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1.
We investigated the cytosolic free Ca2+ concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca2+]i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K+ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca2+]i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca2+]i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca2+]i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca2+]i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane. Received: 23 January 2001/Revised: 15 June 2001  相似文献   

2.
Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca2+ concentration ([Ca2+]i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca2+]i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1 ≈1 μM) induced increase of [Ca2+]i in a dose-dependent manner. This elevation of [Ca2+]i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca2+ stores by the endoplasmic reticulum inhibitor thapsigargin (2 μM), [Ca2+]i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca2+-free condition, orexin A failed to induce any changes of Ca2+ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca2+]i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 μM) and D609 (10 μM), respectively. Selective blockade of L-type Ca2+ channels by nifedipine (5 μM) significantly suppressed the elevation of [Ca2+]i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca2+]i in neurons from deep layers of PFC, which depends on extracellular Ca2+ influx via L-type Ca2+ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.  相似文献   

3.
Intracellular Ca2+ ([Ca2+]i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca2+]i transients vary with Ca2+ load. In addition to a rapid initial decay, large (> 500 nM) [Ca2+]i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca2+]i transients, consistent with stopping uptake of Ca2+ by mitochondria. CCCP causes a small increase of [Ca2+]i at rest. After a large Ca2+ entry the amount is much larger, consistent with release from a mitochondrial Ca2+ pool that fills during cytoplasmic Ca2+ loading. The rate of Ca2+ uptake by mitochondria is dependent upon [Ca 2+]i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca2+]i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca2+-uniporter) in the pipette, GnRH does not induce rhythmic [Ca2+]i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca2+ loads in gonadotropes and participate in GnRH-induced periodic [Ca2+]i oscillations.  相似文献   

4.
Recent studies have provided evidence that depolarization in the absence of extracellular Ca2+ can trigger Ca2+ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca2+ from intracellular stores in response to depolarization, independent of Ca2+ influx. We measured changes in cytosolic ΔF/F0 in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K+ depolarization in the presence (2 mM) and absence of extracellular Ca2+ ([Ca2+]e). Progressive elevations in extracellular [K+]e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca2+]e. Peak amplitude of ΔF/F0 transients in 2 mM [Ca2+]e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F0 in 0 mM [Ca2+]e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca2+]e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F0 transients in 0 mM [Ca2+]e were slower than those of ΔF/F0 transients evoked in 2 mM [Ca2+]e. Rises in ΔF/F0 evoked by high [K+]e in the absence of extracellular Ca2+ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, or the inositol 1,4,5-triphosphate (IP3) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd2+, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca2+ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca2+ from IP3-sensitive internal stores in response to membrane depolarization, independent of Ca2+ influx.  相似文献   

5.
《Journal of biomechanics》2014,47(16):3903-3908
Intracellular calcium transient ([Ca2+]i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca2+]i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca2+]i transient in osteoblasts. Fluorescent dyes DiBAC4(3) and fura-2 AM were respectively used to detect membrane potential and [Ca2+]i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca2+]i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca2+]i transients. Replacing extracellular Na+ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca2+]i transients. However, voltage-activated K+ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca2+ or blocking of L-type voltage-sensitive Ca2+ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca2+]i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca2+ mobilization wherein FSS activated SACs to promote Na+ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca2+ influx though L-VSCCs switched on [Ca2+]i response in osteoblasts.  相似文献   

6.
Summary Neutral-carrier pH-and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pHi or [Ca2+]i in crayfish septate axons uncoupled by acidification. For measuring [Ca2+]i a new neutral carrier sensor sensitive to picomolar [Ca2+] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pHi or [H+]i peaked 40–90 sec before junctional resistance. The difference in shape and peak time between pHi and junctional resistance curves caused significant hysteresis in the pHi versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3–4 times greater than those with fast acidifications of similar magnitude. With acetate, [Ca2+]i, increased by approximately one order of magnitude from basal values of 0.1–0.3 m. The curves describing the time course of changes in [Ca2+]i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca2+]i recovered following a single exponential decay with a time constant of 2 min. Different rates of acidification caused increases in [Ca2+]i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca2+]i than to [H+]i.  相似文献   

7.
The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20–25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based ([Ca2+]i) microfluorimetry. The ET-triggered ([Ca2+]i) transients were mimicked by ET, receptor agonist BO-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca2+-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3 sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca2+-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 μM ATP or 10 μM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.  相似文献   

8.
In the present study, the bombesin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca2+ transients. The average frequency of the Ca2+ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min−1 (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca2+ transient followed by a sustained plateau or by a decrease to basal levels. In Ca2+- free medium, bombesin caused only one or two Ca2+ transients and withdrawal of extracellular Ca2+ abolished the Ca2+ transients. The voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca2+ transients and stopped the Ca2+ transients in some cells. Thapsigargin caused a sustained rise in [Ca2+]i) in the presence of extracellular Ca2+ while in its absence the rise in [Ca2+]i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca2+], by about 50%. Depletion of intracellular Ca2+ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca2+-free medium caused an agonist-independent increase in [Ca2+]i) when extracellular Ca2+ was restored, which was larger than in control cells that had been incubated in Ca2+-free medium for the same period of time. This rise in [Ca2+]i and the thapsigargin-induced increase in [Ca2+]i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca2+ pool enhances Ca2+ influx through VDCC and voltage-independent Ca2+ channels (VICC). In conclusion, the bombesin-induced Ca2+ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca2+ transients. Intracellular Ca2+ is mobilized during each Ca2+ transient, but Ca2+ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca2+ response. Ca2+ influx in whole or part is activated by a capacitative Ca2+ entry mechanism.  相似文献   

9.
In this study, the functional consequences of the pharmacological modulation of the M‐current (IKM) on cytoplasmic Ca2+ intracellular Ca2+concentration ([Ca2+]i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. Kv7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K+]e, the IKM activator retigabine (RT, 10 μM) inhibited [3H]d ‐aspartate ([3H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the IKM blocker XE‐991 (20 μM). The IKM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K+]e‐induced synaptosomal [Ca2+]i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca2+]i transients. The P/Q‐type voltage‐sensitive Ca2+channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca2+]i increase and [3H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca2+]i and the resulting enhancement of [3H]d ‐Asp release induced by [Ca2+]i mobilization from intracellular stores, or by store‐operated Ca2+channel activation. Collectively, the present data reveal that the pharmacological activation of IKM regulates depolarization‐induced [3H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca2+]i occurring through P/Q‐type VSCCs.  相似文献   

10.
The calcium-sensitive fluorescent indicator fura-2 and a microscope equipped for rapidly changing excitation wavelengths were used to look at the effects of growth factors on cytosolic free calcium ([Ca2+]i,) in NRK-49F cells. In these cells bradykinin induced a rapid increase in [Ca2+]i, which generally decayed to near basal [Ca2+]i within 3 minutes. The initial rise in [Ca2+]i in response to bradykinin was relatively independent of extracellular calcium; however, the decay to basal [Ca2+]i was more rapid in the absence of extracellular calcium. Measurements made on individual cells showed a heterogeneity in the response to bradykinin. Epidermal growth factor (EGF) had no effect on [Ca2+]i in NRK-49F cells when added alone in the presence of extracellular calcium. Simultaneous addition of bradykinin and EGF produced a more prolonged increase in [Ca2+]i than bradykinin alone. The prolongation was dependent on the presence of extracellular calcium and did not occur in its absence. Transient increases in [Ca2+]i occurring after the initial peak were occasionally seen in these cells. Our results indicate that there is rapid interaction between the signaling mechanisms for bradykinin and EGF. When this occurs, one effect is the transport of calcium into the cell from the extracellular environment, causing a more prolonged rise in [Ca2+]i. This effect occurs within 1 minute after combined addition of bradykinin and EGF.  相似文献   

11.
2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca2+ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca2+]i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca2+ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca2+]i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca2+]i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca2+]i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca2+ and SOC channel blockers such as SK&F96365, La3+ and Gd3+. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca2+]i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca2+]i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.  相似文献   

12.
The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca2+]i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca2+ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca2+]i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca2+]i after expoxure to 50 mM K+ which was of the same magnitude as the increase in [Ca2+]i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca2+ channels verapamil had no effect on the hyposmolarity induced increase in [Ca2+]i. On the contrary, this increase in [Ca2+]i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca2+ channels Mg2+ and La3+. Dantrolene which prevents release of Ca2+ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca2+ which represents an influx of Ca2+ rather than a release of Ca2+ from internal, dantrolene sensitive stores.  相似文献   

13.
Subtypes of inhibitory GABAergic neurons containing Ca2+-binding proteins play a pivotal role in the regulation of spontaneous synchronous [Ca2+]i transients in a neuronal network. In this study it is shown that: (1) the interneurons that containing Ca2+-binding proteins at buffer concentration can be identified by the shape of Ca2+-signa1 in response to depolarization or activation of ionotropic glutamate receptors; (2) Ca2+-binding proteins are involved in desynchronization of spontaneous Ca2+ transients. At low frequencies of spontaneous synchronous [Ca2+]i transients (less than 0.2 Hz) neurons show quasi-synchronous pulsations. At higher frequencies, synchronization of spontaneous synchronous [Ca2+]i transients occurs in all neurons; (3) it is established that several synchronous oscillations with different frequencies coexist in the network and the amplitude of their depolarizing pulse also varies. This phenomenon is apparently the mechanism that selectively directs information in separate neurons using the same network; and (4) in one population of interneurons at high frequencies of spontaneous synchronous [Ca2+]i transients the inversion of Cl concentration gradient is observed. In this case, the inhibition of GABA(A) receptors suppresses the activity of neurons in this population and excites other neurons in the network. Thus, the GABAergic neurons that contain Ca-binding proteins show different mechanisms to regulate the synchronous neuronal activities in cultured rat hippocampal cells.  相似文献   

14.
We investigated the cytosolic free calcium concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular Ca2+ concentration via the bathing solution ([Ca2+]B). Changing [Ca2+]B had only an effect on [Ca2+]i if the cells were depolarized by raising the extracellular K+ concentration. Surprisingly, raising [Ca2+]B from 2 to 10 mm caused a decrease in [Ca2+]i, and an increase was evoked by reducing [Ca2+]B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca2+]B moderate membrane depolarizations were sufficient to evoke a [Ca2+]i increase, while progressively larger depolarizations were necessary at higher [Ca2+]B. The changes in the relationship between [Ca2+]i and membrane potential upon varying [Ca2+]B could be reversed by changing extracellular pH. We conclude that [Ca2+]B affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels via the electrochemical Ca2+ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in a counteracting way: Raising the extracellular Ca2+ concentration enhances the electrochemical Ca2+ gradient and hence Ca2+ influx, but it attenuates Ca2+ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa. Received: 23 January 2001/Revised: 23 June 2001  相似文献   

15.
Agomelatine, a novel antidepressant exerting its effects through melatonergic and serotonergic systems, implicated to be effective against pain including neuropathic pain but without any knowledge of mechanism of action. To explore the possible role of agomelatine on nociceptive transmission at the peripheral level, the effects of agomelatine on intracellular calcium ([Ca2+]i) signaling in peripheral neurons were investigated in cultured rat dorsal root ganglion (DRG) neurons. Using the fura-2-based calcium imaging technique, the effects of agomelatine on [Ca2+]i and roles of the second messenger-mediated pathways were assessed. Agomelatine caused [Ca2+]i signaling in a dose-dependent manner when tested at 10 and 100 μM concentration. Luzindole, a selective melatonin receptor antagonist, almost completely blocked the agomelatine-induced calcium signals. The agomelatine-induced calcium transients were also nearly abolished following pretreatment with the 100 ng/ml pertussis toxin, a Gi/o protein inhibitor. The stimulatory effects of agomelatine on [Ca2+]i transients were significantly reduced by applications of phospholipase C (PLC) and protein kinase C (PKC) blockers, 10 μM U73122, and 10 μM chelerythrine chloride, respectively. The obtained results of agomelatine-induced [Ca2+]i signals indicates that peripheral mechanisms are involved in analgesic effects of agomelatine. These mechanisms seems to involve G-protein-coupled receptor activation and PLC and PKC mediated mechanisms.  相似文献   

