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1.
Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents 总被引:1,自引:0,他引:1
Secondo A Pannaccione A Cataldi M Sirabella R Formisano L Di Renzo G Annunziato L 《American journal of physiology. Cell physiology》2006,290(1):C233-C243
The role of nitric oxide (NO) in the occurrence of intracellular Ca2+ concentration ([Ca2+]i) oscillations in pituitary GH3 cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca2+]i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca2+]i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca2+ channels (VDCC) blocker nimodipine (1 µM) or in Ca2+-free medium. Conversely, its effect was preserved when Ca2+ release from intracellular Ca2+ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca2+]i oscillations by determining Ca2+ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K+ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (IDR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K+ currents. Similar results were obtained when GH3 cells were treated with L-arginine. The present study suggests that in GH3 cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca2+]i oscillations through an inhibitory effect on IDR and on IERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents 相似文献
2.
Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione 总被引:1,自引:0,他引:1
In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca2+ levels ([Ca2+]i), reactive oxygen species (ROS) generation, and cell death (respective EC50 values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca2+ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca2+]i increase. This indicates that glutamate-induced cell death is attributable to [Ca2+]i increase and ROS generation, and the [Ca2+]i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca2+]i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca2+]i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca2+]i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca2+]i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species 相似文献
3.
We examined the effects of dissolved nitric oxide (NO) gas oncytoplasmic calcium levels ([Ca2+]i) in C6glioma cells under anoxic conditions. The maximum elevation (27 ± 3 nM) of [Ca2+]i was reached at 10 µM NO. Asecond application of NO was ineffective if the first was >0.5 µM.The NO donor diethylamine/NO mimicked the effects of NO. Acute exposureof the cells to low calcium levels was without effect on the NO-evokedresponse. Thapsigargin (TG) increased [Ca2+]iand was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP waswithout effect on the NO-evoked response. If cells were pretreated withTG or exposed chronically to nominal amounts of calcium, NO decreased[Ca2+]i. The results suggest that C6 gliomacells have two receptors for NO. One receptor (NOA)elevates [Ca2+]i and resides on theendoplasmic reticulum (ER). The other receptor (NOB)decreases [Ca2+]i and resides on theplasmalemma or the ER. The latter receptor dominates when the level ofcalcium within intracellular stores is diminished. 相似文献
4.
I. Khalilov X. Leinekugel M. Mukhtarov R. Khazipov 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2014,8(2):162-168
The intracellular blockade of GABAA-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABAA receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABAA receptor channels substitute for Cl? and in which Mg2+ and ATP are omitted to induce rundown of the GABAA receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg2+- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABAA-receptor-mediated synaptic currents and responses induced by the GABAA receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg2+, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities. 相似文献
5.
The contribution of small-conductance (SKCa) and intermediate-conductance Ca2+-activated K+ (IKCa) channels to the generation of nitric oxide (NO) by Ca2+-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca2+ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca2+, as judged by 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl2 (50 µmol/l) + ouabain (100 µmol/l), depleting intracellular Ca2+ stores by thapsigargin or removing external Ca2+ inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SKCa and IKCa channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca2+ transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SKCa and IKCa channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SKCa and IKCa channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca2+ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca2+ stores is to trigger the opening of both KCa channels along with Ca2+ entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SKCa and IKCa channels are essential for NO-mediated vasorelaxation. calcium; endothelium; hyperpolarization; small-conductance calcium-activated potassium channel; intermediate-conductance calcium-activated potassium channel channel 相似文献
6.
Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells. 相似文献
7.
