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1.
Voltage dependence of Ca2+ sparks in intact cerebral arteries 总被引:4,自引:0,他引:4
Jaggar Jonathan H.; Stevenson Andra S.; Nelson Mark T. 《American journal of physiology. Cell physiology》1998,274(6):C1755
Ca2+ sparks have beenpreviously described in isolated smooth muscle cells. Here we presentthe first measurements of local Ca2+ transients("Ca2+ sparks") in an intactsmooth muscle preparation. Ca2+sparks appear to result from the opening of ryanodine-sensitive Ca2+ release (RyR) channels in thesarcoplasmic reticulum (SR). Intracellular Ca2+ concentration([Ca2+]i)was measured in intact cerebral arteries (40-150 µm in diameter) from rats, using the fluorescentCa2+ indicator fluo 3 and a laserscanning confocal microscope. Membrane potential depolarization byelevation of external K+ from 6 to30 mM increased Ca2+ sparkfrequency (4.3-fold) and amplitude (~2-fold) as well as globalarterial wall[Ca2+]i(~1.7-fold). The half time of decay (~50 ms) was not affected bymembrane potential depolarization. Ryanodine (10 µM), which inhibitsRyR channels and Ca2+ sparks inisolated cells, and thapsigargin (100 nM), which indirectly inhibitsRyR channels by blocking the SRCa2+-ATPase, completely inhibitedCa2+ sparks in intact cerebralarteries. Diltiazem, an inhibitor of voltage-dependentCa2+ channels, lowered global[Ca2+]iand Ca2+ spark frequency andamplitude in intact cerebral arteries in a concentration-dependentmanner. The frequency of Ca2+sparks (<1s1 · cell1),even under conditions of steady depolarization, was too low tocontribute significant amounts ofCa2+ to globalCa2+ in intact arteries. Theseresults provide direct evidence that Ca2+ sparks exist in quiescentsmooth muscle cells in intact arteries and that changes of membranepotential that would simulate physiological changes modulate bothCa2+ spark frequency and amplitudein arterial smooth muscle. 相似文献
2.
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
3.
Pei Jian-Ming; Yu Xiao-Chun; Bian Jin-Song; Wong Tak-Ming 《American journal of physiology. Cell physiology》1999,277(3):C492
To study the effects of -opioid receptor stimulation onintracellular Ca2+ concentration([Ca2+]i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca2+]iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca2+]itransient, which results from the influx ofCa2+ and the subsequentmobilization of Ca2+ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca2+]iand inhibited the[Ca2+]itransient induced by caffeine, which mobilizesCa2+ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa2+ from its intracellular poolinto the cytoplasm. The Ca2+responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca2+]iand the electrically induced[Ca2+]itransient were significantly attenuated when the extracellular pH(pHe) was loweredto 6.8, which itself reduced intracellular pH(pHi) and increased[Ca2+]i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa+/H+exchange blocker, or 0.2 mM Ni2+,a putativeNa+/Ca2+exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa+/H+andNa+/Ca2+exchanges. When glucose at 50 mM, known to activate theNa+/H+exchange, was added, both the resting[Ca2+]iand pHi increased. Interestingly,the effects of U-50488H on [Ca2+]iand the electrically induced[Ca2+]itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca2+ (3 mM) solutionwas superfused, the resting[Ca2+]iincreased; the increase was abolished by 0.2 mMNi2+, but thepHi remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca2+]iand electrically induced[Ca2+]itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa2+ from SR. Activation of bothNa+/H+andNa+/Ca2+exchanges, leading to an elevation of[Ca2+]i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation. 相似文献
4.
