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1.
Abstract— Regulations of the increase in intracellular Ca2+concentration ([Ca2+]i) and inositol 1, 4, 5-trisphosphate (IP3) production by increasing intracellular cyclic AMP (cAMP) levels or activating protein kinase C (PKC) were studied in rat frontocortical cultured neurons. Amitriptyline (AMI; 1 mM), a trìcyclic antidepressant, and bradykinin (BK; 1 μM) stimulated IP3 production and caused transient [Ca2+]i increases. Pretreatment with forskolin (100mkUM, 15 min) decreased the AMI-and BK-induced [Ca2+]i increases by 33 and 48%, respectively. However, this treatment had no effect on the AMI-and BK-induced IP3 productions. Dibutyryl-cAMP (2 mM, 15 min) also decreased the AMI-and BK-induced [Ca2+]i increases by 23 and 47%, respectively. H-8 (30 μM), an inhibitor of protein kinase A (PKA), attenuated the ability of forskolin to inhibit the AMI-and BK-induced [Ca2+]i increases, suggesting that the activation of cAMP/PKA was involved in these inhibitory effects of forskolin. On the other hand, forskolin treatment had no effect on 20 mM caffeine-, 10 μM glutamate-, or 50 mM K+-induced [Ca2+]i increases. Pretreatment with phorbol 12-myristate 13-acetate (PMA; 100 nM, 90 min) decreased both the AMI-induced [Ca2+]i increases and the IP3 production by 31 and 25%, respectively. H-7 (200 μM), an inhibitor of PKC, inhibited the ability of PMA to attenuate the [Ca2+]i increases. PMA also inhibited the BK-induced IP3 production and the [Ca2+]i increases. Taken together, these results suggest that activation of cAMP/ PKA may inhibit the IP3-mediated Ca2+ release from internal stores; on the other hand, activation of PKC may inhibit the phosphatidylinositol 4,5-bisphosphate breakdown and consequently reduce the [Ca2+]i increases or inhibit independently both responses. PKA and PKC may differently regulate the phosphatidylinositol-Ca2+ signaling in rat frontocortical cultured neurons.  相似文献   

2.
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   

3.
Abstract: Hyposmotic swelling-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca2+]i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca2+ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca2+]i elevation. Omission of external Ca2+ or addition of Cd2+, Mn2+, or Gd3+ did not reduce RVD, although it was decreased by La3+ (0.1–1 mM). Verapamil did not affect either the swelling-evoked [Ca2+]i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca2+ stores, such as treatment (in Ca2+-free medium) with 0.2 µM thapsigargin (Tg), 10 µM 2,5-di-tert-butylhydroquinone, 1 µM ionomycin, or 100 µM ATP abolished the increase in [Ca2+]i but did not affect RVD. However, prolonged exposure to 1 µM Tg blocked RVD regardless of ER Ca2+ content or cytosolic Ca2+ levels. Ryanodine (up to 100 µM) and caffeine (10 mM) did not modify [Ca2+]i or RVD. BAPTA-acetoxymethyl ester (20 µM) abolished [Ca2+]i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca2+]i rise occurs as a consequence of increased Ca2+ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.  相似文献   

4.
Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca2+ ([Ca2+]i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca2+]i was strictly dependent on extracellular Ca2+ (Ca2+e); similarly, in NG108-15 cells, a component of the elevations in [Ca2+]i was Ca2+e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca2+e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn2+. Using 19F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn2+ concentration ([Zn2+]i). In buffer containing 200 µM EGTA to prevent the Ca2+e-dependent elevations in [Ca2+]i, the [Zn2+]i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn2+]i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn2+]i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn2+]i to 3.99 ± 0.68 nM. No Zn2+ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca2+]i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca2+]i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca2+]i differed significantly from either pretreatment levels or buffer-treated controls.  相似文献   

5.
Regulation of the increases in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP3 formation and caused an initial transient peak of [Ca2+]i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP3 formation and Ca2+ mobilization. Following preincubation, carbachol-induced Ca2+ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP3 formation and increase in [Ca2+]i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The Kd and Bmax of the muscarinic receptor for [3H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.  相似文献   

