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 A2 (PLA2) 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 [14C]choline incorporation into [14C]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-2. 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.
Kim
D K
DK
Department of Pharmacology, Wonkwang University School of Medicine, Iksan, Korea.
Jung
K Y
KY
D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002460:Cell Line; D002794:Choline / Q000378:metabolism; D004285:Dogs; D005997:Glycerylphosphorylcholine / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases / Q000187:drug effects / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology*
D002250:Carbon Radioisotopes; D012433:Ryanodine; D002110:Caffeine; D005997:Glycerylphosphorylcholine; D010741:Phospholipases A; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases; C061931:glycerophosphocholine cholinephosphodiesterase; D002794:Choline; D002118:Calcium