Fmoc-DL-β-Me-Phe-OH

The effect of NPC-14686 (Fmoc-L-homophenylalanine), a novel anti-inflammatory agent on intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) renal tubular cells, was investigated, using fura-2 as a Ca(2+) dye. At concentrations between 10 and 200 microM NPC-14686 increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by 90%. In Ca(2+)-free medium, pretreatment with 100 microM NPC-14686 nearly abolished the [Ca(2+)](i) increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) and abolished the [Ca(2+)](i) increase induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) (a mitochondrial uncoupler). NPC-14686 (100 microM) induced a slight [Ca(2+)](i) increase after pretreatment with 2 microM CCCP and 1 microM thapsigargin. Addition of 3 mM Ca(2+) elicited a [Ca(2+)](i) increase in cells pretreated with 100 microM NPC-14686 in Ca(2+)-free medium.

The effect of the anti-inflammatory compound NPC-14686 on intracellular Ca²⁺ concentration ([Ca²⁺](i)) and viability in OC2 human oral cancer cells was investigated. The Ca²⁺-sensitive fluorescent probe fura-2 was used to examine [Ca²⁺](i). NPC-14686 induced [Ca²⁺](i) rises in a concentration-dependent fashion. The effect was reduced approximately by 10% by removing extracellular Ca²⁺. NPC-14686- elicited Ca²⁺ signal was decreased by nifedipine, econazole, SKF96365, and GF109203X. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished NPC-14686-induced [Ca²⁺](i) rises. Conversely, pretreatment with NPC-14686 abolished thapsigargin or BHQ-induced [Ca²⁺](i) rises. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²⁺](i) rises. At 20-100 μM, NPC-14686 inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-acetoxymethyl ester (BAPTA/AM).

NPC-14686 has been shown to have anti-inflammatory effect in previous studies, but the mechanisms are unclear. The effect of NPC-14686 on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and viability in MG63 human osteosarcoma cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]i. NPC-14686 at concentrations of 100-500 μM induced a [Ca²⁺]i rise in a concentration-dependent manner. The response was reduced by 80% by removing Ca²⁺. NPC-14686 induced Mn²⁺ influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²⁺]i rise. At 20-50 μM, NPC-14686 decreased cell viability, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that NPC-14686 (30-50 μM) induced apoptosis in a concentration-dependent manner.

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in HA22/VGH human hepatoma cells was explored by using fura-2 as a fluorescent Ca(2+) indicator. NPC-14686 at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. The Ca(2+) signal was reduced by removing extracellular Ca(2+) or by 10 microM nifedipine and was not changed by verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) abolished 200 microM NPC-14686-induced Ca(2+) release; and conversely pretreatment with NPC-14686 abolished thapsigargin-induced Ca(2+) release. The Ca(2+) release induced by 200 microM NPC-14686 was not changed by inhibiting phospholipase C with 2 microM U73122. Together, the results suggest that in human hepatoma cells, NPC-14686 induced a [Ca(2+)](i) increase by causing store Ca(2+) release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing nifedipine-sensitive Ca(2+) influx.