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Luke B. Potts, Enoch Kuo, Greg Lu, Yi Ren, Ellen Ngo, Lih Kuo, Travis W. Hein; ROCK-II Isoform and PKC Mediate Porcine Retinal Arteriolar Constriction to Endothelin-1. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5270.
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Endothelin-1 (ET-1) has been shown to play a role in physiologic regulation of retinal blood flow, as well as in several retinal pathologies including diabetic retinopathy and glaucoma. Protein kinase C (PKC) activation has also been implicated in several retinal pathologies. The interrelationship between these two remains unknown. The Rho kinase (ROCK) isoforms (I and II) have been shown to be involved in a diverse set of functional outcomes in blood vessels, including agonist-induced smooth muscle contraction and vascular pathophysiology. The purpose of this study was to elucidate the interrelationships of the ROCK isoforms and PKC in the development of myogenic tone and ET-1-mediated constriction.
We utilized the non-selective ROCK and PKC inhibitors H-1152 (3 µM) and chelerythrine (3 µM), respectively, and siRNA against ROCK I and II isoforms in isolated/pressurized porcine retinal arterioles (~70 µm) to assess the role of ROCK in vascular constriction to ET-1 and the PKC activator phorbol 12,13-dibutyrate (PDBu). Specifically, these pharmacologic and siRNA strategies were used to prevent/reverse myogenic tone and agonist-induced vasoconstriction. Immunoblotting and immunohistochemical methods yielded information on ROCK expression and distribution in retinal tissue and arterioles. RT-PCR was used to determine mRNA levels of the ET-1 precursor pre-pro-ET-1.
Administration of H-1152 significantly inhibited myogenic tone by 94±0.4%. ET-1 and PDBu-mediated vasoconstrictions were both significantly reversed by treatment with H-1152 or chelerythrine. Knockdown of ROCK II, but not ROCK I, prevented development of myogenic tone, as well as ET-1 and PDBu-mediated vasoconstrictions. At the protein level, there was an 8-fold greater ROCK II than ROCK I expression in both retinal neural and vascular tissues. Immunohistochemical staining revealed a predominance of ROCK II expression in vascular smooth muscle cells. Retinal arterioles and neural tissue express pre-pro-ET-1 transcripts.
Taken together, our results suggest a major role of ROCK II in initiating and maintaining myogenic tone and constriction to ET-1 in porcine retinal arterioles, with PKC activation being upstream of this ROCK II activation. ROCK II and PKC likely mediate physiologic and pathophysiologic responses to endogenously released ET-1.
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