Search

Lipid emulsion infusion is a first-line therapy against the toxicity of local anesthetics and is a potential treatment for other drug overdoses, especially for highly lipophilic drugs. Considering the lipophilic property of volatile anesthetics, we hypothesized that lipid emulsion could reverse general anesthesia.Using adult rats, we tested the effect of lipid emulsion infusion on time to emergence after discontinuation of sevoflurane and isoflurane, and further evaluated restoration of righting reflex under continuous sevoflurane anesthesia. Electroencephalogram during lipid emulsion infusion was also investigated under continuous sevoflurane inhalation. The effect of lipid emulsion on sevoflurane-induced respiratory and hemodynamic depressions was evaluated by measuring respiratory rate, PaCOLipid emulsion infusion significantly decreased time to emergence from sevoflurane anesthesia (131 ± 53 vs. 237 ± 69 s) and restored righting reflex during continuous sevoflurane inhalation, by comparing normal saline infusion. Consistent with the behavioral findings, the electroencephalogram under continuous sevoflurane showed decreased power of theLipid emulsion facilitated reversal from volatile anesthetics, as shown by several parameters. As lipid emulsion could bind to volatile anesthetics and simply decrease their effects, our findings suggest that lipid emulsion is a potentially useful agent to reverse general anesthesia.

Background Local anesthetics, particularly potent long acting ones such as bupivacaine, can cause cardiotoxicity by inhibiting sodium ion channels; however, the impact of left ventricular hypertrophy on the cardiotoxicity and the underlying mechanisms remain undetermined. Transient receptor potential canonical (TRPC) channels are upregulated in left ventricular hypertrophy. Some transient receptor potential channel subtypes have been reported to pass relatively large cations, including protonated local anesthetics; this is known as the “pore phenomenon.” The authors hypothesized that bupivacaine-induced cardiotoxicity is more severe in left ventricular hypertrophy due to upregulated TRPC channels. Methods The authors used a modified transverse aortic constriction model as a left ventricular hypertrophy. Cardiotoxicity caused by bupivacaine was compared between sham and aortic constriction male rats, and the underlying mechanisms were investigated by recording sodium ion channel currents and immunocytochemistry of TRPC protein in cardiomyocytes. Results The time to cardiac arrest by bupivacaine was shorter in aortic constriction rats (n =11) than in sham rats (n = 12) (mean ± SD, 1,302 ± 324 s vs. 1,034 ± 211 s; P = 0.030), regardless of its lower plasma concentration. The half-maximal inhibitory concentrations of bupivacaine toward sodium ion currents were 4.5 and 4.3 μM, which decreased to 3.9 and 2.6 μM in sham and aortic constriction rats, respectively, upon coapplication of 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3 channel activator. In both groups, sodium ion currents were unaffected by QX-314, a positively charged lidocaine derivative, that hardly permeates the cell membrane, but was significantly decreased with QX-314 and 1-oleoyl-2-acetyl-sn-glycerol coapplication (sham: 79 ± 10% of control; P = 0.004; aortic constriction: 47± 27% of control; P = 0.020; n = 5 cells per group). Effects of 1-oleoyl-2-acetyl-sn-glycerol were antagonized by a specific TRPC3 channel inhibitor. Conclusions Left ventricular hypertrophy exacerbated bupivacaine-induced cardiotoxicity, which could be a consequence of the “pore phenomenon” of TRPC3 channels upregulated in left ventricular hypertrophy. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That New

Background Tricyclic antidepressants (TCAs) are a major cause of fatal drug poisoning due to their cardiotoxicity. Alkalinization by sodium bicarbonate (NaHCO3) administration, the first-line therapy for TCA-induced cardiotoxicity, can occasionally yield insufficient efficacy in severe cases. Because most TCAs are highly lipophilic, lipid emulsion may be more effective than alkalinization. However, it remains to be determined whether lipid emulsion is more beneficial than alkalinization in reversing amitriptyline-induced cardiotoxicity. Methods Hemodynamic variables were recorded from in vivo guinea pig models and Langendorff-perfused hearts. Whole-cell patch-clamp experiments were conducted on enzymatically isolated ventricular cardiomyocytes to record fast sodium currents (INa). Lipid solutions were prepared using 20% Intralipid. The pH of the alkaline solution was set at 7.55. We assessed the effect of lipid emulsion on reversing amitriptyline-induced cardiotoxicity, in vivo and in vitro, compared to alkalinization. The data were evaluated by Student t test, 1-way repeated-measures analysis of variance, or analysis of covariance (covariate = amitriptyline concentration); we considered data statistically significant when P Results In the in vivo model, intervention with lipids significantly reversed the amitriptyline-induced depression of mean arterial pressure and prolongation of QRS duration on electrocardiogram more than alkalinization (mean arterial pressure, mean difference [95% confidence interval]: 19.0 mm Hg [8.5-29.4]; QRS duration, mean difference [95% confidence interval] -12.0 milliseconds [-16.1 to -7.8]). In the Langendorff experiments, perfusion with 1% and 2% lipid solutions demonstrated significant recovery in left ventricular developed pressure (LVdevP), maximum change rate of increase of LVdevP (dP/dtmax) and rate-pressure product compared with alkaline solution (LVdevP [mm Hg], alkaline 57 ± 35, 1% lipid 94 ± 12, 2% lipid 110 ± 14; dP/dtmax [mm Hg/s], alkaline 748 ± 441, 1% lipid 1502 ± 334, 2% lipid 1753 ± 389; rate-pressure product [mm Hg·beats·minute], alkaline 11,214 ± 8272, 1% lipid 19,025 ± 8427, 2% lipid 25,261 ± 4803 with analysis of covariance). Furthermore, lipid solutions (0.5%-4%) resulted in greater recovery of hemodynamic parameters at 3 μM amitriptyline. Amitriptyline inhibited INa in a dose-dependent manner: the half-maximal inhibitory concentration (IC50) was 0.39 μM. The IC50 increased to 0.75 μM in the alkaline solution, 3.2 μM in 1% lipid solution, and 6.1 μM in 2% lipid solution. Furthermore, the lipid solution attenuated the use-dependent block of sodium channels by amitriptyline more than alkaline solution. On 30 consecutive pulses at 1 Hz, the current decreased to 50.1 ± 2.1, 60.3 ± 1.9, and 90.4% ± 1.8% in standard, alkaline, and 1% lipid solution, respectively. Even 0.5% lipid solution showed greater effects than the alkaline solution in all experiments. Conclusions Lipid emulsion significantly suppressed amitriptyline-induced INa, inhibition, which was likely related to the marked improvement in hemodynamic status observed in vivo and in isolated perfused hearts. These results suggest the superiority of lipid emulsion as the first-line therapy for TCA-induced cardiotoxicity compared to alkalinization therapy.

