Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Phenolic compounds affect intracellular free Ca²⁺ concentration ([Ca²⁺]_i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca²⁺ signaling in PC12 cells using fura-2-based digital Ca²⁺ imaging and whole-cell patch clamping. Treatment with ATP (100 µM) for 90 s induced increases in [Ca²⁺]_i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 µM) for 10 min inhibited the ATP-induced [Ca²⁺]_i response in a concentration-dependent manner (IC₅₀=2.84 µM). Treatment with octyl gallate (3 µM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca²⁺ with nominally Ca²⁺-free HEPES HBSS or depletion of intracellular Ca²⁺ stores with thapsigargin (1 µM). Treatment for 10 min with the L-type Ca²⁺ channel antagonist nimodipine (1 µM) significantly inhibited the ATP-induced [Ca²⁺]_i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca²⁺]_i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 µM) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca²⁺]_i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca²⁺ from extracellular space and P2Y receptor-induced release of Ca²⁺ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca²⁺ responses by inhibiting the secondary activation of voltage-gated Ca²⁺ channels.     

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

The aim of this study was to determine whether ⁴⁵Ca²⁺ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC₅₀ = 45 M), ATPTS (EC₅₀ = 50 M) and 2-meSATP (EC₅₀ = 81 M) but not meATP (I mM) stimulated ⁴⁵Ca²⁺ influx 2–5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADPS did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC₅₀ = 191 M) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells.ATP-stimulated ⁴⁵Ca²⁺ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated ⁴⁵Ca²⁺ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency :- guanidinium (EC₅₀ = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose.A number of P2 purinoceptor antagonists inhibited ATP-stimulated ⁴⁵Ca²⁺ influx. Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (3–300 M), pyridoxal 5phosphate (3–300 M) and d-tubocurarine (30–300 M) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC₅₀. Suramin (100–300 M) and cibacron blue (30–300 M) produced a surmountable antagonism while DIDS (4,4-diisothiocyana-tostilbene-2,2disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 M. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X₂ purinoceptors. These findings suggest that ATP-stimulated ⁴⁵Ca²⁺ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.     

Fenvalerate-induced Ca transients via both intracellular and extracellular way in mouse GC-2spd (ts) cells

Fenvalerate (Fen) is a widely used synthetic pyrethroid insecticide which is considered to impede the male reproductive function. However, little is known about its underlying mechanism. In this study, we found that fenvalerate affected the Ca²⁺ homeostasis, inducing Ca²⁺ transients via both intracellular Ca²⁺ release and extracellular Ca²⁺ influx. Ca²⁺ influx was via store-operated channel (SOC). Therefore, the effects of fenvalerate on Ryanodine receptors (RyRs) and Inositol (1,4,5)-trisphosphate receptors (IP₃Rs) which involved in forming Ca²⁺ transient was assessed by pharmacological way. We also demonstrated that fenvalerate affected the expression of both receptors and hindered cell proliferation as well. In addition, we discovered that 2-APB, an antagonist of IP₃Rs, inhibited GC-2spd (ts) cells (GC-2 cells) proliferation. Cell cycle analysis of GC-2 cells treated with fenvalerate and 2-APB indicated that both of which showed a slight S-phase accumulation. In conclusion, our results demonstrate that fenvalerate-induced Ca²⁺ transients from both calcium release through RyRs or IP₃Rs and calcium influx via SOC. IP₃Rs seem to serve a predominant role in triggering Ca²⁺ transients which could participate to the regulation of GC-2 cell proliferation.     

Ca(2+)-activated K+ current induced by external ATP in PC12 cells

1. The effect of external ATP on the membrane current was investigated in PC12 cells by whole-cell voltage-clamp techniques. 2. Lower concentrations of ATP (1 or 10 mumol/L) induced only an inward current at 1 mmol/L EGTA in the K+ pipette solution, while higher concentrations of ATP (100 mumol/L and 1 mmol/L) induced an outward current following the inward current. 3. Lowering the EGTA concentration in the pipette solution induced a larger outward current following ATP application. The membrane potential at which the outward current crossed with the control before ATP application was more negative at lower concentrations of EGTA in the pipette. 4. The development of the outward current was blocked by a Ca(2+)-free external solution, 5 mmol/L tetraethylammonium and a Cs+ pipette solution instead of K+, indicating that the outward current was a Ca(2+)-activated K+ current. 5. Charybdotoxin (0.1 mumol/L) and iberiotoxin (0.1 mumol/L), but not apamin (0.2 mumol/L) blocked the development of the outward current, indicating the ATP-induced outward current is a BK-type Ca(2+)-activated K+ channel current and not the SK type. 6. UTP had no effect on the membrane current, indicating that the ATP-induced current change was not mediated by P2u but by P2x purinoceptor. 7. In conclusion, stimulation of P2x purinoceptors by ATP induces a Ca(2+)-permeable inward current that results in increases in intracellular Ca2+ concentrations and activation of a BK-type Ca(2+)-activated K+ current in PC12 cells.     

