Thrombin-induced endothelin-1 synthesis and secretion in retinal pigment epithelial cells is rho kinase dependent.
OBJECTIVE
The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood–retinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the blood–retinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1.


METHODS
Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca²+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels.


RESULTS
Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca²+]<]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion.


CONCLUSIONS
Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells.
Narayan
Santosh
S
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center , Fort Worth, Texas, USA. narayans@mskcc.org
Prasanna
Ganesh
G
Tchedre
Kissaou
K
Krishnamoorthy
Raghu
R
Yorio
Thomas
T
D001813:Blood-Retinal Barrier / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D019332:Endothelin-1 / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism* / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000503:physiopathology; D012168:Retinal Pigments / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000819:agonists / Q000378:metabolism* / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000032:analysis / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism / Q000494:pharmacology
D019332:Endothelin-1; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D012168:Retinal Pigments; D054460:rho-Associated Kinases; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D002118:Calcium