16.
The effects of extracellular Mg2+ on both dynamic changes of [Ca2+]i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca2+ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca2+ concentrations ([Ca2+]i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca2+]i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca2+]i (150 nM) for several hours. The initial [Ca2+]i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca2+]i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg2+ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg2+, caused an increase in basal [Mg2+]i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg2+, 1 M thapsigargin induces, in 10 mM Mg2+, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca2+]i and a lower Ca2+ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca2+]i which was inhibited by high extracellular and intracellular Mg2+.  相似文献   

17.
The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca2 + concentration ([Ca2 +]i) and proliferation was examined by using the Ca2 +-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca2 +]i in a concentration-dependent manner. Celecoxib-induced [Ca2 +]i increase was partly reduced by removal of extracellular Ca2 +. Celecoxib-induced Ca2 + influx was independently suggested by Mn2 + influx-induced fura-2 fluorescence quench. In Ca2 +-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2 +-ATPase, caused a monophasic [Ca2 +]i increase, after which celecoxib only induced a tiny [Ca2 +]iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca2 +]i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca2 +]i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca2 +]i increase in renal tubular cells by stimulating both extracellular Ca2 + influx and intracellular Ca2 + release and is highly toxic to renal tubular cells in vitro.  相似文献   

18.
The rise in intracellular Ca2+ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca2+ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca2+ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca2+]i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca2+]i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca2+ permeable AMPA receptors partially inhibited the AMPA induced [Ca2+]i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca2+ influx occurs through Ca2+ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca2+]m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca2+]i but no change was observed in mitochondrial Ca2+ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca2+ permeable AMPA receptors, however the larger part of Ca2+ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.  相似文献   

19.
Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca2+]i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca2+]i in acinar cells. The removal of extracellular Ca2+ or the use of Ca2+ channel blockers partially inhibited the ATP-induced [Ca2+]i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP3 receptors) did not completely inhibit the ATP-induced [Ca2+]i increase. P1 purinoceptor agonists did not induce any changes in [Ca2+]i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca2+]i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca2+]i, although UTP (a P2Y2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y1 played a significant role in the cellular response to ATP. BzATP (a P2X7 receptor agonist) induced a small increase in [Ca2+]i, but α,β-meATP (a P2X1,3 receptor agonist) had no effect. RT-PCR indicated that P2X2,3,4,5,6,7 and P2Y1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y1) as well as by P2X purinoceptors.  相似文献   

20.
In the labouring uterus, millions of myocytes forming the complex geometrical structure of myometrium contract in synchrony to increase intrauterine pressure, dilate the cervix and eventually expel the foetus through the birth canal. The mechanisms underlying the precise coordination of contractions in human myometrium are not completely understood. In the present study, we have characterized the spatio‐temporal properties of tissue‐level [Ca2+]i transients in thin slices of intact human myometrium. We found that the waveform of [Ca2+]i transients and isotonic contractions recorded from thin slices was similar to the waveform of isometric contractions recorded from the larger strips in traditional organ bath experiments, suggesting that the spatio‐temporal information obtained from thin slices is representative of the whole tissue. By comparing the time course of [Ca2+]i transients in individual cells to that recorded from the bundles of myocytes we found that the majority of myocytes produce rapidly propagating long‐lasting [Ca2+]i transients accompanied by contractions. We also found a small number of cells showing desynchronized [Ca2+]i oscillations that did not trigger contractions. The [Ca2+]i oscillations in these cells were insensitive to nifedipine, but readily inhibited by the T‐type Ca2+ channel inhibitor NNC55‐0396. In conclusion, our data suggest that the spread of [Ca2+]i signals in human myometrium is achieved via propagation of long‐lasting action potentials. The propagation was fast when action potentials propagated along bundles of myocytes and slower when propagating between the bundles of uterine myocytes.  相似文献   

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