6.
Abstract: Oxidative insult elicited by hydrogen peroxide (H2O2) was previously shown to increase the basal intracellular Ca2+ concentration in synaptosomes. In the present study, the effect of H2O2 on the depolarization-evoked [Ca2+] signal was investigated. Pretreatment of synaptosomes with H2O2 (0.1–1 mM) augmented the [Ca2+] rise elicited by high K+ depolarization with essentially two alterations, the sudden sharp rise of [Ca2+]i due to K+ depolarization is enhanced and, instead of a decrease to a stable plateau, a slow, steady rise of [Ca2+]i follows the peak [Ca2+]i. H2O2 in the same concentration range lowered the ATP level and the [ATP]/[ADP] ratio. When carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (1 µM) or rotenone (2 µM)/oligomycin (10 µM) was applied initially to block mitochondrial ATP production, the lowered [ATP]/[ADP] ratio was further reduced by subsequent addition of 0.5 mM H2O2. The decline of the [ATP]/[ADP] ratio was parallel with but could not explain the enhanced K+-evoked [Ca2+]i signal, indicated by experiments in which the [ATP]/[ADP] ratio was decreased by FCCP (0.1 µM) or rotenone (2 µM) to a similar value as by H2O2 without causing any alteration in the [Ca2+]i signal. These results indicate that H2O2-evoked oxidative stress, in its early phase, gives rise to a complex dysfunction in the Ca2+ homeostasis and, parallel with it, to an impaired energy status.  相似文献   

7.
《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   

8.
Abstract: The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an “early” Na+/Cl?-dependent component observed within minutes of glutamate exposure, and a “delayed” Ca2+-dependent component (ED50~50 µM) that coincided with progressive degeneration of granule cells 4–24 h after a brief (5–15 min) exposure to 100 µM glutamate. Quantitative analysis of cell viability and morphological observations identify a “window” in which TMB-8 (at >100 µM) protects granule cells from the Ca2+-dependent, but not the Na+/Cl?-dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity.  相似文献   

9.
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   

10.
Abstract: High concentrations of Zn2+ are found in presynaptic terminals of excitatory neurons in the CNS. Zn2+ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn2+ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn2+ concentration ([Zn2+]i). We used fura-2 and magfura-2 to detect the consequences of Zn2+ influx in cultured neurons under conditions that restrict changes in intracellular Ca2+ and Mg2+ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn2+ chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn2+]i. We found that fura-2 was useful in measuring small increases in [Zn2+]i associated with exposure to Zn2+ alone that may be mediated by a Na+/Ca2+ exchanger. Magfura-2, which has a lower affinity for Zn2+, was more useful in measuring larger agonist-stimulated increases in [Zn2+]i. The coapplication of 300 µM Zn2+ and 100 µM glutamate/10 µM glycine resulted in a [Zn2+]i increase that was ~40–100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK-801 (30 µM), or extracellular Na+. This suggests that Zn2+ influx can occur through at least two different pathways, leading to varying increases in [Zn2+]i. These findings demonstrate the feasibility of measuring changes in [Zn2+]i in neurons.  相似文献   

11.
Abstract: It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK2 receptors with features that differ from those of NK2 receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK2 and NK3 receptors, but not of NK1 receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca2+]i) induced by 0.33 µM neurokinin A was completely inhibited by SR 48968, an NK2 receptor antagonist, whereas the partial response to 0.33 µM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, an NK3 receptor antagonist. In addition, the [Ca2+]i increase by 0.33 µM senktide, an NK3 receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK2 and NK3 receptors. It was also demonstrated that NK2 and NK3 receptors can be activated independently by 3.3 µM neurokinin A in the presence of 1.0 µM SR 142801 or 1.0 µM senktide, respectively. Therefore, the mechanisms of Ca2+ signaling mediated by endogenous NK2 and NK3 receptors were investigated. The independent activation of NK2 or NK3 receptors induced not only the [Ca2+]i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK2 and NK3 receptor-mediated [Ca2+]i increase was partially attenuated in the absence of extracellular Ca2+ or in the presence of nickel, an inorganic Ca2+ influx blocker, but was unaffected by nifedipine and ω-conotoxin, L- and N-type voltage-dependent Ca2+ channel blockers, respectively. Furthermore, the depolarization by 60 mM K+ did not affect the [Ca2+]i. These findings suggested that the NK2 and NK3 receptor-mediated [Ca2+]i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca2+ and the entry of extracellular Ca2+ through voltage-independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK2 and NK3 receptors.  相似文献   

12.
Abstract: The effect of replacement of extracellular Na+ with N-methyl-d -glucamine (NMG) on P2 receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 value of 230 µM. Replacement of Na+ with NMG as well as removal of Mg2+ from the bathing buffer potentiated ethidium bromide uptake, [Ca2+]i increase, and 45Ca2+ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg2+ to limit the amount of ATP4?, replacement of Na+ with NMG had no effect on the ATP-induced [Ca2+]i increase but caused a markedly larger [Ca2+]i increase when the calculated concentration of ATP4? was >10 µM. The calculated EC50 value for ATP4? stimulation of the [Ca2+]i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca2+ release was the major pathway for the ATP-induced [Ca2+]i increase; both removal of Mg2+ and replacement of Na+ with NMG did not affect the action of ATP. These data suggest that ATP4?-promoted pores are antagonized by Na+ and Mg2+ in dibutyryl cyclic AMP-differentiated NG108-15 cells.  相似文献   

