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1.
Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca2+]i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and 100.0 μM ATP) induced a rise in the [Ca2+]i. Carbachol evoked an increase in the [Ca2+]i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca2+]i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca2+]i in myocytes. The absence of the slow component in the [Ca2+]i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca2+ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca2+]i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises in the [Ca2+]i induced by both CCh and ATP; therefore, the release of Ca2+ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca2+ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006.  相似文献   

2.
Abstract: Rilmenidine, a ligand for imidazoline and α2-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca2+ concentration ([Ca2+]i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca2+]i, consisting of a transient increase (1–100 µM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1–10 mM rilmenidine). A similar elevation in [Ca2+]i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca2+-free medium, indicating that rilmenidine evokes release of Ca2+ from intracellular stores. However, the sustained elevation of Ca2+ was completely dependent on extracellular Ca2+, consistent with rilmenidine activating Ca2+ influx.Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca2+ influx pathway. The α2-adrenergic antagonist rauwolscine attenuated the increase in [Ca2+]i induced by clonidine (a selective α2 agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca2+ release from intracellular stores and Ca2+ influx by a mechanism independent of α2-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca2+ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.  相似文献   

3.
Abstract: The mechanisms involved in Ca2+ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca2+ concentration ([Ca2+]i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca2+]i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca2+]i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca2+]i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP3) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca2+ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca2+]i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP3 receptors, appear to be crucial to the mAChR-mediated [Ca2+]i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP3-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP3 receptors may not contribute significantly to NMDA-evoked elevation of [Ca2+]i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca2+ stores is proposed.  相似文献   

4.
The mechanism underlying the generation of cytosolic free Ca2+ ([Ca2+i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca2+]i oscillations with variable patterns. The bombesin-induced [Ca2+]i oscillations could last more than 1 h and glucose was required for maintaining these [Ca2+ fluctuations. Bombesin-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+ entry and were attenuated by membrane hype rpolarization or by L-type Ca2+ channel blockers. These [Ca2+]i oscillations were apparently not associated with fluctuations in plasma membrane Ca2+ permeability as monitored by the Mn2+ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca2+ stores, respectively, by inhibiting Ca2+-ATPase of endoplasmic reticulum and by affecting Ca2+-induced Ca2+ release, disrupted bombesin-induced [Ca2+]i oscillations. 4-chloro-m-resol raised [Ca2+]i by mobilizing an intracellular Ca2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca2+]i It raised [Ca2+]i by promoting Ca2+ entry while inhibiting bombesin-elicited [Ca2+]i oscillations. Our results suggest that in bombesin-elicited [Ca2+]i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca2+ stores; and (ii) the Ca2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca2+]i oscillations. The interplay between intracellular Ca2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca2+ entry determines the [Ca2+]i oscillations evoked by bombesin.  相似文献   

5.
Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca2+ sources: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) Intracellular stores of Ca2+ were depleted by soaking cells in Ca2+-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca2+]i and secretion in cells with or without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca2+ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca2+]i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ~20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca2+]i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland.  相似文献   

6.
Measurements of Ca2+ influx and [Ca2+]i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca2+ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca2+]i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca2+]i levels were dependent on extracellular Ca2+. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca2+ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca2+ channels. The trivalent cation La3+, a non-specific blocker of plasma membrane Ca2+ channels, eliminated the rPTTH-stimulated increase of [Ca2+]i levels in PG cells and so did amiloride, an inhibitor of T-type Ca2+ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca2+]i levels, which was also dependent on extracellular Ca2+ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca2+ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca2+]i levels and one agent’s mediated increase of [Ca2+]i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca2+ release from inositol 1,4,5 trisphosphate (IP3)-sensitive Ca2+ stores, blocked the rPTTH-stimulated increases of [Ca2+]i levels, suggesting an involvement of IP3 in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca2+]i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca2+]i levels, filling state of IP3-sensitive intracellular Ca2+ stores and the PTTH-receptor’s-mediated Ca2+ influx.  相似文献   