A proton is a ubiquitous signaling ion. Many transmembrane H+ transport pathways either maintain pH homeostasis or generate acidic compartments. The osteoclast is a bone-resorbing cell, which degrades bone tissues by secreting protons and lysosomal enzymes into the resorption pit. The plasma membrane facing bone tissue (ruffled border), generated partly by fusion of lysosomes, may mimic H+ flux mechanisms regulating acidic vesicles. We identified three electrogenic H+-fluxes in osteoclast plasma membranes—a vacuolar H+-ATPase (V-ATPase), a voltage-gated proton channel (Hv channel) and an acid-inducible H+-leak—whose electrophysiological profiles and regulation mechanisms differed. V-ATPase and Hv channel, both may have intracellular reservoirs, but the recruitment/internalization is regulated independently. V-ATPase mediates active H+ efflux, acidifying the resorption pit, while acid-inducible H+ leak, activated at an extracellular pH

The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet–induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+–induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency–induced skeletal diseases.

Osteoclasts are highly differentiated bone-resorbing cells and play a significant role in bone remodelling. In the resorption pit, inorganic phosphate (Pi) concentrations increase because of degradation of hydroxyapatite. We studied effects of extracellular Pi on voltage-gated H+ channels in osteoclast-like cells derived from a macrophage cell line (RAW264). Extracellular Pi (1.25–20 mM) increased the H+ channel currents dose dependently and reversibly. The Pi-induced increases were attenuated by removal of extracellular Na+ and by phosphonoformic acid, a blocker of Na+-dependent Pi transporters. Pi increased the maximal conductance, decreased activation time constant, increased deactivation time constant, and shifted the conductance-voltage relationship to more negative voltages. The most marked change was enhanced gating which was mainly caused by elevation of intracellular Pi levels. The Pi-induced enhanced gating was partially inhibited by protein kinase C (PKC) inhibitors, GF109203X and staurosporine, indicating that PKC-mediated phosphorylation was involved in part. The increase in the maximal conductance was mainly due to accompanying decrease in intracellular pH. These effects of Pi were not affected by intracellular Mg2+, bafilomycin A1 (V-ATPase inhibitor) and removal of intracellular ATP. Extracellular Pi also upregulated reactive oxygen species (ROS). Diphenyleneiodonium chloride, an inhibitor of NADPH oxidases, decreased ROS production and partially attenuated the enhanced gating. In the cells during later passages where osteoclastogenesis declined, H+ channel activities and ROS production were both modest. These results suggest that, in osteoclasts, ambient Pi is a common enhancer for H+ channels and ROS production and that potentiation of H+ channels may help ROS production.

Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H(+)-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to

To achieve the most accurate assessment of functional Ca

To achieve the most accurate assessment of functional Ca2+ channel or modulator properties and their regulation, a patch clamp technique to record membrane currents is required. This technique has wide applications ranging from recording the activity of native channels in their natural environment to that of recombinant channels expressed in heterologous cells. This chapter introduces the methods that have been used for the detection of calcium release-activated calcium (CRAC) currents, one of the store-operated calcium entry pathways, in human primary T cells. This standard protocol is for laboratories already equipped with a full patch clamp setup or for investigators collaborating with laboratories experienced in patch clamp.

Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (VD3) are important factors in Ca(2+) homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca(2+) level ([Ca(2+)]i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD3 increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca(2+)]i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca(2+) channels and Ca(2+) release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD3-induced RANKL-lv fusion included the membrane-bound VD3 receptor (1,25D3-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD3 will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.

Key points • Voltage-gated H+ channels help to compensate for the pH and voltage disturbances generated by production of reactive oxygen species. • In this study, we investigated how changes in the intracellular pH levels control H+ channel activity in macrophage-lineage cells, osteoclasts and microglia. • An increase in intracellular pH decreased the numbers of H+ channels available at the plasma membrane through facilitation of dynamin-dependent endocytic internalization. • This inhibitory regulation mechanism for H+ channels is novel. • The results help us to understand better the significance of the intracellular pH levels in membrane dynamics and H+ channel availability, which, in turn, may modulate natural immunity. Abstract Voltage-gated proton channels (H+ channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H+ channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H+ channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H+ channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H+ channel conductance were probably due to the decreases in H+ channels available at the plasma membrane. The decreases in the H+ channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H+ channels, pHi increases by these H+-transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H+ channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H+ channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.

Ca2+ release-activated calcium channel 3 (CRACM3) is a unique member of the CRAC family of Ca2+-selective channels. In a non-excitable exocytosis model, we found that the extracellular L3 domain and the cytoplasmic C-terminus of CRACM3 interacted in an activity-dependent manner with the N-peptide of syntaxin4, a soluble N-ethylmaleimide-sensitive factor attachment receptor protein. Our biochemical, electrophysiological and single-vesicle studies showed that knockdown of CRACM3 suppressed functional exocytosis by decreasing the open time of the vesicle fusion pore without affecting Ca2+ influx, the activity-dependent membrane capacitance (Cm) change, and the total number of fusion events. Conversely, overexpressing CRACM3 significantly impaired cell exocytosis independent of Ca2+, led to an impaired Cm change, decreased the number of fusion events, and prolonged the dwell time of the fusion pore. CRACM3 changes the stability of the vesicle fusion pore in a manner consistent with the altered molecular expression. Our findings imply that CRACM3 plays a greater role in exocytosis than simply acting as a compensatory subunit of a Ca2+ channel.

