Role of lysophospholipid growth factors in the modulation of aqueous humor outflow facility.
OBJECTIVE
To investigate the role of lysophospholipid growth factors in the regulation of aqueous humor outflow in the trabecular meshwork (TM).


METHODS
The expression profile of the endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by RT-PCR of human TM (HTM) cell-derived total RNA and by PCR amplification of HTM cell-derived and tissue-derived cDNA libraries. The effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on actin cytoskeleton and focal adhesions and on myosin light-chain (MLC) phosphorylation in HTM cells were evaluated by immunofluorescence microscopy and Western blot analysis, respectively. Activation of Rho GTPase in HTM cells was quantified by "pull-down" assays. Mobilization of intracellular calcium in HTM cells was determined using spectrofluorometric digital-imaging microscopy. The effects of LPA and S1P on aqueous humor outflow facility were evaluated by perfusion of enucleated porcine eyes.


RESULTS
Each of the receptor isoforms Edg1, -2, -3, and -4 was readily detectable in three of four HTM cell-derived libraries, whereas Edg2 was detectable in the HTM tissue library. LPA (20 microM) and S1P (1 microM) stimulated actin stress fiber and focal adhesion formation, increased MLC phosphorylation, and induced marked activation of Rho GTPase in HTM cells. Both LPA (20 microM) and S1P (10 microM) also stimulated increases in intracellular calcium concentration in HTM cells. LPA- and S1P-induced effects on MLC phosphorylation in HTM cells were markedly inhibited by pretreatment with the Rho kinase-specific inhibitor Y-27632 (5 microM). Perfusion of LPA (50 microM) and S1P (5 microM) in enucleated porcine eyes produced a significant decrease in aqueous humor outflow facility from baseline of 37% (n = 6) and 31% (n = 5), respectively.


CONCLUSIONS
These studies demonstrate that LPA and S1P, the physiological agonists of Edg receptors, decrease outflow facility in perfused porcine eyes in association with increased MLC phosphorylation and Rho guanosine triphosphatase (GTPase) activation. These data provide evidence for a novel mechanism for negative regulation of outflow facility, which may contribute to overall physiological homeostasis of aqueous humor outflow facility.
Mettu
Priyatham Sai
PS
Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Deng
Pei-Feng
PF
Misra
Uma K
UK
Gawdi
Govind
G
Epstein
David L
DL
Rao
P Vasantha
PV
D000199:Actins / Q000378:metabolism; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022001:Focal Adhesions / Q000378:metabolism; D005784:Gene Amplification; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D049312:Receptors, Lysophospholipid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology*; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D018994:Myosin Light Chains; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; C060506:sphingosine 1-phosphate; D020741:rho GTP-Binding Proteins; D013110:Sphingosine; C032881:lysophosphatidic acid; D002118:Calcium