2-O-methyl PAF as a Ca²⁺ mobilizer in Madin Darby canine kidney cells

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca ²⁺ concentration ([Ca²⁺]_i) was measured by using the Ca²⁺-sensitive fluorescent dye fura-2. 2-O-methyl PAF (≥15 μM) caused a rapid rise of [Ca²⁺]_i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca²⁺] _i rise was partly reduced by removal of extracellular Ca ²⁺. 2-O-methyl PAF-induced extracellular Ca²⁺ influx was also suggested by Mn²⁺ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca²⁺ influx was blocked by nifedipine, verapamil and diltiazem. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, caused a monophasic [Ca²⁺]_i rise, after which 2-O-methyl PAF failed to increase [Ca²⁺]_i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca²⁺ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca ²⁺]_i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca²⁺]_i in renal tubular cells by stimulating both extracellular Ca²⁺ influx and intracellular Ca²⁺ release.