The purinergic P2X4 receptors (P2X4Rs) of spinal microglia are upregulated after a peripheral nerve injury and play important roles in the pathogenesis of chronic pain. The effects of general anesthetics on chronic pain and the mechanisms are still unclear. The aim of this study is to examine the effects of general anesthetics on microglial P2X4Rs. Currents induced by ATP were recorded by the whole-cell clamp technique using a mouse microglial cell line (MG5). Isoflurane and sevoflurane, ketamine, thiopental, midazolam, and propofol were coapplied with ATP using the U-tube system or added to the external perfusate. ATP-induced two distinct types of current: P2X4R-mediated and P2X7R-mediated currents. P2X4R-mediated currents were identified pharmacologically and isolated. Volatile anesthetics including sevoflurane and isoflurane and intravenous anesthetics including thiopental, ketamine, and midazolam had no effect at clinically relevant concentrations (n=5-8). Propofol showed a dual effect, potentiating at lower concentrations (0.3-3 µM) and inhibiting at higher concentrations (IC50 57 µM). The maximum enhancement was observed at 1 µM propofol (143±5% of control, n=5). Propofol (1 µM) shifted the dose-response curve for the P2X4R currents to lower concentrations of ATP and increased the maximum amplitude. Propofol exerted dual actions on P2X4R-mediated currents at clinically relevant concentrations. This may suggest that the administration of propofol could affect the development of chronic pain through the modulation of microglial P2X4R responses.

Voltage-gated proton channels play crucial roles during the respiratory burst in phagocytes, such as microglia. As local anaesthetics have a variety of anti-inflammatory properties, including inhibition of phagocytosis, they may act on the proton channels. Most local anaesthetics are tertiary amines and may affect proton channels through modification of pH(i) as weak bases. To test these hypotheses, the effects of lidocaine and bupivacaine on proton channels were examined in a rat microglial cell line (GMI-R1) as a function of pH(o) and pH(i). Both lidocaine and bupivacaine reversibly decreased the current, with IC(50) values of ∼1.2 and ∼0.5 mM, respectively, at pH(o)/pH(i) 7.3/5.5. The inhibition was enhanced with either pH(o) increase or pH(i) decrease, suggesting that the protonation of the base forms inside the cell contributed to the inhibitory effects. Both local anaesthetics shifted the reversal potentials to more positive voltages, indicating increases in pH(i). The potencies of inhibition were correlated well with the degree of increase in pH(i). The lidocaine-induced inhibition was eliminated when the pH(i) increases were cancelled by co-application of a weak acid, butyrate. The cytosolic alkalizations by lidocaine and bupivacaine were confirmed using a pH-sensitive fluorescent dye, BCECF, in non-voltage-clamped cells. Furthermore, chemiluminescence measurement proved that both anaesthetics inhibited production of reactive oxygen species by the cells. In conclusion, lidocaine and bupivacaine inhibit proton channels primarily by the weak base mechanism via an increase in pH(i). This is a novel mechanism underlying actions of local anaesthtics.

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (Iratio) were determined. The use of Iratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (DeltaT, -15 to 15 degrees C) were applied over a wide temperature range (4-49 degrees C), and the Q10s for the proton currents were evaluated from the Iratios. Q10 exhibited a high temperature dependence, varying from 2.2 at 10 degrees C to 1.3 at 40 degrees C. This implies that processes with different temperature dependencies underlie the observed Q10. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q10 was decomposed into Q10 of the channel and of the access resistances. Finally, the Q10 for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5 degrees C to 2.2 at 45 degrees C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.

BACKGROUND: Microglial cells play important roles in coordinating the inflammatory brain responses to hypoxia and trauma. Ionotropic P2X receptors and metabotropic P2Y receptors (P2YRs) expressed in microglia can be activated by extracellular adenosine triphosphate (ATP) derived from damaged cells or astrocytes, and participate in the signaling pathways evoked in brain insult. Although several inhaled and IV anesthetics produce neuroprotective effects through neuronal mechanisms, little is known about how general anesthetics modulate microglial responses in the pathological state. We examined the effects of various general anesthetics on purinergic responses in a rat microglial cell line. METHODS: Currents were consistently activated by applications of ATP via a U-tube system under the whole-cell configuration. ATP-induced nondesensitizing currents observed after several applications of ATP exhibited characteristics of P2X 7 receptors. The P2YRs-mediated mobilization of intracellular Ca 2+ was measured using a Ca 2+ -sensitive fluorescent dye (fura-2). RESULTS: Inhaled anesthetics (sevoflurane, isoflurane, and halothane) at doses three times as high as minimum alveolar concentrations had no effect on the P2X 7 Rs-mediated currents. IV anesthetics (ketamine, propofol, and thiopental) enhanced the P2X 7 Rs-mediated currents reversibly. The potencies for activation of P2X 7 Rs were not correlated with the octanol/buffer partition coefficients. Thiopental, at low concentrations, slightly inhibited the P2X 7 Rs-mediated currents, suggesting its dual actions on P2X 7 Rs. The P2YRs-mediated mobilization of intracellular Ca 2+ was not affected by any of the general anesthetics tested. CONCLUSIONS: Our results suggest that IV anesthetics, particularly thiopental and propofol, may modulate microglial functions through P2X 7 Rs in pathological conditions.

