Inhibition of voltage-gated proton channels by local anaesthetics in GMI-R1 rat microglia.

Non-technical summary  Lidocaine and bupivacaine are the most commonly used local anaesthetics in clinical practices such as neuraxial anaesthesia and local infiltration. They are known to suppress phagocytosis and the production of reactive oxygen species in immune cells. Voltage-gated proton channels are abundantly expressed in immune cells, including microglia, and play crucial roles in sustaining phagocytosis. We show that both lidocaine and bupivacaine increase the intracellular pH of microglia by their weak base properties and, consequently, inhibit proton channels. This is a novel mechanism underlying actions of local anaesthetics. Our results also indicate that the proton channel is a useful tool for monitoring the behaviours of lidocaine and bupivacaine across the cellular membrane. 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 pHi 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 pHo and pHi. Both lidocaine and bupivacaine reversibly decreased the current, with IC50 values of ∼1.2 and ∼0.5 mm, respectively, at pHo/pHi 7.3/5.5. The inhibition was enhanced with either pHo increase or pHi 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 pHi. The potencies of inhibition were correlated well with the degree of increase in pHi. The lidocaine-induced inhibition was eliminated when the pHi 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 pHi. This is a novel mechanism underlying actions of local anaesthtics.