In the presentstudy, we examined the ability of adenosine 3',5'-cyclicmonophosphate (cAMP) to reduce elevated levels of cytosolicCa2+ concentration([Ca2+]i)in pancreatic -cells.[Ca2+]iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single-cells by fura 2 and autofluorescence microfluorometry. Sustained[Ca2+]ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activatorforskolin (10 µM), and an incretin glucagon-likepeptide-1-(7-36) amide (109 M), as well as byglucose (16.7 mM). The[Ca2+]i-reducingeffects of cAMP were greater at elevated glucose (8.3-16.7 mM)than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA),H-89, counteracted[Ca2+]i-reducingeffects of cAMP but not those of glucose. Okadaic acid, a phosphataseinhibitor, at 10-100 nM also reduced sustained [Ca2+]ielevation in a concentration-dependent manner. Glucose, but not DBcAMP,increased NAD(P)H in -cells.[Ca2+]i-reducingeffects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitorof the endoplasmic reticulum (ER)Ca2+ pump. In contrast,[Ca2+]i-reducingeffects of cAMP were not altered by ryanodine, an ERCa2+-release inhibitor,Na+-free conditions, or diazoxide,an ATP-sensitive K+ channelopener. In conclusion, the cAMP-PKA pathway reduces[Ca2+]ielevation by sequestering Ca2+ inthapsigargin-sensitive stores. This process does not involve, but ispotentiated by, activation of -cell metabolism. Together with theknown[Ca2+]i-increasingaction of cAMP, our results reveal dual regulation of -cell[Ca2+]iby the cAMP-signaling pathway and by a physiological incretin. 相似文献
5.
Ciglitizone inhibits cell proliferation in human uterine leiomyoma via activation of store-operated Ca2+ channels 总被引:1,自引:0,他引:1
Kim BY Cho CH Song DK Mun KC Suh SI Kim SP Shin DH Jang BC Kwon TK Cha SD Bae I Bae JH 《American journal of physiology. Cell physiology》2005,288(2):C389-C395
This study investigated the acute effects of a peroxisome proliferator-activated receptor (PPAR)- ligand, ciglitizone, on cell proliferation and intracellular Ca2+ signaling in human normal myometrium and uterine leiomyoma. Changes in intracellular Ca2+ concentration ([Ca2+]i) were measured with fura-2 AM, and cellular viabilities were determined by viable cell count and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay. Ciglitizone (100 µM) induced greater inhibition of cell proliferation in uterine leiomyoma than in myometrium. Ciglitizone also dose-dependently increased [Ca2+]i in both myometrium and uterine leiomyoma; these [Ca2+]i increases were inhibited by PPAR- antagonists and raloxifene. Ciglitizone-induced [Ca2+]i increase showed only an initial peak in normal myometrial cells, whereas in uterine leiomyoma there was a second sustained [Ca2+]i increase as well. The initial [Ca2+]i increase in both myometrium and uterine leiomyoma resulted from the release of Ca2+ by the sarcoplasmic reticulum via activation of ryanodine receptors. The second [Ca2+]i increase was observed only in uterine leiomyoma because of a Ca2+ influx via an activation of store-operated Ca2+ channels (SOCCs). Cell proliferation was inhibited and secondary [Ca2+]i increase in uterine leiomyoma was attenuated by cotreatment of ciglitizone with a SOCC blocker, lanthanum. The results suggest that ciglitizone inhibits cell proliferation and increases [Ca2+]i through the activation of SOCCs, especially in human uterine leiomyoma. peroxisome proliferator-activated receptor-; intracellular calcium; uterine cells 相似文献
6.
Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle 总被引:3,自引:0,他引:3
Perez Guillermo J.; Bonev Adrian D.; Nelson Mark T. 《American journal of physiology. Cell physiology》2001,281(6):C1769
The goal of the present study was to testthe hypothesis that local Ca2+ release events(Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitiveK+ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca2+ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca2+ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa2+ concentration ([Ca2+]i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 106). A Ca2+spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca2+ spark, BKchannel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ~30 µM to 4 µM sparkCa2+ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca2+ spark sites are in closeproximity to the BK channels and that local[Ca2+]i reaches micromolar levels to activateBK channels. 相似文献
7.