The vacuolar-type H+-ATPase (V-ATPase) in the plasma membrane of a variety of cells serves as an acid-secreting pathway, and its activity is closely related to cellular functions. Massive proton secretion often leads to electrolyte disturbances in the vicinity of the cell and may in turn affect the activity of the V-ATPase. We characterized, for the first time, the proton currents mediated by plasmalemmal V-ATPase in murine osteoclast-like cells and investigated its activity over a wide range of pH gradients across the membrane (ΔpH = extracellular pH – intracellular pH). The V-ATPase currents were identified as outward H+ currents and were dependent on ATP and sensitive to the inhibitors bafilomycin A1 and N,N′-dicyclohexylcarbodiimide. Although H+ was transported uphill, the electrochemical gradient for H+ affected the current. The currents were increased by elevating ΔpH and depolarization, and were reduced by lowering ΔpH and hyperpolarization. Elevation of extracellular Ca2+ (5–40 mm) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg2+ mimicked the inhibition. With 40 mm Ca2+, the currents decreased to < 40% at 0 mV and to < 10% at about −80 mV. Increases in the intracellular Ca2+ (0.5–5 μm) did not affect the current. The data suggest that acid secretion through the plasmalemmal V-ATPase is regulated by a combination of the pH gradient, the membrane potential and the extracellular divalent cations. In osteoclasts, the activity-dependent accumulation of acids and Ca2+ in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase.

The streaming potential (Vstream) is a signature feature of ion channels in which permeating ions and water molecules move in a single file. Vstream provides a quantitative measure of the ion and water flux (the water–ion coupling ratio), the knowledge of which is a prerequisite for elucidating the mechanisms of ion permeation. We have developed a method to measure Vstream with the whole-cell patch-clamp configuration. A HEK293 cell stably expressing the HERG potassium channel was voltage clamped and exposed to hyperosmotic solutions for short periods of time (

UNLABELLED The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. INTRODUCTION H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings. METHODS To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). RESULTS AND CONCLUSIONS Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (delta(pH)), indicating that the H+ channel changed its activity by sensing delta(pH). The reversal potential (V(rev)) was a valuable tool for the real-time monitoring of delta(pH) in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 microM) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of V(rev), suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K(+)-rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In approximately 80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the delta(pH) changes, implying that both delta(pH)-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward V(rev). These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C.

Ion channels and transporters act as major components that regulate membrane excitability in neurons, muscles, and some secretory glands, but may also contribute to the regulation of proliferation, differentiation, and death in a greater variety of cells including non-excitable ones. The molecular basis of ionic mechanisms underlying the later regulation has been partly identified in the last several years and is a hot issue now. In this short review, some of the molecular mechanisms underlying these regulations and novel compounds acting on the mechanisms were introduced as exciting topics in this area. Several types of transient receptor potential (TRP), identified as Ca(2+)-permeable, non-selective cation channels, may play obligatory roles in functional complexes, which regulate multiple signal transduction pathways triggering proliferation, differentiation, or death of many cell types. In addition, the relation between Cl(-) pump activity and the induction of beta-amyloid protein toxicity for neuronal cell death in Alzheimer disease was described. Unique functions of H(+) channel and pump in osteoclasts in bone mineral homeostasis and remodeling were also discussed. Finally, topics about activation of specific types of Cl(-) channels and K(+) channels, which are responsible for the induction of apoptosis or proliferation in several types of cells, were introduced.

An elevation in the extracellular Ca(2+) concentration ([Ca(2+)](o)) is a key signal for bone remodeling by inhibiting the resorbing activity of osteoclasts. The [Ca(2+)](o)-sensing responses include a variety of morphological and functional changes, but the underlying mechanisms are yet to be defined. This study was aimed at investigating the [Ca(2+)](o)-sensing mechanisms leading to the activation of the Cl(-) channel in murine osteoclasts. A rise in either Ca(2+) or Gd(3+) activated an outwardly rectifying Cl(-) (OR(cl)) channel reversibly and dose-dependently, which was characterized by rapid activation kinetics, little inactivation, and blockage by DIDS. The concentration required for a half-maximal response was estimated to be20-30 mmol/L for Ca(2+). Intracellular dialysis with an ATP-free pipette solution or application of an actin destabilizer, cytochalasin D, decreased the [Ca(2+)](o)-activated OR(cl) current. Substitution of extracellular Na(+) by an impermeable cation, N-methyl-D-glucamine(+), inhibited the [Ca(2+)](o)-activated OR(cl) channel, suggesting that the activation depended on extracellular Na(+). A blocker for the Na(+)-Ca(2+) exchanger, 2'4'-dichlorobenzamil hydrochloride (DCB), inhibited the [Ca(2+)](o)-activated OR(cl) channel as well. Although 10 mmol/L Ca(2+) activated the OR(cl) current only slightly at a standard intracellular pH (7.3), decreasing pH by dialyzing cells with an acidic pipette solution (pH 6.6) enhanced the [Ca(2+)](o)-activated OR(cl) current. This potentiation by cell acidosis was eliminated by amiloride, a blocker for the Na(+)-H(+) exchanger. Zinc ion (0.1 mmol/L) and a polycation, neomycin (0.2 mmol/L), activated the OR(cl) current at intracellular pH 6.6, whereas the effects of those cations were negligible at intracellular pH 7.3. These results suggest that [Ca(2+)](o)-sensing mechanisms, leading to activation of the OR(cl) channel in murine osteoclasts, are regulated by ATP and actin cytoskeletal organization, and are sensitized greatly by cell acidosis. Contributions of Na(+)-dependent transporters in this activating process are examined in the context of a possible intermediate signal of cell swelling caused by Na(+) influx.

