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.     

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.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

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.     

Ca2+ responses to acetylcholine and adenosine triphosphate in the otocyst of chick embryo

The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca²⁺-sensitive fluorescence measurements. Increases in [Ca²⁺]_i were evoked by the bath application of acetylcholine (1 μM or higher). The rise in [Ca²⁺]_i was due to the release of Ca²⁺ from intracellular Ca²⁺ stores, since the Ca²⁺ response occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolisehd the response to 10 μM acetylcholine; muscarine and carbamylcholine (100 μM each) evoked Ca²⁺ rises. Increases in [Ca²⁺]_i were also evoked by the bath application of ATP (10 μM or higher). The Ca²⁺ rise by ATP was evoked even in a Ca²⁺-free medium. Adenosine (500 μM) did not cause any Ca²⁺ response. Suramin and reactive blue 2 (200 μM each) completely blocked the Ca²⁺ response to 500μM ATP. Uridine triphosphate (500 μM) caused comparable Ca²⁺ responses with those to 500 μM ATP. These results suggested the involvement of P_2U purinoceptors. The potentiation of Ca²⁺ rise was observed when acetylcholine and ATP were co-applied at submaximal concentrations (10 μM and 100 μM, respectively). We conclude that undifferentiated cells in the otocyst epithelium have CaCa²⁺ mobilizing systems activated by acetylcholine and ATP. © 1995 John Wiley & Sons, Inc.     

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.     

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-.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

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.     

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.     

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

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.     

Ca2+ - and Nitric Oxide-Dependent Stimulation of Cyclic GMP Synthesis in Neuronal Cell Line Induced by P2-Purinergic/Pyrimidinergic Receptor

Abstract: The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma × rat glioma hybrid cells [108CC15 (NG 108-15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for ～1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5′-(γ-thio)triphosphate > UTP = 2-methylthio-ATP > ADP ? CTP, AMP, α,β-methylene-ATP, 2′- and 3′-O-(4-benzoylbenzoyl)ATP. The EC₅₀ of 1 ± 0.2 µM for UTP was significantly lower than that for ATP (14 ± 8 µM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P_2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC₅₀ = 40–60 µM) or the pyridoxal phosphate analogue pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (IC₅₀ = 20–30 µM). Pretreatment of cells with the Ca²⁺ ionophore ionomycin or with 2,5-di(tert-butyl)-1,4-benzohydroquinone, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca²⁺ stores, suppressed the ATP- or UTP-induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP-induced rise in cyclic GMP level by 60%. The cyclic GMP response caused by ATP was suppressed either by arginine analogues (IC₅₀ for nitroarginine = 1 µM) or by hemoglobin (IC₅₀ = 2 µM). This indicates that ATP/UTP via a P₂-receptor causes formation of nitric oxide, which activates guanylate cyclase. The synthesis of nitric oxide depends on a preceding rise in cytosolic Ca²⁺ level, mostly due to release of Ca²⁺ from internal stores. Bradykinin induces a rise in cyclic GMP level with an amplitude and time course comparable to that caused by ATP. Therefore, we studied cross-desensitization between ATP and bradykinin receptors. Pretreatment with bradykinin completely suppressed a subsequent response to ATP. However, stimulation with ATP reduced a following response to bradykinin by ～40% only. This indicates a heterologous cross-desensitization predominantly in one direction (bradykinin ? ATP).     

Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor-Differentiated PC12 Cells

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.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

A Prototypic Intracellular Calcium Antagonist, TMB-8, Protects Cultured Cerebellar Granule Cells Against the Delayed, Calcium-Dependent Component of Glutamate Neurotoxicity

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.     

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.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

Hydrogen peroxide constricts rat arteries by activating Na+-permeable and Ca2+-permeable cation channels

Oxidative stress is associated with many cardiovascular diseases, such as hypertension and arteriosclerosis. Oxidative stress reportedly activates the L-type voltage-gated calcium channel (VDCC_L) and elevates [Ca²⁺]_i in many cells. However, how oxidative stress activates VDCC_L under clinical setting and the consequence for arteries are unclear. Here, we examined the hypothesis that hydrogen peroxide (H₂O₂) regulates membrane potential (Em) by altering Na⁺ influx through cation channels, which consequently activates VDCC_L to induce vasoconstriction in rat mesenteric arteries. To measure the tone of the endothelium-denuded arteries, a conventional isometric organ chamber was used. Membrane currents and Em were recorded by the patch-clamp technique. [Ca²⁺]_i and [Na⁺]_i were measured with microfluorometry using Fura2-AM and SBFI-AM, respectively. We found that H₂O₂ (10 and 100 µM) increased arterial contraction, and nifedipine blocked the effects of H₂O₂ on isometric contraction. H₂O₂ increased [Ca²⁺]_i as well as [Na⁺]_i, and depolarised Em. Gd³⁺ (1 µM) blocked all these H₂O₂-induced effects including Em depolarisation and increases in [Ca²⁺]_i and [Na⁺]_i. Although both nifedipine (30?nM) and low Na⁺ bath solution completely prevented the H₂O₂-induced increase in [Na⁺], they only partly inhibited the H₂O₂-induced effects on [Ca²⁺]_i and Em. Taken together, the results suggested that H₂O₂ constricts rat arteries by causing Em depolarisation and VDCC_L activation through activating Gd³⁺-and nifedipine-sensitive, Na⁺-permeable channels as well as Gd³⁺-sensitive Ca²⁺-permeable cation channels. We suggest that unidentified Na⁺-permeable cation channels as well as Ca²⁺-permeable cation channels may function as important mediators for oxidative stress-induced vascular dysfunction.     

Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole-induced Ca(2+) influx was insensitive to L-type Ca²⁺ channel blockers and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 20 µM econazole, [Ca²⁺]_i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca²⁺]_i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. This shows that in SIRC cells econazole induces [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca²⁺]_i rise.     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.