thapsigargin and benzamil

The central nervous system (CNS) pericytes play an important role in brain microcirculation. Na(+)/H(+) exchanger isoform 1 (NHE1) has been suggested to regulate the proliferation of nonvascular cells through the regulation of intracellular pH, Na(+), and cell volume; however, the relationship between NHE1 and intracellular Ca(2+), an essential signal of cell growth, is still not known. The aim of the present study was to elucidate the role of NHE1 in Ca(2+) signaling and the proliferation of human CNS pericytes. The intracellular Ca(2+) concentration was measured by fura 2 in cultured human CNS pericytes. The cells showed spontaneous Ca(2+) oscillation under quasi-physiological ionic conditions. A decrease in extracellular pH or Na(+) evoked a transient Ca(2+) rise followed by Ca(2+) oscillation, whereas an increase in pH or Na(+) did not induce the Ca(2+) responses. The Ca(2+) oscillation was inhibited by an inhibitor of NHE in a dose-dependent manner and by knockdown of NHE1 by using RNA interference. The Ca(2+) oscillation was completely abolished by thapsigargin. The proliferation of pericytes was attenuated by inhibition of NHE1. These results demonstrate that NHE1 regulates Ca(2+) signaling via the modulation of Ca(2+) release from the endoplasmic reticulum, thus contributing to the regulation of proliferation in CNS pericytes.

Topics: Amiloride; Brain; Calcium; Calcium Signaling; Cation Transport Proteins; Cell Proliferation; Cells, Cultured; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Microcirculation; Pericytes; RNA Interference; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Thapsigargin; Time Factors

The role of Na/Ca exchange and intracellular mobilized Ca2+ in modifying the depression of defensive behavior command neuron cholinosensitivity induced by the the Na,K pump inhibitor ouabain was studied in common snails using a cellular analog of habituation. Integral transmembrane acetylcholine-evoked currents (ACh currents) were recorded using a two-electrode membrane potential clamping technique. Decreases in neuron cholinosensitivity in the cellular analog of habituation were assessed in terms of the depth of depression of the amplitude of ACh currents during rhythmic local application of acetylcholine (with interstimulus intervals of 2-4 min) to the somatic membrane. The Na/Ca exchange inhibitor benzamyl (applied extracellularly, 15-35 microM) and two specific endoplasmic reticulum Ca-ATPase inhibitors, cyclopiazonic acid and thapsigargin (applied intracellularly. 0.1 mM) prevented modification of depression of the ACh current by ouabain (100 microM). It is concluded that Na/Ca exchange and the release of mobilized Ca2+ from intracellular calcium depots are involved in the mechanism by which the Na,K pump controls the depression of neuron cholinosensitivity in the cellular analog of habituation.

Topics: Acetylcholine; Amiloride; Animals; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Ganglia, Invertebrate; Helix, Snails; In Vitro Techniques; Indoles; Intracellular Fluid; Membrane Potentials; Neurons; Ouabain; Patch-Clamp Techniques; Receptors, Cholinergic; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase; 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