Synergistic inhibition of lysophosphatidic acid signaling by charged and uncharged local anesthetics

We investigated the mechanism of benzocaine (permanently uncharged) and QX3 14 (permanently charged) inhibition of lysophosphatidic acid (LPA) signalin g. To determine their site of action, we studied effects of these drugs, al one and in combination, on LPA-induced Ca2+-dependent Cl currents (I-Cl(Ca) ) in Xenopus oocytes. After 10 min exposure to benzocaine, QX314 (10(-6)-10 (-2) M), or both, we measured effects on I-Cl(Ca), induced by LPA (with and without protein kinase [PKC] activation/inhibition) and on I-Cl(Ca) induce d by the intracellular injection of IP3 and GTP gamma S. LPA application to oocytes resulted in I-Cl(Ca) (50% effective concentration approximately 10 (-8) M). Both anesthetics inhibited LPA signaling concentration-dependently (50% inhibitory concentration [IC50] benzocaine 0.9 mM, QX314 0.66 mM). Th e combination acted synergistically (IC50 benzocaine 0.097 mM/QX314 0.048 m M). Intracellular signaling pathways were not affected. This study shows th at benzocaine and QX314 inhibit LPA signaling and act synergistically, whic h is most easily explained by the existence of two different binding sites. Lack of inhibition of IP3 or GTP gamma S-induced I-Cl(Ca) identifies the r eceptor as a target. Activation of PKC can be excluded as a potential mecha nism. Implications: Lysophosphatidic acid may play a role in wound healing, and its signaling is inhibited by local anesthetics. We identified the mem brane receptor as the local anesthetic site of action and showed that charg ed (QX314) and uncharged (benzocaine) local anesthetics inhibit lysophospha tidic acid signaling synergistically, which can be explained by the presenc e of different binding sites.