Receptor-mediated activation of a Cl(-) current by LPA and S1P in cultured corneal keratocytes.

PURPOSE: This study was designed to examine the effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on Cl(-) currents (ICl(LPA)) in cultured corneal keratocytes isolated from the corneas of New Zealand White rabbits.
METHODS: ICl(LPA) and resting voltages were recorded with the amphotericin perforated-patch technique. Phenotype was determined with antibodies to alpha-smooth muscle actin.
RESULTS: Keratocytes cultured in serum have a phenotype (myofibroblast) and ionic currents similar to those of keratocytes isolated directly from corneas during wound healing. LPA and S1P both activated ICl(LPA) in a dose-dependent manner, and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (DGPP) blocked the LPA response, but not the S1P response. In addition, a relatively inactive form of LPA (LPA 8:0) was relatively ineffective in activating ICl(LPA). Activation of ICl(LPA) significantly depolarized the cells, and this depolarization was reversed by blocking ICl(LPA) with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).
CONCLUSIONS: These results demonstrate that activation of ICl(LPA) by LPA in cultured corneal keratocytes is receptor mediated and that ICl(LPA) can also be activated by S1P. From a functional standpoint, this work confirms that the current, which is typically thought of as purely volume-activated, can be activated through a receptor. In addition, activation of ICl(LPA) results in depolarization of the keratocyte. Finally, this work demonstrates that cultured corneal keratocytes can act as a model for the study of ion channel function in keratocytes during corneal wound healing.