Effects of R(−)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588), a novel cognitive enhancer,on noradrenaline release in rat cerebral cortical slices

We investigated the effects of R(−)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588), a novel cognitive enhancer, on noradrenaline (NA) release from rat cerebral cortical slices in vitro. Addition of T-588 in an assay mixture stimulated [³H]NA release from prelabeled slices in the presence or absence of extracellular CaCl₂, and in the presence of the Ca²⁺/calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. T-588 stimulated NA release with a time lag of about l min, and the high level of release was maintained for at least 10 min, whereas maximal KCl-evoked NA release was observed within l min after the addition of KCl, and the effect declined subsequently. The effect of T-588 was reversible (pretreatment with T-588 showed no effect on NA release after two washes by centrifugation). We also compared the effects of T-588 and N-ethylmaleimide (NEM), a sulfhydryl alkylating agent known to stimulate neurotransmitter release in several types of cells. The addition of NEM stimulated NA release irreversibly from the slices in a Ca²⁺-independent manner, and the effect of NEM, but not that of T-588, was inhibited by the simultaneous addition of dithiothreitol, a sulfhydryl group reducing agent. The addition of T-588, which stimulated NA release by itself, inhibited the NA release by 0.6 mM NEM, although the effect of T-588 was additive in the presence of 0.2 mM NEM. These findings suggest that T-588 stimulates NA release from rat cerebral cortical slices in a Ca²⁺- and calmodulin-independent manner, possibly via an NEM-sensitive factor(s), although the mechanism of the effects of T-588 seems to be different from that of NEM.     

Trimethyltin and triethyltin differentially induce spontaneous noradrenaline release from rat hippocampal slices

The environmental contaminants trimethyltin (TMT) and triethyltin (TET) stimulated the spontaneous release of [(3)H]noradrenaline ([(3)H]NA) from hippocampal slices in a time- and concentration-dependent manner. TMT was the most potent compound, exhibiting an EC50 value 10-fold lower (3.8 microM) than that of TET (39.5 microM). Metal-evoked [(3)H]NA release did not increase in the absence of desipramine and was completely blocked by reserpine preincubation, indicating a vesicular origin of [(3)H]NA release but not a mechanism involving reversal of the transmitter transporter. The voltage-gated Na(+) channel blocker tetrodotoxin (TTX) did not affect metal-evoked [(3)H]NA release. [(3)H]NA release elicited by TMT was partially extracellular Ca(2+)-dependent, since it was significantly decreased in a Ca(2+)-free EGTA-containing medium, whereas TET induced an extracellular Ca(2+)-independent release of [(3)H]NA. Neither inhibitors of Ca(2+)-entry through Na(+)/Ca(2+)exchanger and voltage-gated calcium channels, nor agents that interfere with Ca(2+)-mobilization from intracellular stores affected [(3)H]NA release induced by TMT. TET-evoked [(3)H]NA release was reduced by ruthenium red, which depletes mitochondrial Ca(2+)stores, but was not modified by caffeine and thapsigargin, which interfere with Ca(2+)mobilization from endoplasmic reticulum. The fact that TET effect was also attenuated by DIDS, an inhibitor of anion exchange, indicates that the effect of TET on spontaneous [(3)H]NA release may be mediated by intracellular mobilization of Ca(2+) from mitochondrial stores through a Cl(-) dependent mechanism.     

Involvement of Hg2+-sensitive sulfhydryl groups in regulating noradrenaline release induced by S-nitrosocysteine in rat brain slices

Nitric oxide has been shown to regulate neurotransmitter release. Previously, we reported that S-nitrosothiols such as S-nitrosocysteine (SNC) stimulate noradrenaline (NA) release in rat hippocampus in vivo and in vitro. To examine the role of sulfhydryl groups in SNC-induced NA release, the effects of metal ions such as Hg2+ and N-ethylmaleimide (NEM, a sulfhydryl alkylating agent) on [3H]NA release from labeled rat brain slices (hippocampus and cerebral cortex) were studied and compared with the effects of SNC. The addition of 200 microM HgCl2, but not Pb2+, Zn2+, or Cd2+, stimulated [3H]NA release from both types of slices in the presence of extracellular CaCl2. p-Chloromercuribenzoic acid (p-CMBA) also stimulated [3H]NA release. NEM stimulated [3H]NA release from both types of slices in the presence and absence of extracellular CaCl2. The effect of 200 microM NEM was enhanced, but the effect of 200 microM SNC was inhibited by co-addition of 200 microM p-CMBA in the absence of extracellular CaCl2. The concentration-response curve of SNC shifted to the right after co-addition of 200 microM p-CMBA or 100 microM HgCl2, although the effect of 200 microM NEM was additive to the effect of SNC. These findings demonstrate that SNC acts as a sulfhydryl agent on proteins that regulate NA release, and that SNC may share the same sulfhydryl groups with Hg compounds. The effect of T-588 ?(R)-(-)-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy]eth anol hydrochloride?, a novel cognitive enhancer and a stimulator of NA release, was compared with the effects of sulfhydryl reagents.     

