thapsigargin and methylmercuric-chloride

We previously showed that elevated intracellular Ca(2+) ([Ca(2+)]i) in the molecular layer and granule cells in cerebellar slices is responsible for the initial increases in frequency of spontaneous or miniature inhibitory postsynaptic currents (sIPSCs or mIPSCs) of Purkinje cells following methylmercury (MeHg) treatment. To identify the contribution of different Ca(2+) sources to MeHg-induced stimulation of spontaneous GABA release, we examined sIPSC or mIPSC frequency of Purkinje cells in acutely prepared cerebellar slices using whole-cell patch-clamp recording techniques under conditions of lowered [Ca(2+)]o, pretreatment with caffeine, cyclopiazonic acid (CPA), thapsigargin or ruthenium red (RR) to deplete ryanodine-sensitive and insensitive intracellular Ca(2+) stores or mitochondria, or a combination of lowering [Ca(2+)]o and increased BAPTA buffering. Lowering [Ca(2+)]o significantly reduced sIPSC or mIPSC frequency and amplitudes, but failed to completely prevent MeHg-induced increase in these events frequency. Caffeine, CPA, or thapisgargin also minimized MeHg-induced increase in sIPSC frequency, yet none of them completely blocked MeHg-induced increase in sIPSC frequency. Similarly, the mitochondrial Ca(2+) transport inhibitor RR, or a combination of lowering [Ca(2+)]o and BAPTA buffering reduced but did not prevent MeHg-induced changes in mIPSC frequency. Consistently, confocal Ca(2+) imaging under low [Ca(2+)]o conditions or in the presence of caffeine or CPA exhibited a marked reduction of MeHg-induced increases in [Ca(2+)]i in both molecular and granule layers. Thus, these results verify that a combination of extracellular Ca(2+) influx and Ca(2+) release from different intracellular Ca(2+) pools all contribute to MeHg-induced increase in [Ca(2+)]i and spontaneous GABA release, although extracellular Ca(2+) appears to be the primary contributor.

Topics: Animals; Caffeine; Calcium; Cerebellum; Environmental Pollutants; Female; In Vitro Techniques; Indoles; Inhibitory Postsynaptic Potentials; Male; Methylmercury Compounds; Microscopy, Confocal; Microscopy, Fluorescence; Patch-Clamp Techniques; Purkinje Cells; Rats, Sprague-Dawley; Thapsigargin

The effects of the environmental contaminants methylmercury (MeHg) and inorganic mercury (HgCl(2)) on cell viability, intracellular calcium concentration ([Ca(2+)](i)), and reactive oxygen species (ROS) generation were studied in rat cerebellar granule neuron cultures using fluorescent methods. MeHg exhibited an LC(50) (2.47 microM) tenfold lower than that of HgCl(2) (26.40 microM). To study the involvement of oxidative stress and Ca(2+) homeostasis disruption in mercury-induced cytotoxicity, we tested the neuroprotective effects of several agents that selectively interfere with these mechanisms. After a 24 hr exposure, the cytotoxic effect of both mercury compounds was reduced by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase; the Ca(2+) channel blocker flunarizine; and the Na(+)/Ca(2+) exchanger blocker benzamil. All these compounds decreased the mercury-mediated [Ca(2+)](i) rise. These results indicate that Ca(2+) influx through Ca(2+) channels and the Na(+)/Ca(2+) exchanger and Ca(2+) mobilization from the endoplasmic reticulum are involved in mercury-mediated cytotoxicity. The antioxidants probucol and propyl gallate reduced the HgCl(2) toxicity. Probucol and vitamin E partially inhibited the MeHg toxicity after a 24 hr period, whereas propyl gallate completely prevented this effect. Probucol slightly reduced ROS generation in methylmercury-exposed cultures and decreased mercury-mediated rise of [Ca(2+)](i). Propyl gallate abolished ROS generation and partially inhibited the increase of [Ca(2+)](i) induced by both mercury compounds. Propyl gallate also protected human cerebral cortical neuron cultures from the MeHg effect even after 72 hr of MeHg exposure, thus showing a long-lasting effect. Our data suggest that disruption of redox equilibrium and Ca(2+) homeostasis contribute equally to HgCl(2)-mediated toxicity, whereas oxidative stress is the main cause of MeHg neurotoxicity.

Topics: Amiloride; Animals; Antioxidants; Calcium; Calcium Channel Blockers; Cells, Cultured; Cerebellum; Cerebral Cortex; Disinfectants; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fetus; Flunarizine; Humans; Mercuric Chloride; Methylmercury Compounds; Neurons; Neuroprotective Agents; Oxidative Stress; Propyl Gallate; Rats; Rats, Wistar; Reactive Oxygen Species; Thapsigargin

The environmental contaminants methylmercury (MeHg) and mercuric chloride (HgCl2) stimulated the spontaneous release of [3H]noradrenaline ([3H]NA) from hippocampal slices in a time- and concentration-dependent manner. Both MeHg and HgCl2 were similarly potent, with an EC50 of 88.4 microM and 75.9 microM, respectively. The releasing effects of MeHg and HgCl2 increased in the presence of desipramine, showing that the mechanism does not involve reversal of the transmitter transporter, and were completely blocked by reserpine preincubation, indicating a vesicular origin of [3H]NA release. The voltage-gated Na+ channel blocker tetrodotoxin (TTX) did not affect the response to mercury compounds. [3H]NA release elicited by MeHg was partially dependent on extracellular Ca2+, since it decreased significantly in a Ca2+-free EGTA-containing medium whereas HgCl2 induced a release of [3H]NA independent of extracellular Ca2+. Neither Ca2+-channels blockers, cobalt chloride (CoCl2) and (omega-conotoxin-GVIA, nor the Na+/Ca2+-exchanger inhibitor benzamil reduced MeHg-evoked [3H]NA release. Moreover, thapsigargin or caffeine, endoplasmic reticulum Ca2+-depletors, did not modify metal-evoked [3H]NA release, whereas ruthenium red, which inhibits the mitochondrial Ca2+ transport, decreased the effect of both MeHg and HgCl2. All these data indicate that, in hippocampal slices, mercury compounds release [3H]NA from the vesicular pool by a mechanism involving Ca2+ mobilization from mitochondrial stores.

Topics: Adrenergic Uptake Inhibitors; Amiloride; Animals; Caffeine; Calcium; Calcium Channel Blockers; Chelating Agents; Chromatography, High Pressure Liquid; Cobalt; Desipramine; Egtazic Acid; Enzyme Inhibitors; Hippocampus; Male; Mercuric Chloride; Methylmercury Compounds; Norepinephrine; omega-Conotoxin GVIA; Rats; Rats, Wistar; Reserpine; Ruthenium Red; Sodium Channel Blockers; Sodium-Calcium Exchanger; Synapses; Tetrodotoxin; Thapsigargin