasnot achieved. The pool of intracellularCa2+ mobilized by tunicamycinoverlapped with that mobilized by thapsigargin. Extracellular nickelblocked a full response to tunicamycin when cells were incubated in thepresence of extracellular Ca2+.The effects of tunicamycin on[Ca2+]iwere partially reversed by washing out the drug, and the remainder ofthe response was inhibited by removing extracellularCa2+. These results indicate thattunicamycin mobilizes Ca2+ fromintracellular stores in a manner independent of phospholipase Cactivation and increases the influx ofCa2+ across the plasma membrane. 相似文献
17.
In fura 2-loaded N1E-115 cells, regulationof intracellular Ca2+ concentration([Ca2+]i) following a Ca2+ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa+ dependent and inhibited by 5 mM Ni2+. Incells with normal intracellular Na+ concentration([Na+]i), removal of bath Na+,which should result in reversal of Na+/Ca2+exchange, did not increase [Ca2+]i unlesscell Ca2+ buffer capacity was reduced. When N1E-115 cellswere Na+ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa+/Ca2+ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca2+]ioccurred despite normal cell Ca2+ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa+/Ca2+ exchanger (net efflux ofCa2+) by observing increases (~ 6 mM) in[Na+]i. These Ni2+ (5 mM)-inhibited increases in [Na+]i could onlybe observed when a continuous ionomycin-induced influx ofCa2+ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na+dependent and blocked by 5 mM Ni2+ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na+/Ca2+ exchanger that is capableof regulating [Ca2+]i after release ofCa2+ from cell stores. 相似文献
18.
RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry 总被引:2,自引:0,他引:2
Liang L Zsembery A Schwiebert EM 《American journal of physiology. Cell physiology》2005,289(2):C388-C396
A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca2+-dependent Cl channels via stimulation of Ca2+ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca2+ in human airway epithelial cells that translates into stimulation of sustained secretory Cl transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca2+ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X4, P2X5, and P2X6 subtypes in non-CF (16HBE14o) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca2+ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X4 and P2X6 (but not P2X5) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca2+ entry markedly in fura-2 Ca2+ measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca2+ entry channel subtypes are expressed in airway epithelia. P2X4 and P2X6 may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype. purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy 相似文献
19.
Suzuki Hideaki; Ikeda Katsuhisa; Furukawa Masayuki; Takasaka Tomonori 《American journal of physiology. Cell physiology》1997,273(5):C1533
The biological characteristics of the globular substance, aprecursor of otoconia, are unclear. In the present study, the ATP-induced internal free Ca2+ concentration([Ca2+]i) changes of the globular substanceand the ATP distribution in the vestibular organ were investigatedusing a Ca2+ indicator, fluo 3, and an adeninenucleotide-specific fluorochrome, quinacrine, by means of confocallaser scanning microscopy. [Ca2+]i showed arapid and dose-dependent increase in response to ATP with a 50%effective concentration (EC50) of 16.7 µM. Thisreaction was independent of external Ca2+, indicating thepresence of an internal Ca2+ reservoir. Neither adenosine,,-methylene-ATP, 3'-O-(4-benzoylbenzoyl)-ATP, ADP, norUTP evoked this reaction, whereas 2-methylthio-ATP induced an increaseof [Ca2+]i with an EC50 of 14.4 µM. Moreover, P2 antagonists, reactive blue 2 and suramin, and aphospholipase C inhibitor, U-73122, inhibited the ATP-induced[Ca2+]i increase. These findings indicate thepresence of a P2Y purinoceptor on the globular substance. In addition,granular fluorescence was observed in the quinacrine-stained macularsensory epithelium, indicating the presence of ATP-containing granulesin this tissue. These results suggest that a paracrine mechanisminvolving ATP may exist in the macula and that this mechanism regulatesthe biological behavior of the globular substance. 相似文献
20.
Thrombin-, bradykinin-, and arachidonic acid-induced Ca2+ signaling in Ehrlich ascites tumor cells 总被引:1,自引:0,他引:1
Jorgensen Nanna Koschmieder; Petersen Stine Falsig; Hoffmann Else Kay 《American journal of physiology. Cell physiology》1999,276(1):C26
Stimulation ofsingle Ehrlich ascites tumor cells with agonists (bradykinin, thrombin)and with arachidonic acid (AA) induces increases in the freeintracellular Ca2+ concentration([Ca2+]i)in the presence and absence of extracellularCa2+, measured using theCa2+-sensitive probe fura 2. Sequential stimulation with two agonists elicits sequential increasesin[Ca2+]i,unlike addition of the same agonist twice. Bradykinin and thrombin haveadditive effects on[Ca2+]iin Ca2+-free medium. Thephosphoinositidase C inhibitor U-73122 inhibits the agonist-inducedincreases in[Ca2+]i,whereas ryanodine has no effect. Pretreatment of cells in Ca2+-free medium with thapsigarginabolishes the bradykinin-induced increase in[Ca2+]ibut not the response to thrombin. The AA-induced response is notinhibited by U-73122 and cannot be mimicked by the inactive structuralanalog trifluoromethylarachidonyl ketone. Pretreatment of the cellswith 50 µM AA (but not with 10 µM AA) abolishes the agonist-inducedincrease in[Ca2+]i.Thus bradykinin, thrombin, and AA induce increases in[Ca2+]iin Ehrlich cells due to Ca2+ entryand release from intracellular stores. Thrombin causes release ofCa2+ from an intracellular storethat is insensitive to bradykinin and is not depleted by thapsigarginbut is depleted by AA. 相似文献