7.
A rise in the intracellular concentration of ionized calcium ([Ca2+]i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca2+ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca2+ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca2+]i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP3Rs) is an important determinant of the [Ca2+]i dynamics and recruits neighboring Ca2+-release sites to generate [Ca2+]i waves; (v) [Ca2+]i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca2+-dependent K+ and Cl- channels sense the local changes in [Ca2+]i during a Ca2+ spark and thereby may couple changes in [Ca2+]i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI cat) does not mirror changes in [Ca2+]i, thus reflecting the complexity of [Ca2+]i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca2+ release, either spontaneous or caffeine-induced, does not augment mI cat; (ix) intracellular flash release of Ca2+ is less effective in augmentation of mI cat than flash release of IP3, suggesting that IP3 may sensitize muscarinic cationic channels to Ca2+; (x) intracellular flash release of IP3 fails to augment mI cat in SMCs, in which [Ca2+]i was strongly buffered, suggesting that IP3 exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca2+]i rather than depletion of the IP3-dependent Ca2+ store; and (xi) predominant expression of IP3R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP3R-mediated Ca2+ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.  相似文献   

8.
《Cell calcium》1996,20(3):303-314
In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca2+]i) followed by a sustained rise. In Ca2+-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca2+]i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca2+]i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca2+]i and inhibited the following [Ca2+]i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca2+]i. Muscarine-induced sustained [Ca2+]i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca2+]i rise and Mn2+ influx in response to muscarine were suppressed by a voltage-dependent Ca2+ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca2+ entry, but also Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca2+ channels may function as one of the Ca2+ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.  相似文献   

9.
Extracellular nicotinamide adenine dinucleotide (NAD+) is known to increase the intracellular calcium concentration [Ca2+]i in different cell types and by various mechanisms. Here we show that NAD+ triggers a transient rise in [Ca2+]i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca2+ from IP3-responsive intracellular stores and an influx of extracellular Ca2+. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD+-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD+. The identification of P2Y1 and P2Y11 as receptor subtypes responsible for the NAD+-triggered increase in [Ca2+]i was supported by several lines of evidence. First, specific P2Y1 and P2Y11 receptor antagonists inhibited the NAD+-induced increase in [Ca2+]i. Second, NAD+ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD+ caused an increase in [cAMP]i, prevented by the P2Y11 receptor-specific antagonist NF157.  相似文献   

10.
Summary Electrical uncoupling of crayfish septate axons with acidification has been shown to cause a substantial increase in [Ca2+]i which closely matches in percent the increase in junctional resistance. To determine the origin of [Ca2+]i increase, septate axons have been exposed either to drugs that influence Ca2+ release from internal stores, caffeine and ryanodine, or to treatments that affect Ca2+ entry. A large increase in junctional resistance and [Ca2+]i maxima above controls resulted from addition of caffeine (10–30mm) to acetate solutions, while a substantial decrease in both parameters was observed when exposure to acetate-caffeine was preceded by caffeine pretreatment. In contrast, ryanodine (1–10 m) always caused a significant decrease in junctional resistance and [Ca2+]i maxima when applied either together with acetate or both before and with acetate. Calcium channel blockers such as La3+, Cd2+ and nisoldipine had no effect, while an increase in the [Ca2+] of acetate solutions either decreased junctional resistance and [Ca2+]i maxima or had no effect. The data suggest that cytoplasmic acidification causes an increase in [Ca2+]i by releasing Ca2+ from caffeine and ryanodine-sensitive Ca2+ stores. The increase in [Ca2+]i results in a decrease in gap junction conductance.  相似文献   

