Visualization of Ins(1,4,5)P3 dynamics in living cells: two distinct pathways for Ins(1,4,5)P3 generation following mechanical stimulation of HSY-EA1 cells

Abstract

In the present study, the contribution of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] generation on the mechanical-stimulation-induced Ca2+ response was investigated in HSY-EA1 cells. Mechanical stimulation induced a local increase in the cytosolic concentration of Ins(1,4,5)P3 ([IP3]i), as indicated by the Ins(1,4,5)P3 biosensor LIBRAvIII. The area of this increase expanded like an intracellular Ins(1,4,5)P3 wave as [IP3]i increased in the stimulated region. A small transient [IP3]i increase was subsequently seen in neighboring cells. The phospholipase C inhibitor U-73122 abolished these Ins(1,4,5)P3 responses and resultant Ca2+ releases. The purinergic receptor blocker suramin completely blocked increases in [IP3]1 and the Ca2+ release in neighboring cells, but failed to attenuate the responses in mechanically stimulated cells. These results indicate that generation of Ins(1,4,5)P3 in response to mechanical stimulation is primarily independent of extracellular ATP. The speed of the mechanical-stimulation-induced [IP3]i increase was much more rapid than that induced by a supramaximal concentration of ATP (1 mM). The contribution of the Ins(1,4,5)P3-induced Ca2+ release was larger than that of Ca2+ entry in the Ca2+ response to mechanical stimulation in HSY-EA1 cells.