REGULATION OF ENDOTHELIN-1 IN HUMAN NONPIGMENTED CILIARY EPITHELIAL-CELLS BY TUMOR-NECROSIS-FACTOR-ALPHA

Endothelins (ET) are potent vasoactive peptides present in many ocular structures and are formed from precursor Big endothelins (Big ET-1) b y the action of an endothelin-converting enzyme (ECE). ET-1 is thought to decrease intraocular pressure by contracting the ciliary muscle th us enhancing the outflow of aqueous humor through the Canal of Schlemm and trabecular meshwork. However, the mechanisms involved in the regu lation of endothelin-l (ET-1) synthesis and release in ocular tissues have not been fully characterized. In this study we examined the effec t of tumor necrosis factor-alpha (TNF-alpha; 10 nM), a proinflammatory cytokine, on the cellular mechanisms leading to ET-1 synthesis and re lease in SV-40 transformed human ciliary non-pigmented epithelial cell s (HNPE). ET-1 and Big endothelin-l (Big ET-1) immunoreactivity was ti me-dependently increased following TNF-alpha treatment. Phorbol eaters (PMA), activators of PKC, also raised the immunoreactive levels of ET -1 and Big ET-1 while, staurosporine, a PKC inhibitor (20 nM), decreas ed ET-1 levels in TNF?-alpha-stimulated cells. Pre-treatment with phos phoramidon (1 mu M) an ECE-inhibitor, followed by TNF-alpha-stimulatio n, decreased ir-ET-l levels. Cycloheximide (9 mu M), a protein synthes is inhibitor, decreased TNF-alpha-stimulated levels for ir-ET-l and ir -Big ET-1, suggesting that TNF-alpha may be directly regulating ET-1 e xpression at the ET-1 gene. Our data indicates that TNF-alpha regulate s ET-1 levels in HNPE cells possibly by activating PKC either to stimu late protein synthesis and/or to enhance ET-1 secretion. These results suggest that ET-1 released from the ciliary body may play an importan t role in aqueous humor dynamics following cytokine activation. (C) 19 98 Academic Press Limited.