Participation of a protein phosphatase in the regulation of intracellular calcium concentration in osteoblasts by olpadronate and lidadronate

GRACIELA SANTILLÁN,; KATZ SEBASTIÁN,; STOCKMAN GASTÓN,; SCODELARO BILBAO, PAOLA GABRIELA; MORELLI SUSANA,; RICARDO BOLAND,; ROLDÁN EMILIO,

Rio de Janeiro

Congreso; IOF World Congress on Osteoporosis; 2004

International Osteoporosis Foundation (IOF)

In previous studies we found that the bisphosphonates (BPs) olpadronate (OPD) and NH2-olpadronate (lidadronate; LID) are able to regulate, in the short term, cytosolic Ca2+ levels ([Ca2+]i) in ROS 17/2.8 rat osteoblastic-like cells. This effect is dependent on prestimulation with the osteotropic agent ATP and due mainly to influx of the cation from the outside through voltage-operated calcium channels (VDCC) and purinergic activation of PLC. In the present work, we evaluated the mechanism by which these BPs modulate the Ca2+ response in osteoblasts. By using Fura-2- loaded ROS17/2.8 cells, cytoplasmic Ca2+ changes were recorded by fluorimetry. The bisphosphonate-induced rapid changes in [Ca2+]i were not observed in a Ca2+ )free medium or in medium with 1.5 mM Ca2+ plus 5 lM nifedipine or 5 lM verapamil, involving extracellular Ca2+influx through VDCC channels in BPs effects. The protein phosphatase inhibitors orthovanadate and sodium fluoride mimicked the purinergic-dependent BPs-induced Ca2+ response at low concentrations (1–200 lM) but at higher levels caused a more sustained Ca2+influx blocking the action of BPs. Previous binding assays using [3H]-olpadronate in whole cells showed the presence of a specific, saturable and high affinity binding site for OPD. We now observed that an important proportion of the BPs binder is located in the osteoblast plasma membrane. In addition, like olpadronate and lidadronate, 8 mM p-nitro-phenylphosphate or a´ -naphthylphosphate (phosphatase substrates), compete for binding to this site, whereas purinergic agonists and antagonists, and both protein phosphatase inhibitors (0.2–8 mM) did not displace [3H]OPD. These results suggest the existence of cell membrane target for bisphosphonates, presumably a protein phosphatase, through which BPs modulate the purinergic Ca2+signaling and in turn trigger a cellular response in osteoblasts.