Zinc is a trace element in the mammalian body, and increasing evidence shows its critical role in bone development and osteoclastogenesis. The relationships between zinc and voltage-gated ion channels have been reported; however, the effects of zinc on membrane potential and the related ion channels remain unknown. In this study, we found that zinc-induced hyperpolarization in RAW264.7 cells (RAW) was promoted by inhibition of hyperpolarization-activated cyclic nucleotide modulated channels (HCNs). In electrophysiological experiments with RAW-derived osteoclasts, HCNs were functional and generated hyperpolarization-activated inward currents (Ih) with properties similar to the Ih recorded in excitable cells such as neurons and cardiomyocytes. Quantitative PCR of HCN subunits HCN1 and HCN4 in RAW cells showed detectable levels of HCN1 mRNA and HCN4 expression was the highest of all four subunits. HCN4 knockdown decreased osteoclastic Ih and promoted osteoclastogenesis in the presence of zinc, but not in the absence of zinc. To determine the effect of membrane hyperpolarization on osteoclastogenesis, we developed a light-controllable membrane potential system in RAW cells by stably expressing the light-driven outward proton pump, Archaerhodopsin3 (Arch). Arch activation by yellow-green light hyperpolarizes the cell membrane. Light-induced hyperpolarization accelerated osteoclast differentiation in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL). Thus, HCN activation reduced the hyperpolarization-related promotion of osteoclast differentiation in the presence of zinc. This study revealed the novel role of HCN and membrane potential in non-excitable osteoclasts.

Effects of the extracellular Ca2+ concentration ([Ca2+] o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca2+ was mediated by a Ba2+-sensitive, inwardly rectifying K+ (IRK) current. A rise in [Ca2+] o (5–40 mm) inhibited the IRK current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl− (ORCl) current. The activation of the ORCl current developed slowly and needed higher [Ca2+] o than that required to inhibit the IRK current. The inhibition of the IRK current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the ORCl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca2+ level neither reduced the IRK current nor activated the ORCl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca2+] o -induced changes in the IRK and ORCl conductances. These results suggest that high [Ca2+] o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IRK conductance and an activation of an ORCl conductance. The two conductances modulated by [Ca2+] o may be involved in different phases of bone resorption because they differed in Ca2+ sensitivity, temporal patterns of changes and regulatory mechanisms.

The regulated control of Ca(2+) influx is essential for the activation and function of the adaptive immune response, as Ca(2+) is a key regulator of important transcription factors. To determine whether Ca(2+) release-activated Ca(2+) (CRAC) channels contribute to the abnormal behaviour of T cells in patients with rheumatoid arthritis (RA), we performed a cross-sectional study to characterize the expression and functional status of CRACM1 channels in RA patients. Peripheral blood was obtained from 50 RA patients, 50 osteoarthritis (OA) patients and healthy donors. We measured Ca(2+) influx and CRAC currents in naïve and memory CD4(+) T cells. CRACM1 expression was evaluated in T cells from each of the three groups. These cells were further characterized by flow cytometric analysis of interleukin-4 (IL-4), IL-17, interferon-γ and tumour necrosis factor-α. These cytokines were also measured in naïve CD4(+) T cells following the lentivirus-mediated silencing of CRACM1.There was a significant positive correlation between Ca(2+) influx in naïve T cells and RA activity. Functionally aberrant naïve CD4(+) T cells from patients with active RA showed the different cytokine release pattern and exhibited increased Ca(2+) influx as well as increased CRACM1 protein expression and function. Specific lentiviral-induced gene silencing of CRACM1 reversed the alterations in T-cell cytokine production. The data presented here indicate that an upregulation of CRACM1 expression and function may be responsible for the abnormal cytokine release of naïve CD4(+) T cells in RA patients. CRACM1 might therefore represent a new molecular target for RA therapies.

Voltage-gated proton channels (H(+) channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H(+) channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H(+) channel conductance did not recover fully when the exposure was prolonged (5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H(+) channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H(+) channel conductance were probably due to the decreases in H(+) channels available at the plasma membrane. The decreases in the H(+) channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H(+) channels, pHi increases by these H(+)-transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H(+) channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H(+) channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.

Inositol-1,4,5-trisphosphate (IP3)-activated calcium-permeable channels were recorded from inside-out patches of cultured cerebellar Purkinje cells. When 2-5 microM of IP3 was applied to the internal surface of the inside-out patches, inward Ba2+ currents were activated within 10 sec following the application in 11 out of 24 patches. In the presence of heparin (100 micrograms/ml), activation of Ba2+ currents by IP3 was inhibited. Unitary currents with different amplitudes and kinetics were observed; small and large unitary currents, and rapid fluctuations with various amplitudes. The small unitary currents (single channel conductance; 5.6 pS) were most frequent. Addition of inositol 1,3,4-trisphosphate (2-5 microM) slightly activated Ba2+ currents in 2 out of 10 patches, but the amount of the increment was much smaller than that produced by IP3. These results suggest a possibility that IP3 directly activates Ca(2+)-permeable channels in the plasma membrane of cerebellar Purkinje cells.

Voltage-gated proton channels play crucial roles during the respiratory burst in phagocytes, such as microglia. As local anaesthetics have a variety of anti-inflammatory properties, including inhibition of phagocytosis, they may act on the proton channels. Most local anaesthetics are tertiary amines and may affect proton channels through modification of pH(i) as weak bases. To test these hypotheses, the effects of lidocaine and bupivacaine on proton channels were examined in a rat microglial cell line (GMI-R1) as a function of pH(o) and pH(i). Both lidocaine and bupivacaine reversibly decreased the current, with IC(50) values of ∼1.2 and ∼0.5 mM, respectively, at pH(o)/pH(i) 7.3/5.5. The inhibition was enhanced with either pH(o) increase or pH(i) decrease, suggesting that the protonation of the base forms inside the cell contributed to the inhibitory effects. Both local anaesthetics shifted the reversal potentials to more positive voltages, indicating increases in pH(i). The potencies of inhibition were correlated well with the degree of increase in pH(i). The lidocaine-induced inhibition was eliminated when the pH(i) increases were cancelled by co-application of a weak acid, butyrate. The cytosolic alkalizations by lidocaine and bupivacaine were confirmed using a pH-sensitive fluorescent dye, BCECF, in non-voltage-clamped cells. Furthermore, chemiluminescence measurement proved that both anaesthetics inhibited production of reactive oxygen species by the cells. In conclusion, lidocaine and bupivacaine inhibit proton channels primarily by the weak base mechanism via an increase in pH(i). This is a novel mechanism underlying actions of local anaesthtics.