Desipramine-induced Ca2+ movement and cytotoxicity in PC3 human prostate cancer cells.

The effect of the antidepressant desipramine on intracellular Ca(2+) movement and viability in prostate cancer cells has not been explored previously. The present study examined whether desipramine could alter Ca(2+) handling and viability in human prostate PC3 cancer cells. Cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of cells were measured using fura-2 as a probe. Desipramine at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner. The responses saturated at 300 microM desipramine. The Ca(2+) signal was reduced by half by removing extracellular Ca(2+), but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem or verapamil. In Ca(2+)-free medium, after treatment with 300 microM desipramine, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) failed to release Ca(2+) from endoplasmic reticulum. Conversely, desipramine failed to release more Ca(2+) after thapsigargin treatment. Inhibition of phospholipase C with U73122 did not affect desipramine-induced Ca(2+) release. Overnight incubation with 10-800 microM desipramine decreased viability in a concentration-dependent manner. Chelation of cytosolic Ca(2+) with BAPTA did not reverse the decreased cell viability. Collectively, the data suggest that in PC3 cells, desipramine induced a [Ca(2+)](i) increase by causing Ca(2+) release from endoplasmic reticulum in a phospholipase C-independent fashion and by inducing Ca(2+) influx. Desipramine decreased cell viability in a concentration-dependent, Ca(2+)-independent manner.     

Modulation of dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus.

1. We studied the release of [3H]-dopamine and [3H]-noradrenaline (NA) from hippocampal synaptosomes induced by glutamate receptors and the associated Ca2+ influx through Ca2+ channels. The release of tritiated neurotransmitters was studied by use of superfusion system and the intracellular free Ca2+ concentration ([Ca2+]i) was determined by a fluorimetric assay with Indo-1 as a probe for Ca2+. 2. Presynaptic glutamate receptor activation induced Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA from rat hippocampal synaptosomes. Thus, L-glutamate induced the release of both neurotransmitters in a dose-dependent manner (EC50 = 5.62 microM), and the effect of 100 microM L-glutamate was inhibited by 83.8% in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX), but was not affected by 1 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801). 3. Other glutamate receptor agonists also stimulated the Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA as follows: N-methyl-D-aspartate (NMDA), at 200 microM, released 3.65 +/- 0.23% of the total 3H catecholamines, and this effect was inhibited by 81.2% in the presence of 1 microM MK-801; quisqualate (50 microM), S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA) (100 microM) or kainate (100 microM) released 1.57 +/- 0.26%, 1.93 +/- 0.17% and 2.09 +/- 0.22%, of the total 3H catecholamines, respectively. 4. The ionotropic glutamate receptor agonist, AMPA, induced an increase in the [Ca2+]i which was inhibited by 58.6% in the presence of 10 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by Zn2+ of uridine 5'-triphosphate-induced Ca(2+)-influx but not Ca(2+)-mobilization in rat phaeochromocytoma cells.

1. Uridine 5''-triphosphate (UTP)-evoked increase in intracellular Ca2+ concentration ([Ca]i) and release of dopamine were investigated in rat phaeochromocytoma PC12 cells. UTP (1-100 microM) evoked an increase in [Ca]i in a concentration-dependent manner. This response was decreased to about 30% by extracellular Ca(2+)-depletion, but not abolished. This [Ca]i rise was mimicked by 100 microM ATP but not by 100 microM 2-methyl-thio-ATP or alpha,beta-methylene-ATP in the absence of external Ca2+, suggesting that the response was mediated by P2U purinoceptors, a subclass of P2-purinoceptors. 2. The UTP-evoked [Ca]i rise consisted of two components; a transient and a sustained one. When external Ca2+ was removed, the sustained component was abolished while the transient component was decreased by about 70% but did not disappear. These results suggest that UTP induces Ca(2+)-mobilization and, subsequently, Ca(2+)-influx. 3. The UTP-evoked increase in [Ca]i was not affected by Cd2+ (100 and 300 microM) or nicardipine (30 microM), inhibitors of voltage-gated calcium channels, but was significantly inhibited by Zn2+ (10-300 microM) in the presence of external Ca2+. Zn2+, however, did not affect the Ca2+ response to UTP in the absence of external Ca2+. 4. UTP (30 microM-1 mM) evoked the release of dopamine from the cells in a concentration-dependent manner. This dopamine release was abolished by Ca(2+)-depletion or Zn2+ but not by Cd2+ or nicardipine. 5. Taken together, the data demonstrate that UTP stimulates P2U-purinoceptors and induces a rise in [Ca]i both by Ca(2+)-mobilization and Ca(2+)-influx in PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.     

Effects of (1R,9S)-beta-hydrastine on intracellular calcium concentration in PC12 cells

(1R,9S)-beta-hydrastine (BHS) decreases the basal intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells.(5) This study examined the effects of (1R,9S)-BHS on [Ca(2+)](i) in PC12 cells. (1R,9S)-BHS at 10-100 microM in combination with a high extracellular K+ level (56 mM) inhibited the release of dopamine in a concentration-dependent manner with an IC(50) value of 66.5 microM. BHS at 100 microM inhibited the sustained increase in [Ca(2+)](i) induced by a high K+ level (56 mM), and had an inhibitory effect on the 2 microM nifedipine-induced blockage of the K+ -stimulated sustained increase in [Ca(2+)](i). In addition, (1R,9S)-BHS at 100 microM prevented the rapid and sustained increase in [Ca(2+)](i) elicited by 20 mM caffeine, but did not have an effect on the increase induced by 1 microM thapsigargin, in the presence of external Ca(2+). These results suggest that the active sites of (1R,9S)-BHS are mainly L-type Ca(2+) channels and caffeine-sensitive Ca(2+)-permeable channels in PC12 cells.     

Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells

The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.     

Suppression by cholinesterase inhibition of a Ca(2+)-independent efflux of [3H]acetylcholine from the neuromuscular junction of the isolated rat diaphragm.

Endplate preparations of the left rat hemidiaphragm were incubated with [3H]choline to label neuronal acetylcholine stores. Elevation of the concentration (13.5-135 mmol/l) of extracellular potassium chloride (KCl) stimulated the release of [3H]acetylcholine in a concentration-dependent manner. KCl (27 mmol/l) still caused a significant efflux of [3H]acetylcholine in a Ca(2+)-free medium. Inhibitors of cholinesterase (physostigmine, diisopropylfluorophosphate) suppressed by 80% this Ca(2+)-independent efflux of [3H]acetylcholine. Vesamicol (10 mumol/l), the blocker of the vesicular acetylcholine carrier, also suppressed the stimulated, Ca(2+)-independent efflux of [3H]acetylcholine. The inhibitory effect of physostigmine was not prevented by muscarine or nicotine receptor antagonists, but the inhibitory effect was lost when the stimulus strength was increased (81 mmol/l KCl). The present experiments showed cholinesterase inhibition to suppress a Ca(2+)-independent efflux of [3H]acetylcholine, probably by interference with a membrane-bound acetylcholine carrier.     

3,4-Diaminopyridine-evoked noradrenaline release in rat hippocampus: role of Na+ entry on Ca2+ pools and of protein kinase C.

Slices of rat hippocampus, preincubated with [3H]noradrenaline [(3H]NA), were superfused continuously and stimulated by addition of 3,4-diaminopyridine (3,4-DAP; 100 microM) for 10 min to the superfusion medium. An overflow of 3H evoked by 3,4-DAP (representing [3H]NA release) was measurable not only in the presence but also in the absence of extracellular Ca2+. Both the protein kinase C (PKC) activator 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB) and the PKC inhibitor polymyxin B, affected mainly the evoked release in the absence of extracellular Ca2+ in a facilitatory or inhibitory manner, respectively. Moreover, in the absence of extracellular Ca2+, both the 3,4-DAP-evoked [3H]NA release and the facilitatory effect of 4 beta-PDB were abolished in the presence of tetrodotoxin or in the absence of Na+ in the superfusion medium. Ruthenium red, a blocker of mitochondrial Ca2+ reuptake, potently increased 3,4-DAP-evoked [3H]NA release in Ca(2+)-free EGTA-containing medium. The facilitatory effects of ruthenium red and 4 beta-PDB were additive. From these and earlier observations we conclude (1) that the mechanism of 3,4-DAP-evoked [3H]NA release involves both Ca2+ influx into the nerve terminals and mobilization of intraneuronal Ca2+ pools. Most probably Ca2+ release from cytoplasmic Ca2+ stores (e.g. endoplasmic reticular pools or mitochondria) is induced by Na+ ions entering the nerve endings during 3,4-DAP-evoked repetitive action potentials. (2) The facilitatory effect of phorbol ester on 3,4-DAP-evoked NA release appears to be mediated not by changes in Ca2+ influx, but by enhancement of intraneuronal events distal to Na+ ion entry and increased intracellular Ca2+ availability.     

Effects of baclofen on amino acid release.

This study showed the effects of baclofen on endogenous excitatory (Glu and Asp) and non-excitatory (Tau, Gly and Ala) amino acid release. (A) Release was stimulated by K+ 30 mM in rat frontal cortex slices in vitro (evoked release in ng/g tissue per 5 min): 3739 +/- 215 (Asp), 3429 +/- 357 (Glu), 763 +/- 181 (Tau), 945 +/- 71 (Ala), 468 +/- 44 (Gly). (B) Release was largely Ca(2+)-dependent for all amino acids but Ca(2+)-independent release was observed for Asp and Glu (29 and 32%, of total evoked release respectively). (C) Ca(2+)-dependent release was inhibited by baclofen 100 microM (% of inhibition taking as 100%, the Ca(2+)-dependent release in the absence of baclofen): 46 (Asp), 96 (Glu) (85% inhibition by baclofen 10 microM), 100 (Tau), 77 (Ala). Ca(2+)-independent release was inhibited: 87 and 88% (Asp), 85 and 95% (Glu) by baclofen 10 and 100 microM respectively. It is concluded that baclofen at a high concentration inhibits the evoked release of Glu, Asp, Tau and Ala due to inhibition of the Ca(2+)-dependent fraction and also of the Ca(2+)-independent component in the case of Glu and Asp. Baclofen at a low concentration inhibits only Glu- and Asp-evoked release (total release) due to inhibition of both Ca(2+)-dependent and -independent fractions in the case of Glu and to inhibition of the Ca(2+)-independent fraction in the case of Asp. The possibility that baclofen might affect the Ca(2+)-independent carrier-mediated release of Glu-Asp is discussed.     

A bioactive metabolite of benzo[a]pyrene, benzo[a]pyrene-7,8-dione, selectively alters microsomal Ca2+ transport and ryanodine receptor function

Polycyclic aromatic hydrocarbons are environmental pollutants known to be carcinogenic and immunotoxic. In intact cell assays, benzo[a]pyrene (B[a]P) disrupts Ca(2+) homeostasis in both immune and nonimmune cells, but the molecular mechanism is undefined. In this study, B[a]P and five metabolites are examined for their ability to alter Ca(2+) transport across microsomal membranes. Using a well-defined model system, junctional SR vesicles from skeletal muscle, we show that a single o-quinone metabolite of B[a]P, B[a]P-7,8-dione, can account for altered Ca(2+) transport across microsomal membranes. B[a]P-7,8-dione induces net Ca(2+) release from actively loaded vesicles in a dose-, time-, and Ca(2+)-dependent manner. In the presence of 5 microM extravesicular Ca(2+), B[a]P-7,8-dione exhibited threshold and EC(50) values of 0.4 and 2 microM, respectively, and a maximal release rate of 2 micromol of Ca(2+) min(-1) mg(-1). The mechanism by which B[a]P-7,8-dione enhanced Ca(2+) efflux was further investigated by measuring macroscopic fluxes and single RyR1 channels reconstituted in bilayer lipid membranes and direct measurements of SERCA catalytic activity. B[a]P-7,8-dione (< or = 20 microM) had no measurable effect on initial rates of Ca(2+) accumulation in the presence of ruthenium red to block ryanodine receptor (RyR1), nor did it alter Ca(2+)-dependent (thapsigargin-sensitive) ATPase activity. B[a]P-7,8-dione selectively altered the function of RyR1 in a time-dependent diphasic manner, first activating then inhibiting channel activity. Considering that RyR1 and its two alternate isoforms are broadly expressed in mammalian cells and their important role in Ca(2+)-signaling, the present results reveal a mechanism by which metabolic bioactivation of B[a]P may mediate RyR dysfunction of pathophysiological significance.     

