Effects of general anesthetics on P2X4 receptors in a mouse microglial cell line.

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.

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

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.