Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells.
OBJECTIVE
Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K(+) currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells.


METHODS
Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays.


RESULTS
LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to G(alpha(i)/o/t/z). LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-1, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca(2+)](i) increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca(2+)](i) increases were blocked by PD98059 and by loading cells with antibodies to G(alpha(i)/o/t/z). RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA.


CONCLUSIONS
LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca(2+) from intracellular stores that appears independent of IP(3) and ryanodine receptor activation.
Thoreson
Wallace B
WB
Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5540, USA. wbthores@unmc.edu
Ryan
Jennifer S
JS
Shi
Chanjuan
C
Kelly
Melanie E
ME
Bryson
Eric J
EJ
Toews
Myron L
ML
Ediger
Tracy L
TL
Chacko
David M
DM
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015290:Second Messenger Systems / Q000502:physiology; D015398:Signal Transduction
D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D000242:Cyclic AMP; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium