Inhibition of Na+-Ca2+ Exchange by Antisense Oligodeoxynucleotides in Osteoblasts.

초록

Physiological activity of osteoblast including osteogenesis is known to be related to the increase in intracellular Ca2+ activity ([Ca2+]i) of osteoblast. The cellular regulation of ([Ca2+]i) in non-excitable cells is mainly me diated by me transmembrane Ca2+ movement .via Na+-Ca2+ exchange and store-operated Ca2+ influx, and intracellular Ca2+ stores. In this study, we investigated the role of Na+-Ca2+ exchange transport in regulation of intracellular Ca2+ activity in osteoblasts, employing antisense oligodeoxynucleotide (AS-oligo) technique Olsteoblast-like cells (OLC) were isolated from femur and tibia of neonatal rats. and cultured for 8 days. On the 7th day, a tandem pair of antisense oligodeoxynucleotides 5'AAAAATGGTGAAGAGAGTG3' and 5' GTGAACATGACAAATGTCA3' were added into the culture media. Cultured OLCs were load with Fura-2, and fluorescence images were monitored with a cooled CCD camera. [Ca2+]i decreased as Ca2+ concentrations in the superfusing solutions were lowered, and this Ca2+-dependence was augmented in AS-oligo treated OLCs. The removal of Na+ and Ca2+ from the superfusing solutions elicited spike-like transient [Ca2+]i increase, which was inhibited in the AS-oligo treated cells. The Ca2+ influx following the emptying of intenal Ca2+ stores was enhanced in the AS-oligo treated cells. Na+-dependent decline of [Ca2+]i in the presence of caffeine, cyclopiazonic acid and La3+ was retarded in the AS-oligo treated cells. The results from this study provide me experimental evidences for physiological activity of Na+-Ca2+ exchange in regulating intracellular Ca2+ activity in osteoblasts.