Gabapentin inhibits presynaptic Ca(2+) influx and synaptic transmission in rat hippocampus and neocortex

Gabapentin is a widely used drug with anticonvulsant, antinociceptive and anxiolytic properties. Although it has been previously shown that Gabapentin binds with high affinity to the alpha(2)delta subunit of voltage-operated Ca(2+) channels (VOCC), little is known about the functional consequences of this interaction. Here, we investigated the effect of Gabapentin on VOCCs and synaptic transmission in rat hippocampus and neocortex using whole-cell patch clamp and confocal imaging techniques. Gabapentin (100-300 microM) did not affect the peak amplitude or voltage-dependency of VOCC currents recorded from either dissociated or in situ neocortical and hippocampal pyramidal cells. In contrast, Gabapentin inhibited K(+)-evoked increases in [Ca(2+)] in a subset of synaptosomes isolated from rat hippocampus and neocortex in a dose-dependent manner, with an apparent half-maximal inhibitory effect at approximately 100 nM. In hippocampal slices, Gabapentin (300 microM) inhibited the amplitude of evoked excitatory- and inhibitory postsynaptic currents recorded from CA1 pyramidal cells by 30-40%. Taken together, the results suggest that Gabapentin selectively inhibits Ca(2+) influx by inhibiting VOCCs in a subset of excitatory and inhibitory presynaptic terminals, thereby attenuating synaptic transmission.     

The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in PC12 cells

We studied the effects of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells using fura-2 fluorescence imaging. Carmustine (100 microM) caused a delayed increase in [Ca(2+)](i) that developed within approximately 3 h. This effect was enhanced in cells that were pretreated with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BSO, 200 microM, 24 h), and was suppressed in cells that were treated with an antioxidant deferoxamine (50 microM). The carmustine-induced increase in [Ca(2+)](i) was absolutely dependent on the presence of extracellular Ca(2+) and could be inhibited by dihydropyridine blockers of L-type voltage-gated Ca(2+) channels (nimodipine or nitrendipine, 10 microM). The increase in [Ca(2+)](i) was also suppressed in Cl(-)-free solution and in the presence of the Cl(-) channel blockers, indanyloxyacetic acid 94 (IAA-94, 100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM). The inhibition was complete when the blockers were applied simultaneously with carmustine and was partial when the blockers were applied after the initial increase in [Ca(2+)](i). We conclude that carmustine induces an influx of extracellular Ca(2+) through L-type Ca(2+) channels and that this effect is mediated by oxidative stress that results from the depletion of GSH following the inhibition by carmustine of glutathione reductase.     

Heparin specifically inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned rat aortic smooth muscle cells in primary culture

Summary By measuring the ⁴⁵Ca²⁺ movement in saponin-skinned primary cultured rat aortic smooth muscle cells, we examined the specificity of the inhibitory effect of heparin on the IP₃-induced Ca²⁺ release. IP₃ (100 mol/l) markedly (98%) decreased the MgATP-dependent ⁴⁵Ca²⁺ content in the non-mitochondrial Ca²⁺ stores in the presence of 1 mol/l free Ca²⁺. Heparin (1–100 g/ml) dose-dependently inhibited this Ca²⁺ release by IP₃. In Ca²⁺-free solution, heparin (100 g/ml) inhibited the increases in ⁴⁵Ca²⁺ efflux rate evoked by 10 mol/l IP₃. De-N-sulfated heparin did not inhibit the IP₃-induced Ca²⁺ release. Hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C and 2,6-disulfated d-glucosamine had no inhibitory effects on the IP₃-induced Ca²⁺ release. High concentrations (over 1 mg/ml) of heparin inhibited the ⁴⁵Ca²⁺ influx and decreased the Ca²⁺ content in skinned cells. These results suggest that heparin (1–100 g/ml) specifically inhibits the IP₃-induced increase in Ca²⁺ permeability of Ca²⁺ stores and that three sulfate groups at different locations on the molecule of heparin, two at the d-glucosamine and one at the iduronic acid, may be important for this action, in skinned vascular smooth muscle cells, in culture. Send offprint requests to H. Kanaide at the above address     

