Novel effect of N-palmitoyl-l-serine phosphoric acid on cytosolic Ca ²⁺ levels in human osteoblasts

The effect of N-palmitoyl-L-serine phosphoric acid (L-NASPA), which has been used as an inhibitor of lysophosphatidic acid receptors, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human osteosarcoma MG63 cells was measured by using fura-2. L-NASPA (0.1-10 μM) caused a rapid and transient plateau [Ca²⁺]_i rise in a concentration-dependent marmer (EC₅₀=0.5 μM). The L-NASPA-induced [Ca²⁺]_i rise was partly reduced by removal of extracellular Ca²⁺ but was not altered by L-type voltage-gated Ca²⁺ channel blockers. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, induced a [Ca²⁺]_i rise, after which the increasing effect of L-NASPA on [Ca²⁺]_i was completely inhibited; also, pretreatment with L-NASPA partly reduced thapsigargin-induced [Ca ²⁺]_i rise. U73122, an inhibitor of phospholipase C, abolished histamine (but not L-NASPA)-induced [Ca²⁺]_i rise. Overnight incubation with 1 μM L-NASPA did not affect cell proliferation, but 10-20 μM L-NASPA exerted 4% and 15% inhibition, respectively. Collectively, L-NASPA rapidly increased [Ca²⁺] _i in MG63 cells by evoking both extracellular Ca²⁺ influx and intracellular Ca²⁺ release, and is cytotoxic at higher concentrations.