The vacuolar-type H+-ATPase (V-ATPase) is an electrogenic H+ pump that is distributed widely in living organisms. Voltage-gated proton channels (H+ channels) are found in many types of cells including phagocytes. Although their transport mechanisms are distinct, both depend on pH and voltage and share the outcomes, intracellular alkalinization (extracellular acidification) and hyperpolarization. So far, V-ATPases and H+ channels have been studied separately in different cells, using the different approaches under different conditions. V-ATPases and H+ channels are expressed in the plasma membrane of osteoclasts, multinuclear bone-resorbing cells, which allowed us to investigate the two electrogenic H+ transfer mechanisms quantitatively under the same conditions. The current-voltage relationships of the V-ATPases are linear and outward currents were maintained even when the extracellular space was more acidic than the cell inside. Protons flowed through the V-ATPase continuously. In contrast, H+ channels were silent at voltages lower than the threshold. However, once it opened, the H+ channel current surpassed the V-ATPase current. Consequently, the profile of H+ efflux was composed of two phases, one dominated by the V-ATPase and the other, by the H+ channel. Inhibition of V-ATPases by bafilomycin was often accompanied by decreases in the H+ channel current. These results suggested that the V-ATPase and the H+ channel work over different ranges of pH- and voltage-gradients across the membrane in single cells and co-regulate the pH environments of osteoclasts.

Effects of inhibitors of the arachidonic acid cascade on Ca2+ release from intracellular stores and Ca2+ influx through the plasma membrane during stimulus-secretion coupling were examined using rat peritoneal mast cells loaded with fura-2. Compound 48/80 (48/80) was used as a secretagogue. A phospholipase inhibitor, p-bromophenacyl bromide (PBPB), or a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), inhibited the 48/80 (1 microgram/ml)-induced release of histamine, Ca2+, and Mn2+ influxes, but the cyclooxygenase inhibitor, indomethacin (approximately 50 microM), inhibited neither Ca2+ nor Mn2+ influxes. The Ca2+ release induced by 1 microgram/ml of 48/80 was little inhibited by PBPB, NDGA, or indomethacin. The Ca2+ release was activated and saturated with lower concentrations of 48/80 than was the Ca2+ influx. The percent inhibition of the Ca2+ release by 25 microM PBPB was increased by lowering the concentration of 48/80, but NDGA (10 microM) did not inhibit the Ca2+ release induced by low concentrations of 48/80 (0.03-0.1 microgram/ml). These results suggest that activation of the Ca2+ release and the Ca2+ influx were differently regulated and that full activation of Ca2+ influx needs the arachidonic acid cascade produced by higher concentrations of 48/80 than does the Ca2+ release. Lipoxygenase metabolites of arachidonic acid are potential modulators of the Ca2+ influx.

In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance ( Cm) simultaneously. Cm is a measure of cell surface. Extracellular Ca2+ (2.5–40 mM) decreased Cm and the V-ATPase current simultaneously. The decreased Cm, together with the enhanced uptake of a lipophilic dye (FM1–43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A1, a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C γ2 subunit. Holding the cytosolic Ca2+ at either high (0.5–5 μM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.

Voltage-gated proton (H+) channels play a pivotal role in compensating charge and pH imbalances during respiratory bursts in phagocytes. Lactic acidosis is a clinically important metabolic condition accompanying various tissue disorders in which the extracellular pH and the intracellular pH often change in parallel. In this study, we investigated the responses of the H+ channel in microglia to lactate-induced pH disturbances using the perforated-patch recordings. Na-lactate (pH 6.8) acidified the cells and activated the H+ channel within 5 min. This early activation was correlated with increases in the pH gradient across the plasma membrane (DeltapH) and was dose-dependent over a concentration range of 10-150 mM. At 10 mM, the change in DeltapH was only slight, but the H+ currents continued to increase over an hour after the cell acidosis was stabilized. Prolonged exposure to lactate (10-20 mM,1 h) increased the amplitude by two to threefold. The late activation was not explained by increased DeltapH but by changes in the property of the channel per se. Pretreatment with staurosporine and chelerythrine, inhibitors for protein kinase C, prevented the late activation. These results suggest that the H+ channel could be activated greatly during long-lasting lactic acidosis through both DeltapH-dependent and -independent mechanisms.

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by "gain of function" of glutamate dehydrogenase (GDH). Several missense mutations have been found; however, cell behaviors triggered by the excessive GDH activity have not been fully demonstrated. This study was aimed to clarify electrophysiological mechanisms underlying the dysregulated insulin secretion in pancreatic beta cells with GDH mutations. GDH kinetics and insulin secretion were measured in MIN6 cells overexpressing the G446D and L413V. Membrane potentials and channel activity were recorded under the perforated-patch configuration that preserved intracellular environments. In mutant MIN6 cells, sensitivity of GDH to guanosine triphosphate (GTP) was reduced and insulin secretion at low glucose concentrations was enhanced. The basal GDH activity was elevated in L413V bearing a mutation in the antenna-like structure. The L413V cells were depolarized without glucose, often accompanying by repetitive Ca2+ firings. The depolarization was maintained in the presence of adenosine triphosphate (ATP) and disappeared by depleting ATP, suggesting that the depolarization depended on intracellular ATP. In L413V cells, the ATP-sensitive potassium channel (K(ATP) channel) was suppressed and the nonselective cation channel (NSCC) was potentiated, while sensitivity of the channels to their specific blockers or agonists was not impaired. These data suggest that the L413V cells increase the intracellular ATP/adenosine diphosphate (ADP) ratio, which in turn causes sustained depolarization not only by closure of the K(ATP) channel, but also by opening of the NSCC. The resultant activation of the voltage-gated Ca2+ channel appears to induce hyperinsulinism. The present study provides evidence that multiple channels cooperate in unregulated insulin secretion in pancreatic beta cells of the HI/HA syndrome.

