Inositol 1,4,5-trisphosphate-independent Ca2+ mobilization triggered by a lipid factor isolated from vitreous body

A complex phospholipid from bovine vitreous body with a strong Ca2+-mobiliz ing activity has been recently isolated to homogeneity by our group. In thi s work, a sequential analysis of its transmembrane signaling pathway has be en undertaken to characterize the intracellular mechanisms responsible for the Ca2+ rise. The results show that this phospholipid induces, in a dose-d ependent manner (ED50 of around 0.25 mu g/ml), a Ca2+ mobilization from ino sitol 1,4,5-trisphosphate-insensitive intracellular stores, with no partici pation of extracellular Ca2+. Upon repeated administration, it shows no sig ns of autologous desensitization, does not induce heterologous desensitizat ion of the L-alpha-lysophosphatidic acid (LPA) receptor but is desensitized by the previous administration of LPA. The Ca2+-mobilizing activity requir es a membrane protein, is blocked after pre-incubation of the cells with pe rtussis tcPxin and phorbol eaters, as well as by U73122 tan inhibitor of ph ospholipases C/D, R59022 (a diacylglycerol kinase inhibitor), and D609 (whi ch inhibits phosphatidylcholine-specific phospholipase C). Upon administrat ion of this phospholipid, the intracellular levels of phosphatidic acid (PA ) rise with a time course that parallels that of the Ca2+ mobilization, sug gesting that PA could be responsible for this Ca2+ signal. Exposure to AACO CF(3) (a specific inhibitor of phospholipase A(2)) does not modify the Ca2 rise, ruling out the possibility that the PA generated could be further co nverted to LPA by the action of phospholipase A(2). Based on the experiment al data obtained, a signaling pathway involving a phosphatidylcholine-speci fic phospholipase C coupled to diacylglycerol kinase is proposed. This comp ound may represent a new class of bioactive lipids with a putative role in the physiology of the vitreous body.