Receptor-mediated activation of a Cl(-) current by LPA and S1P in cultured corneal keratocytes.
OBJECTIVE
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.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000502:physiology*; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004594:Electrophysiology; D007150:Immunohistochemistry; D015640:Ion Channel Gating; D008246:Lysophospholipids / Q000494:pharmacology*; D009579:Nitrobenzoates / Q000494:pharmacology; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D018118:Chloride Channels; D008246:Lysophospholipids; D009579:Nitrobenzoates; D011956:Receptors, Cell Surface; C060506:sphingosine 1-phosphate; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D013110:Sphingosine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid