Caffeine causes glycerophosphorylcholine accumulation through ryanodine-inhibitable increase of cellular calcium and activation of phospholipase A2 in cultured MDCK cells

Glycerophosphrylocholine (GPC) is a renal medullary compatible organic osmolyte that is derived from choline via phosphatidylcholine, which is catalyzed in part by phospholipase A(2) (PLA(2)) and its degradation by GPC:choline phosphodiesterase (GPC:choline PDE). We found that caffeine elevated intracellular free calcium ([Ca2+](i)) and GPC level in cultured MDCK cells, canine kidney epithelial cells, and propose a possible biochemical mechanism. When MDCK cells were incubated for 3 h with 1 to 10 mM caffeine, cellular GPC was elevated in a dose-dependent manner, and this occurred independently of the extracellular osmolality. Caffeine stimulated the rate of [C-14]choline incorporation into [C-14]GPC and PLA2 activity. Whereas, GPC:choline PDE activity was accompanied by less of increase. These enzyme changes demonstrate the increased net synthesis of MDCK GPC. In order to identify what triggers the PLA2 activation, [Ca2+](i) was measured by using a fluorescence dye, Fura-P. Caffeine (10 mM) resulted in a typical transient increase in MDCK [Ca2+](i) concentration, and this increase was greatly inhibited by pretreatment of MDCK cells with 10 mM ryanodine for 5 min. Ryanodine (10 mM) also inhibited the caffeine-induced stimulation of PLA2 activity. These findings provide the first evidence that caffeine in MDCK cells causes a ryanodine-inhibitable increase of [Ca2+](i) and PLA2 activity, resulting in cellular GPC accumulation.