NPC-14686, a novel anti-inflammatory agent, increased intracellular Ca2+ concentrations in MDCK renal tubular cells

The effect of NPC-14686 (Fmoc-l-homophenylalanine), a novel anti-inflammatory agent on intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin Darby canine kidney (MDCK) renal tubular cells, was investigated, using fura-2 as a Ca²⁺ dye. At concentrations between 10 and 200 μM NPC-14686 increased [Ca²⁺]_i concentration dependently. The [Ca²⁺]_i signal comprised an initial rise and a sustained phase. Ca²⁺ removal inhibited the Ca²⁺ signals by 90%. In Ca²⁺-free medium, pretreatment with 100 μM NPC-14686 nearly abolished the [Ca²⁺]_i increase induced by 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) and abolished the [Ca²⁺]_i increase induced by 2 μM carbonylcyanide m-chlorophenylhydrazone (CCCP) (a mitochondrial uncoupler). NPC-14686 (100 μM) induced a slight [Ca²⁺]_i increase after pretreatment with 2 μM CCCP and 1 μM thapsigargin. Addition of 3 mM Ca²⁺ elicited a [Ca²⁺]_i increase in cells pretreated with 100 μM NPC-14686 in Ca²⁺-free medium. Inhibition of inositol-1,4,5-trisphosphate (IP₃) production by suppressing phospholipase C with 2 μM U73122 did not alter NPC-14686-induced Ca²⁺ release. Trypan blue exclusion revealed that incubation with 10 or 200 μM NPC-14686 for 1–30 min decreased cell viability by 10–20% concentration dependently. Collectively, the results demonstrate that, in MDCK tubular cells, NPC-14686 induced Ca²⁺ release followed by Ca²⁺ entry, with the latter playing a major role. NPC-14686 appears to release intracellular Ca²⁺ in an IP₃-uncoupled manner. NPC-14686 may be of mild cytotoxicity.