Osteoclasts secrete a large amount of proton (H+) ions and proteolytic enzymes into bone resorption pit to degrade bone matrix. In addition to H+ pumps and exchangers, voltage-gated H+ channels, which are H+ conductive pathways, are expressed in osteoclasts. H+ channels are distinct in their strong H+ extrusion ability, but the functional role is not clear. This is the first study of H+ channels in murine osteoclasts generated from mononuclear precursors in the presence of a soluble form of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). The H+ channel was characterized by voltage- and pH-dependent activation, slow activation kinetics, and outward rectification. The reversal potential (Vrev) was shifted to more positive potentials by decreasing the pH gradient across the plasma membrane (deltapH). Employing Vrev as a real time monitor of pH in clamped cells, it is revealed that the H+ channel activation could decrease deltapH by approximately 0.43 unit/s. Decline in the current during prolonged depolarizations was accompanied by a positive shift in Vrev. This implies that the H+ channel activity is auto-regulated by sensing deltapH, to compensate for pH imbalance. The H+ current-density in cells with 36 nuclei was significantly smaller than that in cells withor =5 nuclei, although the activation rate was unaltered. Thus, the H+ channel activity may alter during osteoclastogenesis. These data suggest the H+ channel is a powerful mechanism for pH homeostasis of osteoclasts that are exposed to drastic change in pH environments during the bone resorption cycle.

Structural rearrangement and synaptic reorganization are known to occur in the brain after seizures. If neuronal rearrangement after seizures always results in abnormal hyperexcitability, it would provide an accurate pathway to the appropriate target and as a result, it may be the mechanism of epileptogenesis. This study examined the mechanism of axon guidance in the mature rat brain after seizures by evaluating the expression of the axonal guidance molecule, neuropilin-2. We assessed the expression of neuropilin-2 by northern blotting and immunohistochemistry in rat with seizures created by kindling stimulation and kainate injection.The neuropilin-2 mRNA level was increased in the whole brain of the rats at 24 h after either type of seizure. Neuropilin-2 mRNA was not increased at 2 weeks after the last stimulation. Immunohistochemistry demonstrated that neuropilin-2 protein was increased in the dentate gyrus and the entorhinal cortex in the both seizure models. These findings suggested that there was overexpression of neuropilin-2 in the brains of mature rats with different types of seizure. Accordingly, neuropilin-2 might regulate remodeling after seizures as it does during the development of the hippocampal formation. Our findings suggest that axons may not project and outgrow 'aberrantly' after seizures, but may be regulated by the chemorepellent effect through neuropilin-2.

Osteoclasts possess extracellular Ca2+-sensing machinery to regulate bone-resorbing activity. In murine osteoclasts, an elevation of the extracellular Ca2+ concentration activated a volume-sensitive outwardly rectifying Cl- (OR(Cl)) channel. Exposure to 40 mM Ca2+ activated the OR(Cl) current in association with an increase in the planar cell area. An actin-destabilizer (cytochalasin D), removal of a major extracellular osmolyte (Na+), and a blocker for the Na+/Ca2+ exchanger (2'4'-dichlorobenzamil hydrochloride) inhibited both swelling and activation of the OR(Cl) channel by extracellular Ca2+. There was a positive correlation between the current density and increment in the cell area. The extracellular Ca2+-induced swelling was confirmed in intact (unclamped) cells by three-dimensional analysis using confocal scanning microscopy with a fluorescent dye (BCECF) in the extracellular medium. These results suggest that swelling is an intermediate signal for extracellular Ca2+ sensing of osteoclasts, which leads to activation of the OR(Cl) channel. Cell volume may be a second-message in the regulation of osteoclast function.

Voltage-gated proton (H(+)) channels are unique mechanisms to extrude a massive amount of H(+), and are proposed to regulate intracellular pH of microglia during respiratory bursts. Temporal variations of the H(+) current were studied in rat spinal microglia cultivated on the glial cell layer using the voltage-ramp protocol. Repetitive applications of the large and long-lasting depolarization decreased the amplitudes of the H(+) current transiently and reversibly. This decrease was accompanied by a shift of the reversal potential to a more positive direction, indicating that a drop in the transmembrane pH gradient (delta pH) by the H(+) efflux through the channel reduced the current. The decline of the H(+) current during depolarizations was also observed in a rat microglial cell line (GMI-R1). An increase in the extracellular buffer suppressed the reduction of the current, suggesting that H(+) secreted into the extracellular space contributed to the drop in delta pH. On the other hand, the amplitudes of the H(+) current often fluctuated greatly at intervals of 5-20 min without changes in delta pH. These results suggest that the H(+) current of microglia is tuned via both delta pH-dependent and -independent mechanisms, which may regulate both microglial behavior and the pH environments of the surrounding neural tissue.

Microglia are equipped with a strong proton (H(+)) extrusion pathway, a voltage-gated H(+) channel, probably to compensate for the large amount of H(+) 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(+) current of rat spinal microglia in acidic environments. More than 90% of round/amoeboid microglia expressed the H(+) current, which was characterized by slow activation kinetics, dependencies on both intracellular and extracellular pH, and blockage by Zn(2+). 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(+). The H(+) 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(+) current required nonhydrolytic actions of intracellular ATP and were inhibited by agents affecting actin filaments (phalloidin and cytochalasin D). The H(+) current was also potentiated by swelling caused by hypotonic stress. These findings suggest that the H(+) 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.