2-Benzyloxybenzaldehyde inhibits formyl peptide-stimulated increase in intracellular Ca2+ in neutrophils mainly by blocking Ca2+ entry

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca²⁺] ([Ca²⁺]_i) in rat neutrophils. The late plateau phase, but not the initial Ca²⁺ spike, of the fMLP-induced [Ca²⁺]_i change was inhibited by CCY1a. In the absence of external Ca²⁺, CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca²⁺]_i elevation. CCY1a failed to inhibit [Ca²⁺]_i changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca²⁺ signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca²⁺ readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca²⁺]_i response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn²⁺ influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70–85 kDa molecular mass range. CCY1a had no effect on the basal [Ca²⁺]_i level, the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca²⁺ entry into neutrophils probably by blocking the relevant Ca²⁺ channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

Epigallocatechin-3-gallate increases intracellular [Ca<Superscript>2+</Superscript>] in U87 cells mainly by influx of extracellular Ca<Superscript>2+</Superscript> and partly by release of intracellular stores

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [³H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca²⁺] ([Ca²⁺]_i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca²⁺]_i. The EGCG-induced [Ca²⁺]_i increases were reduced to 20.9% of control by removal of extracellular Ca²⁺. The increases were also inhibited markedly by treatment with the non-specific Ca²⁺ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca²⁺ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca²⁺-ATPase thapsigargin (1 µM) also significantly inhibited the EGCG-induced [Ca²⁺]_i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 µM) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 µM) had no effect. EGCG increased [³H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 µM) and flufenamic acid (100 µM), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca²⁺]_i increase in non-treated and thapsigargin-treated cells but indomethacin (100 µM) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca²⁺]_i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca²⁺ and partly by mobilizing intracellular Ca²⁺ stores by PLC activation. The EGCG-induced [Ca²⁺]_i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.     

Lysophospholipids elevate [Ca2+]i and trigger exocytosis in bovine chromaffin cells

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are responsible for many physiological functions, including angiogenesis, neuronal survival, and immunity. However, little is known about their effects in modulating the stimulus-secretion coupling in bovine chromaffin cells. The result of PCR showed that at least two receptors (S1P(3) and LPA(1)) were expressed in bovine chromaffin cells. The elevation of [Ca(2+)](i) by S1P was fast and sustaining; but the elevation by LPA was slow and transient. The EC(50) for S1P and LPA in elevating the [Ca(2+)](i) were 0.55+/-0.01 and 0.54+/-0.40microM, respectively. This elevation could be totally blocked by thapsigargin, 2-APB, and U73122. Pertussis toxin pretreatment inhibited about half of the elevation in [Ca(2+)](i) suggesting the involvement of G(i) and other G-proteins. Repetitive [Ca(2+)](i) elevations elicited by S1P, but not LPA, were inhibited by ryanodine. S1P was more effective than LPA in triggering exocytosis as measured by the changes in membrane capacitance. The whole-cell Ca(2+) current was inhibited by both lysophospholipids but Na(+) current was inhibited by S1P only. These results suggest the differential effects of LPA and S1P in releasing Ca(2+) from the intracellular Ca(2+) stores and modulating the stimulus-secretion coupling in bovine chromaffin cells.     

NPY and carbachol raise Ca2+ in SK-N-MC cells by three different mechanisms

Summary We have compared the mechanism of NPY-and carbachol-stimulated Ca²⁺ increases in SK-N-MC cells. NPY stimulated Ca²⁺ mobilization via a pertussis toxin-sensitive mechanism. Carbachol stimulated Ca²⁺ mobilization and influx via pertussis toxin-insensitive and -sensitive mechanisms, respectively. Carbachol but not NPY stimulated inositol phosphate accumulation by a pertussis toxin-insensitive mechanism. We conclude that carbachol promotes Ca²⁺ influx via a pertussis toxin-sensitive G protein and Ca²⁺ mobilization via a pertussis toxin-insensitive G-protein coupling to inositol phosphate generation; NPY stimulates Ca²⁺ mobilization via a pertussis toxin-sensitive G protein without apparent involvement of inositol phosphates.     

