Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with spontaneous calcium oscillations and enhanced calcium responsesfollowing endothelin-1 stimulation

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) and i nositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P-4) and thereby regulates c ellular transformation, To investigate the role Ins(1,4,5)P-3-mediated Ca2 oscillations play in cellular transformation, we studied Ins(l,4,5)P3-medi ated Ca2+ responses in cells underexpressing the 43 kDa 5-phosphatase, Chro nic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fol d increase in the resting Ins(1,4,5)P3 concentration and a 4.1-fold increas e in basal intracellular Ca2+. The increased Ins(1,4,5)P-3 Levels resulted in partial emptying (40%) of the Ins(l,il,5)P-3-sensitive Ca2+ store, howev er, store-operated Ca2+ influx remained unchanged. In addition, Ins(1,4,5)P -3 receptors were chronically down-regulated in unstimulated cells, as show n by a 53% reduction in [H-3]Ins(1,4,5)P-3 binding to microsomal receptor s ites. Agonist stimulation with endothelin-l resulted in the rapid rise and fall of Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4 levels, with no significant diffe rences in the rates of hydrolysis of these second messengers in antisenseor vector-transfected cells. These studies indicate, in contrast to its predi cted action, the 43 kDa 5-phosphatase does not metabolise Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4 post agonist stimulation. Cells with decreased 43 kDa 5-ph osphatase activity exhibited spontaneous Ca2+ oscillations in the absence o f any agonist stimulation, and increased sensitivity and amplitude of intra cellular Ca2+ responses to both high and low dose endothelin-l stimulation. me conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1, 4,5)P-3-induced Ca2+ spiking, both in the unstimulated cell and following a gonist stimulation. We propose the enhanced Ca2+ oscillations may mediate c ellular transformation in cells underexpressing the 43 kDa 5-phosphatase.