11.
Intracellular calcium, [Ca2+]i, can regulate meiotic progression of mammalian oocytes. However, the role of [Ca2+]i in the regulation of the spermatogenic process and its cellular homeostatic mechanisms in spermatogenic cells has not been elucidated. Using intracellular fluorescent probes for Ca2+ and immunodetection of plasma membrane (PM) Ca2+-ATPases, we report that: a) rat round spermatids maintain [Ca2+]i levels of 60 ± 5 nM (SEM), as estimated with fluo-3 in single cells or fura-2 in cells in suspension; b) these cells regulate [Ca2+]i by actively extruding it using a PM Ca2+-ATPase; c) rat spermatids also actively transport Ca2+ by sarco-endoplasmic reticulum type ATPases (SERCA); d) rat spermatids possess non-mitochondrial intracellular Ca2+i stores insensitive to thapsigargin but releasable by ionomycin; and e) rat spermatids do not activate Ca2+ entry mechanisms by the release of Ca2+ from SERCA-regulated stores. These results demonstrate that rat round spermatids can generate modulated intracellular Ca2+ signals upon activation of Ca2+ channels or Ca2+ release from intracellular stores.  相似文献   

12.
The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+] i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+] i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+] i transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mm Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 μm ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+] i transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+] i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997  相似文献   

13.
Abstract: The ability of antidepressant drugs (ADs) to increase the concentration of intracellular Ca2+ ([Ca2+]i) was examined in primary cultured neurons from rat frontal cortices using the Ca2+-sensitive fluorescent indicator fura-2. Amitriptyline, imipramine, desipramine, and mianserin elicited transient increases in [Ca2+]i in a concentration-dependent manner (100 μM to 1 mM). These four AD-induced [Ca2+]i increases were not altered by the absence of external Ca2+ or by the presence of La3+ (30 μM), suggesting that these ADs provoked intracellular Ca2+ mobilization rather than Ca2+ influx. All four ADs increased inositol 1,4,5-trisphosphate (IP3) contents by 20–60% in the cultured cells. The potency of the IP3 production by these ADs closely correlated with the AD-induced [Ca2+]i responses. Pretreatment with neomycin, an inhibitor of IP3 generation, significantly inhibited amitriptyline- and imipramine-induced [Ca2+]i increases. In addition, by initially perfusing with bradykinin (10 μM) or acetylcholine (10 μM), which can stimulate the IP3 generation and mobilize the intracellular Ca2+, the amitriptyline responses were decreased by 76% and 69%, respectively. The amitriptyline-induced [Ca2+]i increases were unaffected by treatment with pertussis toxin. We conclude that high concentrations of amitriptyline and three other ADs mobilize Ca2+ from IP3-sensitive Ca2+ stores and that the responses are pertussis toxin-insensitive. However, it seems unlikely that the effects requiring high concentrations of ADs are related to the therapeutic action.  相似文献   

14.
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   

15.
Abstract: Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP3) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca2+ ([Ca2+]i) with an EC50 value of 35 µM. In addition, carbachol produced a 1.3–3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E2 (PGE2). The M3 antagonist p-fluorohexahydrosiladifenidol (IC50 = 0.5–0.8 µM) inhibited the increases in [Ca2+]i, IP3, and cAMP more effectively than the M1 antagonist pirenzepine (IC50 = 5–9 µM) and the M2 antagonist methoctramine (IC50 = 20–30 µM). The involvements of [Ca2+]i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 µM) inhibited significantly the synergistic effects of carbachol and PGE2 on the production of cAMP, whereas the Ca2+ ionophore ionomycin (1 µM) clearly enhanced PGE2-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE2-stimulated cAMP production. These data suggest that phospholipase C-linked M3 receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca2+-dependent system in the neuronal cells.  相似文献   

