Abstract
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.