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

NPC-14686 (Fmoc-L-homophenylalanine), a novel antiinflammatory agent, increases intracellular Ca²⁺ concentrations ([Ca²⁺](i)] in T24 bladder tumor cells. Using fura-2 as a Ca²⁺ probe, NPC-14686 (10-200 μM) increased [Ca²⁺](i) in a concentration-dependent manner. The [Ca²⁺](i) increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca²⁺ removal partly inhibited the Ca²⁺ signals. In Ca²⁺-free medium, pretreatment with 100 μM NPC-14686 abolished the [Ca²⁺](i) increases induced by 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor] and 2 μM carbonylcyanide m- chlorophenylhydrazone (a mitochondrial uncoupler). However, 100 μM NPC-14686 still slightly increased [Ca²⁺](i) after Ca²⁺ stores had been depleted by pretreating with 2 μM CCCP and 1 μM thapsigargin. These results suggest that NPC-14686 released Ca²⁺ from multiple pools. Adding 3 mM Ca²⁺ increased [Ca²⁺](i) in cells pretreated with 100 μM NPC-14686 in Ca²⁺-free medium, indicating that NPC-14686 activated capacitative Ca²⁺ entry. Inhibiting formation of inositol-1,4,5-trisphosphate (IP₃] by blocking phospholipase C with 2 μM U73122 had little effect on NPC-14686-induced Ca²⁺ release. Activating protein kinase C with phorbol 12-myristate 13-acetate (PMA) significantly potentiated NPC-14686-induced [Ca²⁺](i) increase. NPC-14686 (100 μM) also increased [Ca²⁺](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 Ca²⁺ release followed by Ca²⁺ entry. The Ca²⁺ release was unlinked to IP₃ and the [Ca²⁺](i) signal could be modulated by protein kinase C. (C) 2000 Wiley-Liss, Inc.