Murphy Robyn M.; Snow Rodney J.; Lamb Graham D. 《American journal of physiology. Cell physiology》2006,290(1):C116
µ-calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that µ-calpain becomes proteolytically active in the presence of 2200 µM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; 1 µM) than the levels at which µ-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 µM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for µ-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with 15% autolysis observed after 1-min exposure to 2.5 µM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze µ-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% O2 peak until fatigue, n = 3) on the amount of autolyzed µ-calpain or calpain-3 in human muscle. No significant autolysis of µ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo. Ca2+-dependent proteases; proteolysis 相似文献
8.
Conceicao Isaltino M.; Lebrun Ivo; Cano-Abad Maria; Gandia Luis; Hernandez-Guijo Jesus M.; Lopez Manuela G.; Villarroya Mercedes; Jurkiewicz Aron; Garcia Antonio G. 《American journal of physiology. Cell physiology》1998,274(6):C1745
Toxin- (T)from the Brazilian scorpion Tityusserrulatus venom caused a concentration- andtime-dependent increase in the release of norepinephrine andepinephrine from bovine adrenal medullary chromaffin cells. T was~200-fold more potent than veratridine judged fromEC50 values, although the maximalsecretory efficacy of veratridine was 10-fold greater than that of T(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+concentration([Ca2+]o),when 30 µM veratridine plus 0.45 µM T were used. T (0.45 µM) doubled the basal uptake of45Ca2+,whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seenwhen T and veratridine were combined; this was particularlypronounced at 5 mM[Ca2+]o.Veratridine induced oscillations of cytosolicCa2+ concentration([Ca2+]i)in single fura 2-loaded cells without elevation of basal levels. Incontrast, T elevated basal[Ca2+]ilevels, causing only small oscillations. When added together, T andveratridine elevated the basal levels of[Ca2+]iwithout causing large oscillations. T shifted the current-voltage (I-V) curve forNa+ channel current to the left.The combination of T with veratridine increased the shift of theI-V curve to the left, resulting in agreater recruitment of Na+channels at more hyperpolarizing potentials. This led to enhanced andmore rapid accumulation of Na+ inthe cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, andCa2+ entry and secretion. 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.
Role of calcium channels in carboxyl-terminal parathyroid hormone receptor signaling 总被引:1,自引:0,他引:1
Selim AA Mahon M Juppner H Bringhurst FR Divieti P 《American journal of physiology. Cell physiology》2006,291(1):C114-C121
Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(184) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([Ca2+]i) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca2+]i was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca2+]i was observed in OC59 cells in response to the CPTH fragment hPTH-(5384) (250 nM). No [Ca2+]i signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(134) nor a mutant CPTH analog, [Ala5557]hPTH-(5384), that does not to bind to CPTHRs, increased [Ca2+]i in OC59 cells. The [Ca2+]i response to hPTH-(5384) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), -agatoxin IVA (10 nM), and -conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs. calcium influx; osteocytes 相似文献
11.
Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle 总被引:1,自引:0,他引:1
Prakash Y. S.; Kannan Mathur S.; Walseth Timothy F.; Sieck Gary C. 《American journal of physiology. Cell physiology》1998,274(6):C1653
The purpose ofthe present study was to determine whether cyclic ADP-ribose (cADPR)acts as a second messenger forCa2+ release through ryanodinereceptor (RyR) channels in tracheal smooth muscle (TSM). Freshlydissociated porcine TSM cells were permeabilized with -escin, andreal-time confocal microscopy was used to examine changes inintracellular Ca2+ concentration([Ca2+]i).cADPR (10 nM-10 µM) induced a dose-dependent increase in [Ca2+]i,which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 µM) and by the RyR blockers ruthenium red (10 µM) and ryanodine (10 µM), but not by the inositol 1,4,5-trisphosphate receptor blockerheparin (0.5 mg/ml). During steady-state[Ca2+]ioscillations induced by acetylcholine (ACh), addition of 100 nM and 1 µM cADPR increased oscillation frequency and decreased peak-to-troughamplitude. ACh-induced[Ca2+]ioscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR didnot block the[Ca2+]iresponse to a subsequent exposure to caffeine. These results indicatethat cADPR acts as a second messenger forCa2+ release through RyR channelsin TSM cells and may be necessary for initiating ACh-induced[Ca2+]ioscillations. 相似文献
12.
