Activation of phospholipase C and guanylyl cyclase by endothelins in human trabecular meshwork cells

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Abstract
PURPOSE. To characterize effects of endothelins on activities of phospholipase C (PLC) and nucleotide cyclases in human trabecular meshwork (TM) cells. METHODS. Cultured simian virus 40-transformed human TM (HTM-3) or non-transformed (HTM-16) cells were used. Changes in the PLC activity were determined by assaying the production of [ 3 H] inositol phosphates. Accumulation of cyclic GMP or cyclic AMP in cell lysate was measured by radioimmunoassay. RESULTS. Endothelin-1 (ET-1; 1 µM) stimulated PLC in HTM-16 cells, but Sarafotoxin S6c (SRTX), an ET B receptor subtype- selective agonist (1 µM), did not. Similar results were obtained in HTM-3 cells: ET-1, but not ET-3 or SRTX, activated PLC in a dose-dependent manner, with a calculated EC 50 of 646 pM. The peptide also stimulated the accumulation of cGMP in a concentration-dependent manner with an EC 50 of 37.2 pM. ET-3 or SRTX was not effective except at much higher concentrations. Both the PLC and guanylyl cyclase stimulation induced by ET-1 (10 nM) were completely inhibited by pretreating the cells with BQ-123 (<10 µM), an ET A receptor selective antagonist, but not by BQ-788 (10 µM), an ET B receptor subtype-specific antagonist. Neither ET-1 nor ET-3 stimulated adenylyl cyclase activity in HTM-3 cells at concentration as high as 1 µM. COCLUSIONS. ET-1 activates PLC and guanylyl cyclase in TM cells. Potency profiles of ET receptor agonists and antagonists suggest that the ET A receptor subtype is involved in both actions of ET-1. The effects of the ET peptides in TM cells are interesting and could be part of the mechanism of their IOP-lowering effect.