Abstract
Purpose: 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.
| Original language | English |
|---|---|
| Pages (from-to) | 389-397 |
| Number of pages | 9 |
| Journal | Journal of Ocular Pharmacology and Therapeutics |
| Volume | 26 |
| Issue number | 5 |
| DOIs | |
| State | Published - 1 Oct 2010 |
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Thrombin-induced endothelin-1 synthesis and secretion in retinal pigment epithelial cells is Rho kinase dependent. / Narayan, Santosh; Prasanna, Ganesh; Tchedre, Kissaou; Krishnamoorthy, Raghu; Yorio, Thomas.
In: Journal of Ocular Pharmacology and Therapeutics, Vol. 26, No. 5, 01.10.2010, p. 389-397.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Thrombin-induced endothelin-1 synthesis and secretion in retinal pigment epithelial cells is Rho kinase dependent
AU - Narayan, Santosh
AU - Prasanna, Ganesh
AU - Tchedre, Kissaou
AU - Krishnamoorthy, Raghu
AU - Yorio, Thomas
PY - 2010/10/1
Y1 - 2010/10/1
N2 - Purpose: 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.
AB - Purpose: 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.
UR - http://www.scopus.com/inward/record.url?scp=77957958695&partnerID=8YFLogxK
U2 - 10.1089/jop.2010.0072
DO - 10.1089/jop.2010.0072
M3 - Article
VL - 26
SP - 389
EP - 397
JO - Journal of Ocular Pharmacology and Therapeutics
JF - Journal of Ocular Pharmacology and Therapeutics
SN - 1080-7683
IS - 5
ER -