Nakao Miki; Ono Kyoichi; Fujisawa Susumu; Iijima Toshihiko 《American journal of physiology. Cell physiology》1999,276(1):C238
A fluid streamthrough a microtube was applied to cultured human aortic endothelialcells to investigate the endothelial responses of both the ioniccurrents and intracellular Ca2+concentration([Ca2+]i)to mechanical stimulation. The fluid stream induced an increase in[Ca2+]ithat was dependent on both the flow rate and the extracellular Ca2+ concentration.Gd3+ and niflumic acid inhibitedthe fluid stream-induced increase in[Ca2+]i,whereas Ba2+ andtetraethylammonium ion exhibited no effect. The fluid stream-induced [Ca2+]iincrease was accompanied by the activation of an inward current at52.8 mV. The reversal potential of the fluid stream-induced current shifted to positive potentials when the externalCl concentration wasreduced but was not affected by variation of the externalNa+ concentration. During theexposure to the fluid stream,[Ca2+]iwas voltage dependent, i.e., depolarization decreased[Ca2+]i.We therefore conclude that the fluid stream-induced current is largelycarried by Cl and that theCl current may thus play arole in modulating the Ca2+ influxby altering the membrane potential of endothelial cells. 相似文献
13.
TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation 总被引:1,自引:0,他引:1
Expression of TNF-, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF- regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF- regulates the diameter of resistance-sized (150-µm diameter) cerebral arteries by modulating local and global intracellular Ca2+ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF- increased Ca2+ spark and Ca2+ wave frequency but reduced global intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells of intact arteries. TNF- elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (40 mV) cells, TNF- increased the frequency and amplitude of Ca2+ spark-induced, large-conductance, Ca2+-activated K+ (KCa) channel transients 1.7- and 1.4-fold, respectively. TNF--induced transient KCa current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF- induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase blocker that inhibits Ca2+ sparks, and iberiotoxin, a KCa channel blocker, reduced TNF--induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF- activates NAD(P)H oxidase, resulting in an increase in intracellular H2O2 that stimulates Ca2+ sparks and transient KCa currents, leading to a reduction in global [Ca2+]i, and vasodilation. cerebrovascular circulation; ryanodine-sensitive Ca2+ release channel; Ca2+-activated K+ channel; reactive oxygen species; vasodilation 相似文献
14.
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
15.
The hypothesisthat vascular protection in females and its absence in males reflectsgender differences in [Ca2+]i andCa2+ mobilization mechanisms of vascular smooth musclecontraction was tested in fura 2-loaded aortic smooth muscle cellsisolated from intact and gonadectomized male and female Wistar-Kyoto(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca2+), the resting length and[Ca2+]i were significantlydifferent in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than inintact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,phenylephrine (Phe, 105 M) caused transient increasein [Ca2+]i to 428 ± 13 nMfollowed by maintained increase to 201 ± 8 nM and 32% cellcontraction. In intact female WKY, the Phe-induced [Ca2+]i transient was notsignificantly different, but the maintained [Ca2+]i (159 ± 7 nM) and cellcontraction (26%) were significantly less than in intact male WKY. InCa2+-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)caused transient increases in[Ca2+]i and contraction that werenot significantly different between males and females. Membranedepolarization by 51 mM KCl caused 31% cell contraction and increased[Ca2+]i to 259 ± 9 nM in intactmale WKY, which were significantly greater than a 24% contraction and214 ± 8 nM [Ca2+]i in intactfemale WKY. Maintained Phe- and KCl-stimulated cell contraction and[Ca2+]i were significantly greaterin SHR than WKY in all groups of rats. Reduction in cell contractionand [Ca2+]i in intact femalescompared with intact males was significantly greater in SHR (~30%)than WKY (~20%). No significant differences in cell contraction or[Ca2+]i were observed betweencastrated males, ovariectomized (OVX) females, and intact males, orbetween OVX females with 17-estradiol implants and intact females.Exogenous application of 17-estradiol (108 M) tocells from OVX females caused greater reduction in Phe- and KCl-inducedcontraction and [Ca2+]i in SHR thanWKY. Thus the basal, maintained Phe- and depolarization-induced [Ca2+]i and contraction of vascularsmooth muscle triggered by Ca2+ entry from theextracellular space exhibit differences depending on gender and thepresence or absence of female gonads. Cell contraction and[Ca2+]i due to Ca2+release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca2+]i in vascular smoothmuscle of female rats is greater in SHR than WKY rats. 相似文献
16.