Arachidonic acid release and prostaglandin F(2alpha) formation induced by anandamide and capsaicin in PC12 cells

Anandamide, an endogenous agonist of cannabinoid receptors, activates various signal transduction pathways. Anandamide also activates vanilloid VR(1) receptor, which was a nonselective cation channel with high Ca(2+) permeability and had sensitivity to capsaicin, a pungent principle in hot pepper. The effects of anandamide and capsaicin on arachidonic acid metabolism in neuronal cells have not been well established. We examined the effects of anandamide and capsaicin on arachidonic acid release in rat pheochromocytoma PC12 cells. Both agents stimulated [3H]arachidonic acid release in a concentration-dependent manner from the prelabeled PC12 cells even in the absence of extracellular CaCl(2). The effect of anandamide was neither mimicked by an agonist nor inhibited by an antagonist for cannabinoid receptors. The effects of anandamide and capsaicin were inhibited by phospholipase A(2) inhibitors, but not by an antagonist for vanilloid VR(1) receptor. In PC12 cells preincubated with anandamide or capsaicin, [3H]arachidonic acid release was marked and both agents were no more effective. Co-addition of anandamide or capsaicin synergistically enhanced [3H]arachidonic acid release by mastoparan in the absence of CaCl(2). Anandamide stimulated prostaglandin F(2alpha) formation. These findings suggest that anandamide and capsaicin stimulated arachidonic acid metabolism in cannabinoid receptors- and vanilloid VR(1) receptor-independent manner in PC12 cells. The possible mechanisms are also discussed.     

Monensin causes transient calcium ion influx into mouse splenic lymphocytes in a sodium ion-independent fashion

Monensin, a Na(+) ionophore, can increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in many cell types, but no studies have investigated the mechanism underlying a monensin-induced increase in [Ca(2+)](i) in immune cells. In view of this, we investigated the effect of monensin on [Ca(2+)](i) and cytosolic free Na(+) concentration ([Na(+)](i)) in mouse splenic lymphocytes using a fluorescence Ca(2+) indicator, fura-2, and a fluorescence Na(+) indicator, sodium-binding benzofuran isophthalate (SBFI), respectively. Monensin (1-100 microM) caused transient and sustained increases in [Ca(2+)](i) and [Na(+)](i), respectively, in a concentration-dependent manner. The monensin-induced increase in [Ca(2+)](i) was abolished by the omission of extracellular Ca(2+) or 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100-150 microM), and was largely inhibited by Ni(2+) (2-5 mM). The omission of extracellular Na(+) failed to inhibit the monensin-induced increases in [Ca(2+)](i). Furthermore, tetrodotoxin (1-10 microM), 5-(N,N-dimethyl)-amiloride (DMA, 10-20 microM), 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400, 3-10 microM), verapamil (10-200 microM), nifedipine (10-200 microM), omega-agatoxin IVA (0.2-10 microM), omega-conotoxin GVIA (1-10 microM), omega-conotoxin MVIIC (0.5-10 microM), and nordihydroguaiaretic acid (NDGA, 1-10 microM) had no effect on the increases in [Ca(2+)](i). Monensin-induced Mn(2+) influx into splenic lymphocytes. The Mn(2+) influx was completely inhibited by SKF-96365. These results suggest that monensin transiently increases [Ca(2+)](i) in mouse splenic lymphocytes by stimulating Ca(2+) entry via non-selective cation channels in a Na(+)-independent manner.     

The anti-anginal drug fendiline increases intracellular Ca(2+) levels in MG63 human osteosarcoma cells

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored by using fura-2 as a Ca(2+) indicator. Fendiline at concentrations between 1 and 200 microM increased [Ca(2+)](i) in a concentration-dependent manner and the signal saturated at 100 microM. The Ca(2+) signal was inhibited by 65+/-5% by Ca(2+) removal and by 38+/-5% by 10 microM nifedipine, but was unchanged by 10 microM La(3+) or verapamil. In Ca(2+)-free medium, pre-treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store inhibited fendiline-induced intracellular Ca(2+) release. The Ca(2+) release induced by 50 microM fendiline appeared to be independent of IP(3) because the [Ca(2+)](i) increase was unaltered by inhibiting phospholipase C with 2 microM U73122. Collectively, the results suggest that in MG63 cells fendiline caused an increase in [Ca(2+)](i) by inducing Ca(2+) influx and Ca(2+) release in an IP(3)-independent manner.     

