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1.
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   

2.
To study changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular solution decreased. The action of ATP induced a dose-dependent increase in the [Ca2+]i; the EC50 was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results in a rise in the [Ca2+]i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the [Ca2+]i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in the control of functioning of this gland. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005.  相似文献   

3.
Experiments were carried out on isolated neurons of the thalamic nucleus lateralis dorsalis (LD) from 12-day-old rats. According to the morphological characteristics, LD neurons were classified as relay thalamo-cortical units and interneurons. The concentration of free Ca2+ ions in the cytoplasm ([Ca2+] i ) was measured by a fluorescent calcium indicator, fura-2AM. Application of 30 mM caffeine caused a transient change in the [Ca2+] i in 8 of 15 and in 6 of 11 of the thalamo-cortical units and interneurons under study, respectively. After stimulation of a cell with application of 50 mM KCl, a caffeine-induced increase in the [Ca2+] i was observed in all tested neurons. To study the contribution of Ca2+-induced Ca2+ release (CICR) to the calcium transient evoked by depolarization of the neuronal membrane, caffeine in a subthreshold concentration was pre-applied. After 50 mM KCl had been added to the medium following pre-application of 0.5 mM caffeine, the calcium transient amplitude in thalamo-cortical neurons increased by 51 ± 7% (n = 16). In interneurons this effect was not observed (n = 11). The data obtained allow us to hypothesize that CICR contributes to the depolarization-evoked calcium transient only in the relay (thalamo-cortical) neurons. Differences in the pattern of calcium signalling, which were detected in two types of neurons of the thalamic LD, can be a factor determining distinctions in the physiological characteristics of these neurons.  相似文献   

4.
Peptidesecreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca2+ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium ([Ca2+]i) have been implicated in regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal [Ca2+]i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological tapes can extend out processes over a [Ca2+]i range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high k+ saline led to increases in [Ca2+]i in soma, neurite, and lamellipodium of veiling neurons. Increase were great for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb [Ca2+]i were used to assess the role of [Ca2+]i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested addition of 100 μMCD2+, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd2+. Branching neurons were unaffected by Cd2+. Cd2+ at lower levels (10 μM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 μM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal [Ca2+]i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing [Ca2+]i with respect to regenerative outgrowth, but not its form. 1994 John Wiley & Sons, Inc.  相似文献   

5.
The ryanodine-sensitive intracellular Ca2+ stores are known to play a major role in excitation-contraction coupling in muscles. Although these stores are also abundantly present in central neurons, their functional role in these cells remains unclear. Using fluorometric digital imaging of the intracellular Ca2+ concentration ([Ca2+] i ) in rat hippocampal slices, we investigated the dynamic properties of the ryanodine-sensitive Ca2+ stores inCA1 hippocampal pyramidal cells. We found that at rest the ryanodine-sensitive Ca2+ stores are functioning predominantly as a “sink” for Ca ions responding to an increase in [Ca2+] i with an increase in the amount of Ca ions accumulated inside the stores. If, however, [Ca2+] i increases significantly, as happens during strong neuronal discharges, the ryanodine-sensitive Ca2+ stores respond with Ca2+ release, thus acting as an amplifier of the intracellular Ca2+ signal.  相似文献   

6.
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   

7.
Abstract

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by high-K+ (30?mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1?µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p?<?0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300?µM) inhibited the Ca2+ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca2+]i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca2+]i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca2+]i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.  相似文献   

8.
This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca2+]i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl3). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF- 4), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca2+ resulted in a transient rise in [Ca2+]i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca2+]i . These results suggest that dephostatin can mobilize Ca2+ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.  相似文献   

