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Microglia are equipped with a strong proton (H ϩ ) extrusion pathway, a voltage-gated H ϩ channel, probably to compensate for the large amount of H ϩ generated during phagocytosis; however, little is known about how this channel is regulated in pathological states. Because neural damage is often associated with intracellular and extracellular acidosis, we examined the regulatory mechanisms of the H ϩ current of rat spinal microglia in acidic environments. More than 90% of round/amoeboid microglia expressed the H ϩ current, which was characterized by slow activation kinetics, dependencies on both intracellular and extracellular pH, and blockage by Zn 2ϩ . Extracellular lactoacidosis, pH 6.8, induced intracellular acidification and cell swelling. Cell swelling was also induced by intracellular dialysis with acidic pipette solutions, pH 5.5-6.8, at normal extracellular pH 7.3 in the presence of Na ϩ . The H ϩ currents were increased in association with cell swelling as shown by shifts of the half-activation voltage to more negative potentials and by acceleration of the activation kinetics. The acidosis-induced cell swelling and the accompanying potentiation of the H ϩ current required nonhydrolytic actions of intracellular ATP and were inhibited by agents affecting actin filaments (phalloidin and cytochalasin D). The H ϩ current was also potentiated by swelling caused by hypotonic stress. These findings suggest that the H ϩ channel of microglia can be potentiated via cell swelling induced by intracellular acidification. This potentiation might operate as a negative feedback mechanism to protect microglia from cytotoxic acidification and hence acidosis-induced swelling in pathological states of the CNS. Key words: H ϩ channel; lactoacidosis; cell swelling; microglia; pH regulation; ATP; cytochalasin D; cytoskeleton; spinal cordMicroglia are activated in response to various disorders of the CNS, including infection, ischemia, trauma, and neurodegenerative diseases, and they participate in both neuroprotective and neuropathological events (Streit, 1996). They are sensitive to the minor changes in their microenvironment present during very early stages of brain damage, such as subtle imbalances in ion homeostasis, and transform rapidly from the resting to the activated state (Gehrmann et al., 1993;Kreutzberg, 1996). Various ion channels in microglia are considered to contribute to the high responsiveness to pathological events and to be involved in maintenance of the neural microenvironment (Kettenmann et al., 1990;Nörenberg et al., 1994;Schlichter et al., 1996;Eder, 1998).A voltage-gated proton (H ϩ ) channel, first found in snail neurons (Thomas and Meech, 1982), has been suggested to be the mechanism for H ϩ extrusion responsible for compensation of intracellular acidification and for the dissipation of depolarization found in phagocytes that generate a massive amount of H ϩ during respiratory bursts (Lukacs et al., 1993;DeCoursey and Cherny, 1994). Similar H ϩ currents have been described in murine (Eder et al., 19...

Proton (H+) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow–derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly activated a voltage-dependent outward conductance with Q10 of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24°C and Q10 was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K+, Cl−, and Na+), but depended on the pH gradient, suggesting that H+ (equivalents) is a major ion species carrying the current. The H+ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H+ current was reversibly inhibited by Zn2+ but not by bafilomycin A1, an inhibitor for a vacuolar type H+-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn2+, and depletion of extracellular K+, but not by bafilomycin A1. These results suggest that the H+ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H+ conductance.

An outwardly rectifying Cl− (ORCl) current of murine osteoclasts was activated by hypotonic stimulation. The current was characterized by rapid activation, little inactivation, strong outward rectification, blockage by DIDS and permeability to organic acids (pyruvate and glutamate). The hypotonically activated ORCl current was inhibited by intracellular dialysis with an ATP‐free pipette solution, but not by replacement of ATP with a poorly hydrolysable ATP analogue adenosine 5′‐O‐(3‐thiotriphosphate). The current amplitude was reduced when intracellular alkalinity increased over the pH range 6.6–8.0. Intracellular application of cytochalasin D occasionally activated the ORCl current without hypotonic stress, but inhibited activation of the ORCl current by hypotonic stimulation. The hypotonically activated ORCl current was unaffected by a non‐actin‐depolymerizing cytochalasin, chaetoglobosin C, but partially inhibited by deoxyribonuclease I. Removal of extracellular Ca2+ inhibited activation of the ORCl current by hypotonic shock, but did not reduce the current once activated. The hypotonically activated ORCl current was partially decreased by intracellular dialysis with 20 mm EGTA. With 10 mm Ca2+ in the extracellular medium, the ORCl current was activated in response to more minor decreases in osmolarity than with 1 mm Ca2+. The increased sensitivity to hypotonicity was mimicked by increasing the intracellular Ca2+ level (pCa 6.5). These results suggest that hypotonic stimulation and a rise in the extracellular Ca2+ level synergistically activate the ORCl channel of murine osteoclasts, and that the activating process is modified by multiple intracellular factors (pH, ATP and actin cytoskeletal organization).