13.
Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca2+]e) with increases in intracellular calcium concentration ([Ca2+]i) that include single-cell Ca2+ oscillations and propagated intercellular Ca2+ waves. The rate and pattern of propagation of low [Ca2+]e-induced intercellular Ca2+ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca2+ wave propagation. The low [Ca2+]e-induced Ca2+ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca2+]e-induced response is also blocked by replacement of extracellular Ca2+ with Ba2+, Zn2+, or Ni2+, and by 100 µM La3+. Glial cells in lowered [Ca2+]e(0.1–0.5 mM) show an increased [Ca2+]i response to bath application of ATP, whereas glial cells in increased [Ca2+]e (10–15 mM) show a decreased [Ca2+]i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca2+]e and the release of Ca2+ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca2+ such as seizures or ischemia.  相似文献   

14.
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   

15.
Stimulation of A2A receptors (A2A R) coupled to Gs/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A2A R in the modulation of free cytosolic Ca2+ concentration ([Ca2+]i) involving IP3, cAMP and PKA was investigated in HEK 293-A2A R. The levels of IP3 and cAMP were observed by enzyme immunoassay detection method and [Ca2+]i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A2A R agonist NECA showed increased levels of cAMP, PKA, IP3 and [Ca2+]i levels. However, the reverse effect was observed with A2A R antagonists (ZM241385 and caffeine). Blocking the Gαq/PLC/DAG/IP3 pathway with neomycin, a PLC inhibitor did not affect the modulation of IP3 and [Ca2+]i levels in HEK 293-A2A R cells. To investigate the Gαi/AC/cAMP/PKA, HEK 293-A2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A2A R agonist (NECA) showed no effect on cAMP levels. Further, Gαs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP3 mediated [Ca2+]i modulation. In the HEK 293-A2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP3 and [Ca2+]i levels. The study distinctly demonstrated that A2A R modulates IP3 levels to release the [Ca2+]i via cAMP-dependent PKA. The role of A2A R mediated Gαs pathway inducing IP3 mediated [Ca2+]i release may open new avenues in the therapy of neurodegenerative disorder.  相似文献   

16.
Abstract: The relationship between elevations in intracellular free Ca2+ concentration ([Ca2+]i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K+ concentration triggered rapid and sustained increases in [Ca2+]i from a basal level of ~50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP3). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP3 or inhibition of Ca2+-ATPase, rapidly elevated [Ca2+]i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca2+]i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca2+]i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca2+]i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca2+]i, there are important differences among them in terms of the magnitude of elevated [Ca2+]i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca2+]i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.  相似文献   

17.
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   

18.
Abstract: We found that extracellular ATP can increase the intracellular Ca2+ concentration ([Ca2+]i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca2+]i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca2+]i rise. Raising the extracellular Mg2+ concentration to 10 mM decreases the ATP-and UTP-induced [Ca2+]i rise, because the responses depend on the ATP4? and UTP4? concentrations, respectively. The P2U purinoceptor-selective agonist UTP and the P2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ~100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca2+]i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca2+]i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P2U and P2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.  相似文献   

19.
The effect of the natural product diindolylmethane on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells was explored. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Diindolylmethane at concentrations of 20–50 µM induced [Ca2+]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca2+. Diindolylmethane-evoked Ca2+ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca2+, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca2+]i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca2+]i rise. At concentrations of 50–100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca2+]i rise in PC3 cells by evoking phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via phospholipase A2-sensitive store-operated Ca2+ channels. Diindolylmethane caused cell death in which apoptosis may participate.  相似文献   

20.
Abstract: The direct effect of melatonin and related agonists on Li+-amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo-[2-3H]-inositol. The melatonin receptor agonist 6-chloromelatonin (10–100 µM) increased, in a concentration-dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6-chloromelatonin (10 µM) was rapid as transient increases in IP3/IP4 (maximal increase, 29% at 20 s) and IP2 levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP1 level (30% at 5 min), when the amount of IP3/IP4 and IP2 had already been decreased to the control level. The phosphoinositide response elicited by 6-chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6-chloromelatonin (10 µM) in isolated myo-[2-3H]inositol-prelabeled optic tectum membranes was dependent on the presence of guanosine-5′-O-(3-thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration-dependent manner the IP1 accumulation stimulated by 6-chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5-hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5-hydroxytryptamine (10 µM)-, but not 6-chloromelatonin (10 µM)-, induced IP1 accumulation. Melatonin receptor agonists increased IP1 accumulation in a concentration-dependent manner reaching different maximal responses. N-Acetyl-5-hydroxytryptamine was more potent than melatonin in increasing IP1 accumulation, suggesting activation of a melatonin receptor site other than the ML-1 melatonin receptor (i.e., N-acetyl-5-hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML-1 subtype leads to activation of the phospholipase C-mediated signal transduction pathway.  相似文献   

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