Effect of NPC-15199 on Ca ²⁺ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca ²⁺ concentration ([Ca ²⁺ ] _i ) was investigated by using fura-2. NPC-15199 (100-1000 μM) caused a rapid and sustained increase of [Ca ²⁺ ] _i in a concentration-dependent manner (EC ₅₀ =500 μM). NPC-15199-induced [Ca ²⁺ ] _i rise was prevented by 70% by removal of extracellular Ca ²⁺ , but was not changed by dihydropyridines, verapamil and diltiazem. In Ca ²⁺ -free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and thapsigargin (1 μM), an inhibitor of the endoplasmic reticulum (ER) Ca ²⁺ -ATPase, caused a monophasic [Ca ²⁺ ] _i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca ²⁺ ] _i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca ²⁺ ] _i rises. U73122, an inhibitor of phoispholipase C, abolished 10 μM ATP (but not 1 mM NPC-15199)-induced [Ca ²⁺ ] _i rise. These results suggest that NPC-15199 rapidly increases [Ca ²⁺ ] _i by stimulating both extracellular Ca ²⁺ influx and intracellular Ca ²⁺ release via as yet unidentified mechanism(s).