9.
The dynamics of intracellular Ca2+ signal in response to NMDA (N-methyl-D-aspartate, 30 μM) or KA (kainite, 30 μM), its dependence on extracellular Ca2+ and the mechanisms of KA-triggered Ca2+ entry into neurons have been tested in neurons of rat cortical primary cultures. The level of intracellular free Ca2+ concentrations ([Ca2+] i ) was evaluated on Leica SP5 MF confocal microscope using Fluo-3 fluorescent dye, which resolves changes in [Ca2+] i in the micromolar range. The dynamics of [Ca2+] i increase in response to NMDA and KA was different but in both cases the [Ca2+] i increase required the presence of Ca2+ in the extracellular solution. The neuronal population was found to be heterogeneous, based on the response to KA applied together with either L-type calcium channel blocker nifedipine (3 μM) or IEM-1460 (3 μM), a blocker of Ca2+-permeable AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) lacking GluR2 subunit. Experiments exhibited three types of calcium responses, characteristically belonging to interneurons (expressing Ca2+-permeable AMPAR), pyramidal neurons (with AMPAR containing GluR2, making them impermeable to Ca2+), and intermediate type of cells expressing both AMPAR types. Thus, we have demonstrated the role of AMPAR and L-type calcium channels in KA-triggered Ca2+ entry into neurons. The dynamics of [Ca2+] i during the KA treatment was shown to depend on subunit composition of particular AMPAR subtype expressed in neurons. The data suggest that neuronal types existing in adult cortical tissue are probably presented in primary culture, too.  相似文献   

10.
11.
Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca2+]i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca2+]i transients for stimulation with 30?mM KCl (Hi K+) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca2+]i transients in a reversible manner. [Ca2+]i increase evoked by Hi K+ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K+-induced [Ca2+]i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K+-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.  相似文献   

12.
Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca2+] in ), the properties of caffeine-induced Ca2+ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca2+ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca2+] in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca2+; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La3+-sensitive plasmalemmal Ca2+-ATPases. Caffeine-induced Ca2+ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca2+] in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca2+] in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca2+-induced Ca2+ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca2+] in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca2+ content of internal stores was low, but the stores could be charged by transmembrane Ca2+ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca2+ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca2+ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca2+] in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba2+, thus sharing the basic properties of the Ca2+-induced Ca2+ release from endoplasmic reticulum.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 16–25, January–February, 1994.  相似文献   

13.
We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca2+] i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca2+] i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca2+] i increase was reduced by 50% in the absence of extracellular Ca2+, while the prolonged [Ca2+] i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca2+] i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca2+] i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca2+] i transient peak. [Ca2+] i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca2+-free solution, or when Ca2+ stores were depleted by thapsigargin in Ca2+-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca2+] i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca2+ store. Received: 24 March 1997/Revised: 21 July 1997  相似文献   

14.
In the sensory ganglia, neurons are devoid of synaptic contacts, and ganglion neurons surrounded by one of glial cells, satellite cells. Recent studies suggest that neurons and satellite cells interact through neurotransmitters. In the present study, intracellular Ca2+ ([Ca2+]i) dynamics of neurons and satellite cells from one of viscerosensory ganglia, nodose ganglion (NG), were investigated by stimulation with glutamate and its agonist and/or the antagonist of the GABAA receptor bicuculline. In the specimens containing neurons with satellite cells, glutamate and a metabotropic glutamate receptor (mGluR) agonist t-ACPD evoked [Ca2+]i increases in both neurons and surrounding satellite cells. Moreover, bicuculline also induced [Ca2+]i increases in neurons and satellite cells. However, in the isolated neurons, bicuculline did not cause an increase in [Ca2+]i, suggesting that satellite cells are equipped with the ability to release GABA. In the neurons associated with satellite cells, the delay time until the onset of a response was shorter in the case of glutamate stimulation with bicuculline than that without bicuculline (107.3 ± 93.4 vs. 231.8 ± 97.0 s, p < 0.01). Furthermore, immunoreactivities for glutamate transporter, GLAST, and GABA transporter, GAT-3, were observed in both neurons and satellite cells of NG. In conclusion, the levels of [Ca2+]i of NG neurons and surrounding satellite cells are increased by glutamate through at least mGluRs, and endogenous GABA modulates these responses; GABA inhibition is dependent on a close association between neurons and satellite cells. Such neuron–glia interaction in the nodose ganglion may regulate sensory information from visceral organs.  相似文献   