Inhibition of phospholipase A(2) activity by S-nitroso-cysteine in a cyclic GMP-independent manner in PC12 cells

Arachidonic acid and nitric oxide (NO) act as retrograde and intercellular messengers in the nervous system. Regulation of cyclooxygenase is well established, but regulation of phospholipase A(2), the enzyme responsible for the liberation of arachidonic acid, by NO has not been thoroughly investigated. Using the PC12 cell line as a neuronal model, we studied the effects of exogenous NO compounds on arachidonic acid release. Incubation with Ca(2+) ionophores or mastoparan (wasp venom peptide) stimulated [3H]arachidonic acid release from prelabeled PC12 cells. [3H]Arachidonic acid release was inhibited by cytosolic phospholipase A(2) inhibitors, but not by dithiothreitol. A cytosolic phospholipase A(2) protein band with a molecular mass of approximately 100 kDa was detected by immunoblotting. S-Nitroso-cysteine inhibited basal and stimulated [3H]arachidonic acid release in concentration-dependent manners. Other NO compounds such as sodium nitroprusside and S-nitroso-N-acetylpenicillamine did not affect [3H]arachidonic acid release. N-Ethylmaleimide also inhibited [3H]arachidonic acid release. The inhibitory effects of S-nitroso-cysteine and N-ethylmaleimide were irreversible, because [3H]arachidonic acid release from PC12 cells preincubated with S-nitroso-cysteine or N-ethylmaleimide was much lower than that from nontreated cells. These findings suggest (a) cytosolic phospholipase A(2) is activated by Ca(2+) or mastoparan, and inhibited by S-nitroso-cysteine in a cyclic GMP-independent manner, (b) N-ethylmaleimide also inhibits cytosolic phospholipase A(2) and arachidonic acid release in PC12 cells. S-Nitroso-cysteine can regulate the production of other retrograde messenger arachidonic acid.     

Dual effect of tamoxifen, an anti-breast-cancer drug, on intracellular Ca(2+) and cytotoxicity in intact cells

The effect of tamoxifen on Ca(2+) signaling and viability in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Tamoxifen evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 1 and 50 microM with an EC50 of 10 microM. The response was decreased by extracellular Ca(2+) removal. In Ca(2+)-free medium, pretreatment with 5 microM tamoxifen abolished the [Ca(2+)](i) increase induced by the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM), but pretreatment with brefeldin A (50 microM; a Ca(2+) mobilizer of the Golgi complex), thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) pump), and carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler), only partly inhibited tamoxifen-induced [Ca(2+)](i) increases. This suggests that tamoxifen released Ca(2+) from multiple pools. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 5 microM tamoxifen in Ca(2+)-free medium. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) did not alter 5 microM tamoxifen-induced Ca(2+) release. The [Ca(2+)](i) increase induced by 5 microM tamoxifen was not altered by La(3+), nifedipine, verapamil, or diltiazem. Tamoxifen (1-10 microM) decreased cell viability in a concentration- and time-dependent manner. Tamoxifen (5 microM) also increased [Ca(2+)](i) in neutrophils, bladder cancer cells, and prostate cancer cells from humans and glioma cells from rats. Collectively, it was found that tamoxifen increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol 1,4, 5-trisphosphate and also by triggering Ca(2+) influx from extracellular space. The [Ca(2+)](i) increase was accompanied by cytotoxicity.     

Novel effect of Y-24180, a presumed specific platelet-activating factor receptor antagonist, on Ca2+ levels and growth of human osteosarcoma cells.

In human osteosarcoma MG63 cells, the effect of Y-24180, a presumed specific platelet-activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) and proliferation was measured by using fura-2 and tetrazolium as fluorescent dyes, respectively. Y-24180 (1-5 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was inhibited by 35% by dihydropyridines or removal of extracellular Ca(2+), but was not altered by verapamil and diltiazem. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which 5 microM Y-24180 failed to increase [Ca(2+)](i); conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited histamine-induced, but not 5 microM Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with 0.1-5 microM Y-24180 inhibited cell proliferation in a concentration-dependent manner. Together, these findings suggest that Y-24180 acts as a potent and cytotoxic Ca(2+) mobilizer in human osteosarcoma cells, by inducing both extracellular Ca(2+) influx and intracellular Ca(2+) release. Alterations in cytosolic Ca(2+) regulation may lead to interferences of various cellular functions; thus, attention should be exercised in using Y-24180 as a selective PAF receptor antagonist.