Na(+)/Ca(2+) exchange and its role in intracellular Ca(2+) regulation in guinea pig detrusor smooth muscle 总被引:3,自引:0,他引:3
The role of Na+/Ca2+ exchange inregulating intracellular Ca2+ concentration([Ca2+]i) in isolated smooth muscle cellsfrom the guinea pig urinary bladder was investigated. Incrementalreduction of extracellular Na+ concentration resulted in agraded rise of [Ca2+]i; 50-100 µMstrophanthidin also increased [Ca2+]i. Asmall outward current accompanied the rise of[Ca2+]i in low-Na+ solutions(17.1 ± 1.8 pA in 29.4 mM Na+). The quantity ofCa2+ influx through the exchanger was estimated from thecharge carried by the outward current and was ~30 times that which isnecessary to account for the rise of [Ca2+]i,after correction was made for intracellular Ca2+ buffering.Ca2+ influx through the exchanger was able to loadintracellular Ca2+ stores. It is concluded that the levelof resting [Ca2+]i is not determined by theexchanger, and under resting conditions (membrane potential 50 to60 mV), there is little net flux through the exchanger. However, asmall rise of intracellular Na+ concentration would besufficient to generate significant net Ca2+ influx. 相似文献
17.
Huang L Bhattacharjee A Taylor JT Zhang M Keyser BM Marrero L Li M 《American journal of physiology. Cell physiology》2004,286(2):C213-C221
Chronic exposure of pancreatic -cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of -cell basal intracellular free Ca2+ concentration ([Ca2+]i). We have investigated the relationship between the basal intracellular free Ca2+ and the L-type (Cav1.3) Ca2+ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca2+ influx, which was attenuated when the basal [Ca2+]i was elevated. Using voltage-clamp techniques, we found that changing [Ca2+]i could regulate the amplitude of the Ca2+ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Cav1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca2+]i conditions by deconvolution microscopy. The [Ca2+]i-dependent translocation of Cav1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca2+]i. These results suggest that Cav1.3 channel trafficking is involved in glucose desensitization of pancreatic -cells. internalization; intracellular free calcium; glucose desensitization 相似文献
18.
Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells 总被引:1,自引:0,他引:1
Awumey EM Howlett AC Putney JW Diz DI Bukoski RD 《American journal of physiology. Cell physiology》2007,292(5):C1895-C1905
The rat dorsal root ganglion (DRG) Ca2+-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca2+ concentration ([Ca2+]i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca2+] ([Ca2+]e) from 0.5 to 1 mM resulted in increases in [Ca2+]i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca2+]e-response studies indicate a Ca2+ sensitivity with an EC50 of 1.75 ± 0.10 mM. NPS R-467 and Gd3+ activated the CaR. When [Ca2+]e was successively raised from 0.25 to 4 mM, peak [Ca2+]i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca2+, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd3+, indicating desensitization of the response. Furthermore, Ca2+ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca2+ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Cae2+. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca2+]i transients by 49.9 ± 5.2% (at 1 mM Ca2+) and 40.5 ± 6.5% (at 2 mM Ca2+), compared with controls. The findings suggest involvement of PKC in the pathway for Ca2+ mobilization following CaR activation. desensitization; protein kinase C 相似文献
19.
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
20.
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