Intracellular calcium concentrations in human bladder tumor cells could be increased by NPC-14686, a novel antiinflammatory agent

Abstract

NPC-14686 (Fmoc-L-homophenylalanine), a novel antiinflammatory agent, increases intracellular Ca 2+ concentrations ([Ca 2+ ] i ] in T24 bladder tumor cells. Using fura-2 as a Ca 2+ probe, NPC-14686 (10-200 μM) increased [Ca 2+ ] i in a concentration-dependent manner. The [Ca 2+ ] i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca 2+ removal partly inhibited the Ca 2+ signals. In Ca 2+ -free medium, pretreatment with 100 μM NPC-14686 abolished the [Ca 2+ ] i increases induced by 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor] and 2 μM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). However, 100 μM NPC-14686 still slightly increased [Ca 2+ ] i after Ca 2+ stores had been depleted by pretreating with 2 μM CCCP and 1 μM thapsigargin. These results suggest that NPC-14686 released Ca 2+ from multiple pools. Adding 3 mM Ca 2+ increased [Ca 2+ ] i in cells pretreated with 100 μM NPC-14686 in Ca 2+ -free medium, indicating that NPC-14686 activated capacitative Ca 2+ entry. Inhibiting formation of inositol-1,4,5-trisphosphate (IP 3 ] by blocking phospholipase C with 2 μM U73122 had little effect on NPC-14686-induced Ca 2+ release. Activating protein kinase C with phorbol 12-myristate 13-acetate (PMA) significantly potentiated NPC-14686-induced [Ca 2+ ] i increase. NPC-14686 (100 μM) also increased [Ca 2+ ] i in MDCK renal cells, BFTC bladder tumor cells, and MS-1 endothelial cells. Together, the findings suggest that in T24 bladder tumor cells NPC-14686 induced Ca 2+ release followed by Ca 2+ entry. The Ca 2+ release was unlinked to IP 3 and the [Ca 2+ ] i signal could be modulated by protein kinase C.