Muscarinic acetylcholine responses in the early embryonic chic retina

The action of acetylcholine on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in early embryonic chick retinae. Whole neural retinae were isolated from embryonic day 3 (E3) chicks and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). Increases in [Ca²⁺]_i were evoked by the puff application of acetylcholine at concentration than 0.1 μM. The Ca²⁺ response became larger in dose–dependant manner up to 10 μM of acetylcholine applied. The rise in [Ca²⁺]_i was not due to the influx of Ca⁺² through calcium channels, but to the release of Ca²⁺ from internal stores. A calcium channel antagonist, nifedipine, which completely blocks the Ca²⁺ rise caused by depolarization with 100 mM K⁺, had no effects on the acetylcholine response and the Ca²⁺ response to acetylcholine occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolished the response to 10 μM acetylcholine, whereas d-tubocurarine of 100 μM had no effects. Two muscarinic agonists, muscarine and carbamylcholine (100 μM each), evoked comparable responses with that to 10 μM acetylcholine. The developmental change of the muscarinic response was examined from E3 to E13. The Ca2+ response to 100 μM carbamylcholine was intense at E3-E5, then rapidly declined until E8. The muscarinic Ca²⁺ mobilization we found in the early embryonic chick retina may be regarded as a part of the “embryonic muscarinic system” proposed by Drew's group, which appears transiently and ubiquitously at early embryonic stages in relation to organogenesis. 1994 John Wiley & Sons, Inc.     

Lysophosphatidic acid‐induced Ca2+ mobilization in the neural retina of chick embryo

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca²⁺ concentration ([Ca²⁺]_i) with Fura‐2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1–100 μM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca²⁺]_i in a dose‐dependent manner, with an EC₅₀ value of 9.2 μM. The Ca²⁺ rise was also evoked in a Ca²⁺‐free medium, suggesting that release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) was induced by LPA. U‐73122, a blocker of phospholipase C (PLC), inhibited the Ca²⁺ rise to LPA. Pertussis toxin partially inhibited the Ca²⁺ rise to LPA, indicating that G_i/G_o protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca²⁺ rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca²⁺ response to LPA were the same as those of the Ca²⁺ response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca²⁺ rise in the E3 chick retina. Our results show that LPA induces Ca²⁺ mobilization in the embryonic chick retina during neurogenesis. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 495–504, 1999     

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

Abstract— ATP-induced changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca²⁺indicator fura-2, we have shown that the [Ca²⁺]_i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Ca^z+]_i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca²⁺]_i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca²⁺]_i at 3 mM. In the absence of extracellular Ca²⁺, the effect of ATP was inhibited totally, but could be restored by the addition of Ca²⁺ to the cells. Upon removal of Mg²⁺, the maximum increase in [Ca²⁺]_i induced by ATP was enhanced by about 42%. Ca²⁺-channel blockers partially inhibited the ATP-induced [Ca²⁺]_i rise. The ATP-induced [Ca²⁺]_i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca²⁺]_i rise completely. No heterologous desensitization of [Ca²⁺]_i rise was observed between ATP and bradykinin. The magnitude of the [Ca²⁺]_i rise induced by ATP increased between 1.5 and 3.1 times when external Na⁺was replaced with Tris, N-methyl-d -glucamine, choline, or Li⁺. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca²⁺]_i to the basal level in Na⁺-containing or Na⁺-free Tris solution. Our results suggest that ATP stimulates Ca²⁺influx via at least two pathways: ion channels that are permeable to Ca²⁺ and Na⁺, and pores formed by ATP^4-.     

Osmotic Swelling-Induced Changes in Cytosolic Calcium Do Not Affect Regulatory Volume Decrease in Rat Cultured Suspended Cerebellar Astrocytes

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.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

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.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

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 ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y 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 P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Enhanced Depolarization-Evoked Calcium Signal and Reduced [ATP]/[ADP] Ratio Are Unrelated Events Induced by Oxidative Stress in Synaptosomes

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.     

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals

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 ET_B 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.     

Ca2+ Release from Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Store by Antidepressant Drugs in Cultured Neurons of Rat Frontal Cortex

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.     

Peculiarities of phospholipase C-dependent release of CA2+ from intracellular stores upon activation of choline and purine receptors in myocytes of the guinea-pig small intestine

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.     

