Thrombin stimulates inositol phosphate formation, intracellular calcium fluxes and DNA synthesis in cultured fetal human non-pigmented ciliary epithelial cells

Abstract

Thrombin at concentrations as low as 20 p m (0·002 U ml −1) was found to stimulate inositol phosphate levels in cultured human non-pigmented ciliary epithelial cells. Several other proteases, including trypsin and plasmin, had little or no effect, of several protease inhibitors tested, only those with specificity for thrombin blocked the effect. Studies with active site-blocked thrombin suggested that the esterolytic active site of thrombin is required for inositol phosphate stimulation, while γ-thrombin, which has reduced binding affinity to fibrinogen also showed reduced effectiveness in stimulating inositol phosphates. In the presence of 10 m m LiCl, thrombin stimulated inositol monophosphate, inositol bisphosphate and inositol trisphosphate formation, with a prolonged rise of the first and transient early rises in the latter two species. Thrombin also elevated intracellular Ca 2+ levels as measured with the fluorescent calcium probe, indo-1-AM. This elevation could be blocked by prior addition to cells of the thrombin inhibitor, hirudin, and was dependent upon extracellular Ca 2+ for the maintenance of an elevated level in the presence of thrombin. Incorporation of thymidine into DNA in confluent cultures was also stimulated by thrombin, with a four-fold increase in incorporation at 35 hr in thrombin-treated cells compared to controls. The half-maximal concentration for this process was 0·25 U ml −1. Pretreatment with 100 ng ml −1 pertussis toxin greatly reduced the thrombin effect, which is consistent with a role for a G-protein in stimulation of DNA synthesis by thrombin.