Biphasic effects of oxethazaine,a topical anesthetic,on the intracellular Ca(2+) concentration of PC12 cells

There have been few reports on the mechanism(s) of action of oxethazaine (OXZ) despite its potent local anesthetic action. Generally, local anesthetics (LAs) not only inhibit Na(+) channels but also affect various membrane functions. In the present study, using PC12 cells as a nerve cell model, the effects of OXZ on intracellular Ca(2+) concentration ([Ca(2+)](i)) were examined in relation to cytotoxicity and dopamine release. [Ca(2+)](i) was determined by the quin2 method. In resting cells, (6-10)x10(-5)M OXZ produced lactate dehydrogenase leakage, which was Ca(2+)-dependent, inhibited by metal Ca(2+) channel blockers, and preceded by a marked increase in [Ca(2+)](i). Some other LAs showed no cytotoxicity at these concentrations. In K(+)-depolarized cells, however, lower concentrations of OXZ (10(-6)-10(-7)M), that had no effect on resting [Ca(2+)](i), inhibited both the dopamine release and the increase of [Ca(2+)](i) in parallel. The inhibitory potency against the [Ca(2+)](i) increase was in the order of nifedipine>OXZ approximately verapamil>diltiazem, and OXZ acted additively on the Ca(2+) channel blockers. OXZ showed the least effect on K(+)-depolarization as determined by bisoxonol uptake. OXZ also inhibited the increase in [Ca(2+)](i) induced by S(-)-BAY K 8644, a Ca(2+) channel agonist. These observations suggested that low concentrations of OXZ interact with L-type Ca(2+) channels. The biphasic effects of OXZ on Ca(2+) movement may be due to a unique chemical structure, and may participate in and complicate the understanding of the potent pharmacological and toxicological actions of OXZ.     

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Calmidazolium induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was insensitive to L-type Ca²⁺ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), calmidazolium-induced [Ca²⁺]_i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca²⁺]_i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.     

Orai1-STIM1 formed store-operated Ca2+ channels (SOCs) as the molecular components needed for Pb2+ entry in living cells

Heavy metal lead (Pb2+) is a pollutant and causes severe toxicity when present in human tissues especially the nervous system. Recent reviews have suggested that Pb2+ can target Ca2+-related proteins within neurons and that Ca2+ channels might be a candidate for Pb2+ entry. This study's main aim was to identify the functional entry pathway of Pb2+ into living cells. We firstly characterized the endogenous expression of Orai1 and STIM1 mRNA together with the level of thapsigargin (TG) stimulated capacitative Ca2+ entry in PC12 and HeLa cells; this was done by RT-PCR and time-lapse Ca2+ imaging microscopy, respectively. Our data supported Orai1 and STIM1 as contributing to store-operated Ca2+ channel (SOC) basal activity. Secondly, using the indo-1 quenching method with the SOC blocker 2-APB, we observed that Pb2+ was able to enter cells directly through unactivated SOCs without TG pretreatment. Thirdly, we further demonstrated that co-expression of Orai1 and STIM1 differentially enhanced SOC functional activity (4-fold with PC12 and 5-fold with HeLa cells) and Pb2+ entry (5- to 7-fold with PC12 and 2-fold with HeLa cells). Furthermore, after a 1 h of Pb2+ exposure, the depolarization- and histamine-induced Ca2+ responses were significantly decreased in both PC12 and HeLa cells in a dose-dependent manner. This result indicated that the decreased Ca2+ responses were, in part, due to Pb2+ entry. In summary, our results suggest that SOCs are responsible for Pb2+ permeation and that the Orai1-STIM1 protein complex formed by functional SOCs is one of the molecular components involved in Pb2+ entry.     

Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells

The inhibitory effects of liriodenine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced dopamine content increases in PC12 cells were investigated. Treatment of PC12 cells with 5-10 microM liriodenine significantly decreased the intracellular dopamine content in a concentration-dependent manner (IC50 value, 8.4 microM). Liriodenine was not cytotoxic toward PC12 cells at concentrations up to 20 microM. Tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities were inhibited by 10 microM liriodenine to 20-70% and 10-14% of control levels at 3-12 h, respectively; TH activity was more influenced than AADC activity. The levels of TH mRNA, intracellular cyclic AMP and basal Ca2+ concentration were also decreased by 10 microM liriodenine. In addition, 10 microM liriodenine reduced L-DOPA (20-100 microM)-induced increases in dopamine content. However, 10 microM liriodenine resulted in a protective effect against L-DOPA (50-100 microM)-induced cytotoxicity. These results suggest that liriodenine regulates dopamine biosynthesis by partially reducing TH activity and TH gene expression and has protective effects against L-DOPA-induced cytotoxicity in PC12 cells.     

The role of calcium in paracetamol (acetaminophen) cytotoxicity in PC12 cells transfected with CYP4502E1

Paracetamol-induced toxicity is mainly due to the accumulation of its CYP450-mediated N-hydroxylation product - N-acetylimidoquinone. We examined cell viability, proliferation rates and intracellular calcium in PC12 cells and in a PC12 cell line transfected with cytochrome P4502E1 exposed to paracetamol. This drug had a concentration-related effect on cell survival and a LD₅₀ which was significantly different between both cell types. A 48% decrease of PC12 cells was found following application of 5 mmol/L paracetamol for 48 h. A total 73% decrease in cell numbers was found in cells metabolizing the drug. Culture protein levels were diminished in a similar manner. Paracetamol increased intracellular calcium (by 662%) only in CYP4502E1-transfected cells. The protective role of EGTA and verapamil modulating calcium homeostasis was more evident in CYP4502E1-transfected cells. These results suggest that biotransformation of paracetamol by CYP2E1 increases its cytotoxicity and that a calcium imbalance may have a key role in the initiation of cell injury.     

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca²⁺ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Celecoxib-induced Ca²⁺ influx was not blocked by L-type Ca²⁺ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺-free medium, 30 μM of celecoxib failed to induce a [Ca²⁺]_i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca²⁺ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca²⁺]_i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca²⁺ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca²⁺]_i rises by causing phospholipase C–independent Ca²⁺ release from the ER and Ca²⁺ influx via non-L-type, phospholipase A₂-regulated Ca²⁺ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.     

Involvement of Ca(2+)-induced Ca2+ releasing system in interleukin-1beta-associated adenosine release

Interleukin-1beta (IL-1beta) plays an important role in neuroprotective and neurodegenerative events in the central nervous system. To clarify the mechanism of controversial actions of IL-1beta, we determined the effect of IL-1beta, as well as the interaction between IL-1beta and Ca(2+)-induced Ca2+ releasing system (CICR), on adenosine releases in mice hippocampus using mini-slices method. Basal and K(+)-stimulated adenosine releases were regulated by two types of CICRs, including inositol-1,4,5-trisphosphate (IP3) receptor and ryanodine receptor. Lower concentration of IL-1beta increased both adenosine releases, whereas higher concentration did not affect their releases. The stimulatory effect of IL-1beta on basal adenosine release was reduced by removal of extracellular Ca2+ and IP3 receptor inhibitor, while the stimulatory effect of IL-1beta on K(+)-stimulated adenosine release was reduced by ryanodine receptor inhibitor. These results suggest that the potent effect of IL-1beta upon adenosine release might contribute to the neuroprotective action of IL-1beta, whereas IL-1beta-induced neurodegeneration might be due to the overload response of Ca2+ mobilization and the inactivation of adenosine exocytosis.     

异丙酚对PC12细胞膜流动性及[Ca~(2+)]_i的影响

目的　研究异丙酚对PC12细胞膜流动性及 [Ca2 + ]i的影响。方法　以PC12细胞作为神经细胞模型 ,加入不同浓度的异丙酚 ,用荧光偏振法测定细胞膜微粘度 η的动态变化 ,用激光扫描共聚焦显微镜检测 [Ca2 + ]i 随时间变化曲线。结果　①异丙酚各剂量组的 η值均降低 ,并呈现剂量依赖性 ;② 30、10 0mg·L-1的异丙酚在加药后 2 0～ 30s使[Ca2 + ]i 短暂升高 ,[Ca2 + ]i的峰值分别比加药前增加了119%和 14 0 % ,但在 5 0s内均恢复到加药前水平。结论　异丙酚的全麻作用机制可能与细胞膜流动性增高有关