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

We investigated the mechanism of benzocaine (permanently uncharged) and QX314 (permanently charged) inhibition of lysophosphatidic acid (LPA) signaling. To determine their site of action, we studied effects of these drugs, alone 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) induced 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). The combination acted synergistically (IC50 benzocaine 0.097 mM/QX314 0.048 mM). Intracellular signaling pathways were not affected. This study shows that benzocaine and QX314 inhibit LPA signaling and act synergistically, which 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 receptor as a target. Activation of PKC can be excluded as a potential mechanism. Implications: Lysophosphatidic acid may play a role in wound healing, and its signaling is inhibited by local anesthetics. We identified the membrane receptor as the local anesthetic site of action and showed that charged (QX314) and uncharged (benzocaine) local anesthetics inhibit lysophosphatidic acid signaling synergistically, which can be explained by the presence of different binding sites.