Evidence for multiple phospholipid receptors activating Cl- channels in rabbit keratocytes from injured corneas

Purpose. We recently described the activation of Cl^- channels in corneal keratocytes from wounded rabbit corneas by the growth factor-like phospholipid mediator lysophosphatidic acid (LPA). LPA induced currents were not present in cells from control corneas. Recent evidence from work in frog oocytes indicated the expression of multiple subtypes of the LPA receptor. These receptor subtypes are all activated by LPA but show selectivity to an as yet uncharacterized sphingomyelin-derived lipid (SMDL; MW=422) and to 1-acyl-(sn)-2,3 cyclic glycerol phosphate (cLPA), a naturally occurring LPA analog. The purpose of the present study was to determine if the different subtypes of LPA receptors are expressed in keratocytes from wounded rabbit corneas. Methods. Corneas from anesthetized rabbits were wounded with a liquid N₂ cooled brass probe. After 90 h, rabbits were sacrificed and keratocytes were dissociated from the wound region. These cells were placed in a microscope chamber and cells were patch clamped using the amphotericin-perforated patch whole cell technique. Results. Overall, 14 of 20 cells examined were positive for SMDL induced Cl^- currents, and 3 of 21 cells were positive for cLPA induced Cl^- currents. In experiments where cLPA was ineffective at stimulating Cl^- currents, 9 of 11 cells were stimulated by SMDL. In 2 of 3 of cells from this same group which were stimulated by SDML, addition of LPA caused an additional increase in the Cl^- current. LPA was still able to activate the Cl^- currents following elevation of internal Ca⁺⁺ by addition of A23187 to the bath or removal of external Ca⁺⁺ by EGTA buffering of the bath. UV light release of caged GTPls was ineffective at stimulating the current. Conclusions. These results indicate the presence of multiple receptor subtypes responsible for activation of the LPA induced Cl^- current in corneal keratocytes from wounded rabbit corneas.