INTRACELLULAR CA2-TIME CA2+ RESPONSE OF BONE-CELLS EXPERIENCING FLUID-FLOW( STORES AND EXTRACELLULAR CA2+ ARE REQUIRED IN THE REAL)

In this study, we sought to determine if there is a requirement for ca lcium entry from the extracellular space as well as calcium from intra cellular stores to produce real-time intracellular calcium responses i n cultured bone cells subjected to fluid flow. Understanding calcium c ell signaling may help to elucidate the biophysical transduction mecha nism(s) mediating the conversion of fluid dow to a cellular signal. An experimental design which utilized a scheme of pharmacological blocke rs was employed to distinguish between the biochemical pathways involv ed in this cell signaling. A parallel-plate dow chamber served as the cell stimulating apparatus and a fluorescence microscopy system using the calcium-sensitive dye fura-2 measured the intracellular calcium ch anges. In the present study, evidence for a role by the inositol-phosp holipid biochemical pathway, specifically inositol trisphosphate (IP3) was obtained using neomycin which completely inhibited the calcium re sponse to flow. Additionally, a concomitant role of extracellular calc ium was demonstrated through experiments performed in calcium-free med ium which also eliminated the dow response. Experiments conducted with gadolinium, a stretch-activated channel blocker, partially Inhibited (similar to 30%) the dow response while verapamil, a type-L voltage se nsitive channel blocker, had no effect on the flow response. These res ults suggest a requirement of extracellular calcium (or calcium influx ) as well as IP3-induced calcium release from intracellular stores for generating the intracellular calcium response to flow in bone cells. Copyright (C) 1996 Elsevier Science Ltd.