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), as measured with the Ca²⁺-dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P₁ receptors in mediating this response. The increase in [Ca²⁺]_i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca²⁺]_i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′-[γ-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) ～ adenosine 5′-[β-thio]diphosphate ? adenosine 5′-[αβ-methylene] triphosphate, adenosine 5′-[βγ-imido]triphosphate. This profile is not characteristic of either the P_2X or the conventional P_2Y receptors. The Ca²⁺ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P_2Y nucleotide receptors (P_2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley-Liss, Inc.     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

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.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

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.     

Endothelin-Mediated Calcium Response and Inositol 1,4,5-Trisphosphate Release in Neuroblastoma-Glioma Hybrid Cells (NG108-15): Cross Talk with ATP and Bradykinin

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108-15) induced increases in cytosolic free Ca²⁺ ([Ca²⁺]_i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. The increases in [^Ca2+]_i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca²⁺ in the medium by adding excess EGTA decreased the ET-mediated Ca²⁺ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca²⁺]_i was due to influx from an extracellular source. However, the increase in [Ca²⁺]_i was not affected by verapamil or nifedipine (10^?5M). A rank order potency of ET-1 ET-2 ET-3 is shown for the stimulated increase in [Ca²⁺]_i, as well as labeled inositol phosphates, in these cells. ATP (10^?4M) and bradykinin (10^?7M) also induced the increases in [Ca²⁺]_i and Ins(1,4,5)P₃ in NG108-15 cells, albeit to a different extent. When compared at 10^?7M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P₃, but less than a twofold higher increase in [Ca²⁺]_i than those induced by ET-1. Additive increases in both Ins(1,4,5)P₃ and [Ca²⁺]_i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization to the Ca²⁺ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.     

Forskolin and Phorbol Myristate Acetate Inhibit Intracellular Ca2+ Mobilization Induced by Amitriptyline and Bradykinin in Rat Frontocortical Neurons

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.     

Characteristics of cytosolic Ca2+ elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig

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.     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

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.     

Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor-Associated Pores in Dibutyryl Cyclic AMP-Differentiated NG108-15 Cells

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 ATP^4?, 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 ATP^4? was >10 µM. The calculated EC₅₀ value for ATP^4? 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 ATP^4?-promoted pores are antagonized by Na⁺ and Mg²⁺ in dibutyryl cyclic AMP-differentiated NG108-15 cells.     

Acceleration of Sodium-Calcium Exchange Activity during ATP-induced Calcium Release in Transfected Chinese Hamster Ovary Cells

The P_2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca²⁺]_i due to Ca²⁺ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca²⁺ release was accompanied by a Ca²⁺- dependent regulatory activation of Na⁺/Ca²⁺ exchange activity: Addition of extracellular Ca²⁺ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca²⁺]_i in Na⁺-free media (Li⁺ substitution) compared to Na⁺-containing media; no differences between Na⁺- and Li⁺-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na⁺ and Ca²⁺, the ATP-evoked rise in [Ca²⁺]_i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca²⁺]_i which eventually came to exceed the declining [Ca²⁺]_i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba²⁺ influx, consistent with regulatory activation of the Na⁺/Ca²⁺ exchanger. The acceleration of Ba²⁺ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na⁺ or when the Ca²⁺ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca²⁺]_i under Na⁺-free conditions, but did not inhibit the ATP-evoked stimulation of Ba²⁺ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca²⁺ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca²⁺ release from intracellular stores through regulatory activation by increased [Ca²⁺]_i. In the absence of extracellular Ca²⁺, the stimulation of exchange activity is short-lived and follows the time course of the [Ca²⁺]_i transient; in the presence of extracellular Ca²⁺, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca²⁺ entry.     

Temperature dependency of calcium responses in mammary tumour cells

Changes of intracellular calcium concentration ([Ca²⁺]_i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24°C and 35°C. ATP (0·1–100 μM ) and bradykinin (0·1 nM –1 μM ) induced the increase of [Ca²⁺]_i at both temperatures and Ca²⁺-depletion did not affect these [Ca²⁺]_i responses. Both [Ca²⁺]_i responses became more sensitive at 35°C than at 24°C. A clear latency of [Ca²⁺]_i increased after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35°C. These results suggested the involvement of a regenerative or threshold process in the [Ca²⁺]_i responses in mammary tumour cells. © 1997 John Wiley & Sons, Ltd.