Regulation of P2X7-induced pore formation and cell death in pericyte-containing retinal microvessels 总被引:4,自引:0,他引:4
Sugiyama T Kawamura H Yamanishi S Kobayashi M Katsumura K Puro DG 《American journal of physiology. Cell physiology》2005,288(3):C568-C576
The purpose if this study was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X7 and P2Y4 purinoceptors, this nucleotide can kill microvascular cells of the retina. Because P2X7 receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for ATP's lethality. To test this hypothesis, we isolated pericyte-containing microvessels from rat retinas, assessed cell viability using Trypan blue dye exclusion, detected pores by determining the uptake of the fluorescent dye YO-PRO-1, measured intracellular Ca2+ with the use of fura-2, and monitored ionic currents via perforated patch pipettes. As predicted, ATP-induced cell death required P2X7 receptor activation. However, we found that pore formation was minimal because ATP's activation of P2Y4 receptors prevented P2X7 pores from forming. Rather than opening lethal pores, ATP kills via a mechanism involving voltage-dependent Ca2+ channels (VDCC). Our experiments suggest that when high concentrations of ATP caused nearly all microvascular P2X7 receptor channels to open, the resulting profound depolarization opened VDCC. Consistent with lethal Ca2+ influx via VDCC, ATP-induced cell death was markedly diminished by the VDCC blocker nifedipine or a nitric oxide (NO) donor that inhibited microvascular VDCC. We propose that purinergic vasotoxicity is normally prevented in the retina by NO-mediated inhibition of VDCC and P2Y4-mediated inhibition of P2X7 pore formation. Conversely, dysfunction of these protective mechanisms may be a previously unrecognized cause of cell death within the retinal microvasculature. calcium channels; capillaries; purinoceptors; vasotoxicity 相似文献
8.
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
9.
Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP 总被引:1,自引:0,他引:1
Williams BA Liu C Deyoung L Brock GB Sims SM 《American journal of physiology. Cell physiology》2005,288(3):C650-C658
Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca2+ concentration ([Ca2+]i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca2+]i in the presence and absence of extracellular Ca2+, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca2+]i, the PE-induced rise of [Ca2+]i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca2+]i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca2+ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca2+ release from intracellular stores. Reduction of intracellular Ca2+ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor 相似文献
10.
To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels. 相似文献
11.
To clarify the contribution of intracellularCa2+ concentration([Ca2+]i)-dependent and -independentsignaling mechanisms in arteriolar smooth muscle (aSM) to modulation ofarteriolar myogenic tone by nitric oxide (NO), released in response toincreases in intraluminal flow from the endothelium, changes in aSM[Ca2+]i and diameter of isolated rat gracilismuscle arterioles (pretreated with indomethacin) were studied byfluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca2+]i significantly increased andmyogenic tone developed in response to elevations of extracellularCa2+ concentration. The Ca2+ channelinhibitor nimodipine substantially decreased[Ca2+]i and completely inhibited myogenictone. Dilations to intraluminal flow (that were inhibited byN-nitro-L-arginine methyl ester)or dilations to the NO donorS-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were notaccompanied by substantial decreases in aSM[Ca2+]i. 8-Bromoguanosine cGMP and thecGMP-specific phosphodiesterase inhibitor zaprinast significantlydilated arterioles yet elicited only minimal decreases in[Ca2+]i. Thus flow-induced endothelialrelease of NO elicits relaxation of arteriolar smooth muscle by acGMP-dependent decrease of the Ca2+ sensitivity of thecontractile apparatus without substantial changes in thepressure-induced level of [Ca2+]i. 相似文献
12.
Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells 总被引:1,自引:0,他引:1
In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca2+ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100500 µM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca2+ uptake and Ca2+ extrusion rates, whereas low concentrations of Sper/NO (<100 µM) had no effect on mitochondrial Ca2+ levels ([Ca2+]mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca2+ uptake, whereas inhibition of mtNOS with 10 µM L-N5-(1-iminoethyl)ornithine resulted in a significant increase of [Ca2+]mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca2+-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100500 µM) induced PTP opening, whereas lower concentrations (<100 µM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca2+ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca2+ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca2+ overload under pathological conditions where cellular [NO] can reach very high levels. nitric oxide synthase; permeability transition pore; endothelium 相似文献
13.
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
14.
Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system 总被引:1,自引:0,他引:1
Uezono Y Kanaide M Kaibara M Barzilai R Dascal N Sumikawa K Taniyama K 《American journal of physiology. Cell physiology》2006,290(1):C200-C207
Coupling of functional GABAB receptors (GABABR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABAB1a receptor (GB1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABAB2 receptor (GB2R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB1aR-Cerulean and GB2R-Venus form a heterodimer. The GABABR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K+ currents in a concentration-dependent manner in oocytes expressing GB1aR and GB2R, or GB1aR-Cerulean and GB2R-Venus, together with G protein-activated inward-rectifying K+ channels (GIRKs), but not in oocytes expressing GB1aR alone or GB2R alone together with GIRKs. Oocytes coexpressing GB1aR + Gi2-fused GB2R (GB2R-Gi2) caused faster K+ currents in response to baclofen. Furthermore, oocytes coexpressing GB1aR + GB2R fused to Gqi5 (a chimeric Gq protein that activates PLC pathways) caused PLC-mediated Ca2+-activated Cl currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB1aR-Gi2 or GB1aR-Gqi5 together with GB2R. BHK cells and Xenopus oocytes coexpressing GB1aR-Cerulean + a triplet tandem of GB2R-Venus-Gqi5 caused FRET and Ca2+-activated Cl currents, respectively, with a similar potency in BHK cells coexpressing GB1aR-Cerulean + GB2R-Venus and in oocytes coexpressing GB1aR + GB2R-Gqi5. Our results indicate that functional GABABR forms a heterodimer composed of GB1R and GB2R and that the signal transducing G proteins are directly coupled to GB2R but not to GB1R. fluorescence resonance energy transfer 相似文献
15.