Osteoclasts secrete protons and various enzymes to degrade bone matrix, and play a crucial role in bodily Ca2+ homeostasis. Both organic and inorganic bone degradation products are transcytosed through osteoclasts and liberated into the extracellular space (Nesbitt & Horton, 1997; Salo et al. 1997). During the bone resorption cycle, osteoclasts face dynamic changes in diverse extracellular/intracellular environmental factors, such as osmolarity, Ca2+ levels, pH, metabolic activities and organization of cytoskeletons (Zaidi et al. 1993; Lakkakorpi & Vaananen, 1996). The ruffled membrane proton secretion via a vacuolar-type H+-ATPase requires anion efflux through the Cl− channel. Resultant accumulation of HCO3− and reduction of intracellular Cl− are compensated for via the HCO3−-Cl− exchanger in the basal membrane (Schlesinger et al. 1994). Transmembrane Cl− flux is thus closely related to the functional state of osteoclasts. Chloride channels have been demonstrated in rat (Sims et al. 1991), avian (Blair & Schlesinger, 1990), rabbit (Kelly et al. 1994) and murine osteoclasts (Shibata et al. 1997), but still little is known about their role in osteoclast functions. So far, the only known activating extracellular stimulus for Cl− channels of osteoclasts is either a rise in the extracellular Ca2+ concentration ([Ca2+]o) (Fujita et al. 1996; Shibata et al. 1997) or hypotonic stress (Kelly et al. 1994). Resorbing activities are inhibited by a rise in [Ca2+]o possibly via a low-affinity receptor-like [Ca2+]o-sensing molecule (Zaidi et al. 1995). Cell swelling induced by hypotonic shock may also lead to changes in various cellular events, such as ion transport through the plasma membrane, organization of the cytoskeleton and metabolic activities (Lang et al. 1998). Osteoclasts would be exposed to both a rise in [Ca2+]o and an osmotic imbalance during the bone resorption. We have shown that a rise in [Ca2+]o activates an outwardly rectifying Cl− (ORCl) channel in in vitro-generated murine osteoclasts (Shibata et al. 1997) which have the characteristics of mature osteoclasts (Akatsu et al. 1992; Shioi et al. 1994), but the hypotonic effects on their Cl− channels remain to be solved. This study aimed at characterizing hypotonically activated Cl− channels in murine osteoclasts and studying the co-operative action of the two stimuli, a rise in [Ca2+]o and osmotic perturbation. We show that the hypotonically activated Cl− current shares common electrophysiological features with the ORCl current activated by a rise in [Ca2+]o, and that a rise in [Ca2+]o shifts the osmolarity set-point for the current activation to respond to a minor change in osmolarity. In addition, we provide evidence that the hypotonically activated ORCl current requires extracellular Ca2+ and intracellular ATP and is inhibited by intracellular alkalization and perturbation of the actin cytoskeleton. A preliminary report has been published (Sakai et al. 1997).

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 degrees 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 degrees 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.

The effects of general anesthetics on P2X7 receptors in rat microglia were examined by using patch-clamp experiments. In contrast to inhalational anesthetics, intravenous anesthetics enhanced P2X7Rs response in rat microglia.

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg2+, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca2+-high Mg2+ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence of d -(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg2+. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg2+ in the solution and in 2 of 17 cells with 4 mM Mg2+, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg2+ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba2+, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K+, thereby indicating that K+ mediated the IR currents. The negative conductance region was seen at potentials positive to EK. The OR currents did not apparently depend on the extracellular K+ concentration, but were reduced by lowering the extracellular Cl− concentration. The OR currents were partially blocked by 1 mm Ba2+, and were further blocked by a Cl− channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl− concentrations, suggesting that Cl− was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K+ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K+ channel and the outwardly rectifying Cl− channel seem to contribute to the regulation of the membrane potential.

The vacuolar-type H+-ATPase (V-ATPase) is an electrogenic H+ pump that is distributed widely in living organisms. Electrogenicity contributes to the proton motive force, but its precise evaluation is difficult mostly because V-ATPases are abundant at the intracellular membranes of acidic vesicles where fluxes of counter ions and H+-leakage could not be fully controlled. In osteoclasts, bone-resorbing cells, V-ATPases are recruited to the plasma membrane (the ruffled membrane) by exocytotic fusion of lysozomes. The electogenicity of the plasma membrane V-ATPases was evaluated under the whole-cell current-clamp recordings in the absence of Na+, K+ and Cl-, where H+ was a major determinant of the membrane potentials. Under pHo/pHi 7.3/5.5 (ΔpH 1.8), the membrane potential varied greatly among cells, from −250 - −7 mV. Bafilomycin, a specific blocker for V-ATPases, depolarized cells by several to 220 mV. The depolarization was dependent on the amplitudes of the V-ATPase currents and eliminated by replacing intracellular ATP by ADP. The V-ATPase-mediated potential was reduced by decreasing ΔpH and disappeared at near ΔpH −2.5. As the whole-cell leak conductance was ∼0.23 nS (the area resistance of the plasma membrane: 6 x 105 Ω cm2), 10 pA of V-ATPase currents could yield the membrane potential of ∼ 40 mV. Small ΔpH-dependent potential due to H+ leakage currents remained in the presence of bafilomycin. Potentials generated by V-ATPases and H+ leak are thus identified successfully. The ruffled membrane could provides a useful model for investigating H+ fluxes across the membrane energized by V-ATPases.

We analyzed glutamate-induced enhancement of the amplitude of monosynaptic excitatory postsynaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Low concentrations (0.1-0.3 mM) of glutamate, which produced small depolarization, often enhanced EPSP associated with inhibition of a paired pulse facilitation and increased occurrence of spontaneous EPSPs. With 1 mM glutamate, transient enhancement of EPSP was seen in some cells during the early phase or prior to large depolarization, even when input conductance was increased. Transient or sustained enhancement of EPSP was occasionally seen with N-methyl-D-aspartate, kainate and quisqualate, but not with L-2-amino-4-phosphonobutyrate. The results suggest that glutamate enhanced release of excitatory transmitters at low concentrations that apparently did not affect the postsynaptic membrane.