15.
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   

16.
To gain a better understanding of Ca2+-induced Ca2+ release in central neurons, we have studied the increase in intracellular Ca2+ concentration ([Ca2+]i) induced by application of caffeine to cells cultured from embryonic mouse telencephalon (hippocampus or cortex). The magnitudes and distributions of changes in [Ca2+]i in neuron somata were measured by quantitative video microscopy. We observed that application of caffeine to pyramidally shaped neurons typically initiated an increase in [Ca2+]i in the cytoplasmic region between the nucleus and the base of a major dendrite. [Ca2+] in this region increased over a period of 3 to 6 s and was followed by, with a slight delay, a surge of Ca2+ that moved across the soma and into or over the nucleus. Similar Ca2+ that moved across the soma and into or over the nucleus. Similar Ca2+ responses to caffeine were observed in Ca2+-containing and nominally Ca2+-free external solutions, suggesting that caffeine was inducing Ca2+ release from intracellular stores. Ca2+ responses to caffeine were potentiated by inducing a tonic Ca2+ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors activated by 0.3 μM glutamate and multiple responses to caffeine could be elicited by using this Ca2+ influx to refill the intracellular stores. Ryanodine inhibition of caffeine-induced Ca2+ release was use- and concentration-dependent; the median effective concentration EC50 for ryanodine declined from 22 μM for the first application of caffeine to 20 nM for the fourth. We conclude, based on these responses to caffeine, that ryanodine-sensitive mechanisms of intracellular Ca2+ release are active in hippocampal and cortical neurons and may be involved in generation of directed Ca2+ waves that engulf the nucleus. © 1995 John Wiley & Sons, Inc.  相似文献   

17.
The roles of Ca2+ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca2+]i) that declined to a steady-state level. The peak increase in [Ca2+]i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca2+]i did not. Removal of extracellular Ca2+ had little effect on peak [Ca2+]i but greatly reduced steady-state increases in [Ca2+]i and tension. Verapamil inhibited steady-state [Ca2+]i only at [ACh]<1 µM. After depletion of internal Ca2+ stores by 10 min exposure to ACh in Ca2+-free solution and then washout of ACh for 5 min in Ca2+-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca2+ increased [Ca2+]i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca2+]i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca2+]i is necessary for tension maintenance and is dependent on Ca2+ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.  相似文献   

18.
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   

19.
An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca 2+]i). This study analyzed the [Ca 2+]i wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca 2+]i wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca 2+]i wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca 2+]i wave area in the first bout it was likely to have a large [Ca 2+]i wave area in the second bout (r 2 = 0.933). These findings support the idea that individual bone cells have “calcium fingerprints” (i.e., a unique [Ca 2+]i wave profile that is reproducible for repeated exposure to a given stimulus).  相似文献   

20.
Bovine trophoblasts actively proliferate to elongate blastocysts before implantation. The trophoblast at this stage secretes cytokines and starts to differentiate into an endocrine cell (binucleate cell) for successful pregnancy. Intracellular calcium ([Ca2+]i) may act as a second messenger in the trophoblast response. In this study, we investigated [Ca2+]i signals in a bovine trophoblast cell line (BT-1) using fura-2 fluorescence. We found that an application of ATP (1 M) induced a transient increase in [Ca2+]i in BT-1 cells. The ATP-induced increase was not affected by the removal of extracellular Ca2+, but was suppressed by suramin (100 M), an antagonist of P2 receptors. Pretreatment with pertussis toxin (0.1 or 1 g/ml) partially inhibited the response to ATP. The order of potency to increase [Ca2+]i was ATP=UTP>ADP. ATP-induced [Ca2+]i responses preferentially occurred in cells at the periphery of the colony. The reduced responses at the center of the colony were associated with an increase in cell density and decrease in bromodeoxyuridine incorporation. These results indicated that ATP stimulated P2Y receptors coupled to pertussis toxin-sensitive and -insensitive G proteins, leading to an increase in [Ca2+]i as a result of release of Ca2+ from intracellular stores in BT-1 cells. The occurrence of ATP-induced [Ca2+]i signals depended on the cell confluence and reflected the high proliferative activity of the trophoblast cell population.This work was supported by grants from the Bio-oriented Technology Research Advancement Institution (BRAIN), and the Organized Research Combination System in the Science and Technology Agency of Japan. H.N. is a domestic research fellow supported by Japan Society for the Promotion of Science. A.S. is supported by a post-doctoral fellowship from the Japan Science and Technology Corporation.  相似文献   

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