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
16.
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
17.
Karl MO Fleischhauer JC Stamer WD Peterson-Yantorno K Mitchell CH Stone RA Civan MM 《American journal of physiology. Cell physiology》2005,288(4):C784-C794
Intraocular pressure is directly dependent on aqueous humor flow into, and resistance to flow out of, the eye. Adenosine has complex effects on intraocular pressure. Stimulation of A1 and A2A adenosine receptors changes intraocular pressure oppositely, likely through opposing actions on the outflow of aqueous humor. While the cellular sites regulating outflow resistance are unknown, the cells lining the inner wall of Schlemm's canal (SC) are a likely regulatory site. We applied selective adenosine receptor agonists to SC cells in vitro to compare the responses to A1 and A2A stimulation. Parallel studies were conducted with human inner-wall SC cells isolated by a novel enzyme-assisted technique and with cannula-derived mixed inner- and outer-wall SC cells. A1 agonists increased whole cell currents of both inner-wall and cannula-derived SC cells. An A2A agonist reduced currents most consistently in specifically inner-wall SC cells. Those currents were also increased by A2B, but not consistently affected by A3, stimulation. A1, A2A, and A3 agonists all increased SC-cell intracellular Ca2+. The electrophysiological results are consistent with the possibility that inner-wall SC cells may mediate the previously reported modulatory effects of adenosine on outflow resistance. The results are also consistent with the presence of functional A2B, as well as A1, A2A, and A3 adenosine receptors in SC cells. intraocular pressure; aqueous humor outflow; ion transport; adenosine agonists 相似文献
18.
Adenosine stimulates depolarization and rise in cytoplasmic [Ca2+] in type I cells of rat carotid bodies 总被引:1,自引:0,他引:1
During hypoxia, the level of adenosine in the carotid bodies increases as a result of ATP catabolism and adenosine efflux via adenosine transporters. Using Ca2+ imaging, we found that adenosine, acting via A2A receptors, triggered a rise in cytoplasmic [Ca2+] ([Ca2+]i) in type I (glomus) cells of rat carotid bodies. The adenosine response could be mimicked by forskolin (but not its inactive analog), and could be abolished by the PKA inhibitor H89. Simultaneous measurements of membrane potential (perforated patch recording) and [Ca2+]i showed that the adenosine-mediated [Ca2+]i rise was accompanied by depolarization. Ni2+, a voltage-gated Ca2+ channel (VGCC) blocker, abolished the adenosine-mediated [Ca2+]i rise. Although adenosine was reported to inhibit a 4-aminopyridine (4-AP)-sensitive K+ current, 4-AP failed to trigger any [Ca2+]i rise, or to attenuate the adenosine response. In contrast, anandamide, an inhibitor of the TWIK-related acid-sensitive K+-1 (TASK-1) channels, triggered depolarization and [Ca2+]i rise. The adenosine response was attenuated by anandamide but not by tetraethylammonium. Our results suggest that adenosine, acting via the adenylate cyclase and PKA pathways, inhibits the TASK-1 K+ channels. This leads to depolarization and activation of Ca2+ entry via VGCC. This excitatory action of adenosine on type I cells may contribute to the chemosensitivity of the carotid body during hypoxia. O2 sensing; A2A receptor; cAMP; protein kinase A; TWIK-related acid-sensitive K+ channel 相似文献
19.
Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells 总被引:2,自引:0,他引:2
Dedkova EN Ji X Lipsius SL Blatter LA 《American journal of physiology. Cell physiology》2004,286(2):C406-C415
Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]mt). Stimulation of mitochondrial Ca2+ uptake by exposure to different cytoplasmic Ca2+ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]mt was sensitive to the NOS antagonist L-N5-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca2+ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca2+ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria 相似文献
20.
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