16.
Li B  Dong L  Fu H  Wang B  Hertz L  Peng L 《Cell calcium》2011,50(1):42-53
Primary cultures of mouse astrocytes were used to investigate effects by chronic treatment (3-21 days) with fluoxetine (0.5-10 μM) on capacitative Ca2+ influx after treatment with the SERCA inhibitor thapsigargin and on receptor agonist-induced increases in free cytosolic Ca2+ concentration [Ca2+]i, determined with Fura-2. The agonists were the 5-HT2B agonist fluoxetine, the α2-adrenergic agonist dexmedetomidine, and ryanodine receptor (RyR) and IP3 receptor (IP3R) agonists. In untreated sister cultures each agonist distinctly increased [Ca2+]i, but in cultures treated for sufficient length of time or with sufficiently high doses of fluoxetine, acute administration of fluoxetine, dexmedetomidine, or RyR or IP3R agonists elicited reduced, in some cases abolished, effects. Capacitative Ca2+ entry, meditated by TRPC1 channels, was sufficiently inhibited to cause a depletion of Ca2+ stores, which could explain the reduced agonist effects. All effects of chronic fluoxetine administration could be replicated by TRPC1 channel antibody or siRNA. Since increases in astrocytic [Ca2+]i regulate release of gliotransmitters, these effects may have profound effects on brain function. They may be important for therapeutic effects of all 5 conventional ‘serotonin-specific reuptake inhibitors’ (SSRIs), which at concentrations used therapeutically (∼1 μM) share other of fluoxetine's chronic effects (Zhang et al., Neuron Glia Biol. 16 (2010) 1-13).  相似文献   

17.
《Life sciences》1994,56(5):PL103-PL108
We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca2+]i, and IP3 in β-TC3 cells. Carbachol (0.1–100 μM) increased [Ca2+]i and carbachol (0.1–1000 μM) increased insulin release dose-dependently. Carbachol (100 μM) also increased inositol 1,4,5-trisphosphate (IP3) production. U-73122 (2–12 νM) inhibited the effects of carbachol on [Ca2+]i and insulin release in a dose-dependent manner, and at the highest dose studied (12 μM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 μM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca2+]i by 20% and did not inhibit the effect of carbachol on insulin release. Since carbachol increased IP3, [Ca2+]i, and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-depenent processes in β-TC3 cells.  相似文献   

18.
Changes in cytosolic Ca2+ concentration ([Ca2+]i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca2+, the increase in [Ca2+]i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca2+ wave toward the basolateral region. The [Ca2+]i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca2+ was eliminated from the extracellular medium, the Ca2+ wave was a result of Ca2+ release from intracellular stores. The magnitude and velocity of the Ca2+ wave decreased with decreasing concentration of CCh, and the increase in [Ca2+]i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca2+ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca2+ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca2+ pump, caused a slow and homogeneous increase in [Ca2+]i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca2+]i suggesting that the inositol-1,4,5-trisphosphate (InsP3) and ThG-sensitive Ca2+ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP3 is essential to trigger the Ca2+ wave and that the development of the Ca2+ wave may be attributed to regional differences in InsP3 sensitivity of Ca2+ stores. The agonist-induced Ca2+ wave is probably a general phenomenon in exocrine acinar cells.  相似文献   

19.
Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca2+ indicator dye Fura-2 was used to measure Ca2+ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca2+ responses were evoked by kainic acid (KA), α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D -aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca2+]i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca2+ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca2+]i were carried by Ca2+-permeable receptor channels. Significantly smaller changes in [Ca2+]i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca2+ free-EGTA–buffered media, including l -cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclopentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca2+]i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca2+]i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca2+ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca2+]i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca2+]i to increase in NM neurons. Presumably, each route represents a means by which Ca2+ can alter cellular processes. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 321–337, 1998  相似文献   

20.
Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated with such transport has been relatively tedious, even though significant progress has been made in the characterization of the intracellular trafficking of lipid/DNA complexes. Surprisingly little is known about the effects of these delivery vectors on cell functioning. In this report, we show that both cationic lipids and cationic lipid/DNA complexes mobilize the intracellular calcium. Removal of extracellular calcium did not significantly abolish this effect and preincubating cells with thapsigargin led to a decrease in [Ca2+]i, indicating that calcium was released mainly from internal calcium stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122, blocked the [Ca2+]i rise, suggesting an inositol dependent mechanism.  相似文献   

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