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

ABSTRACT The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5tetrakisphosphate (Ins(1,3,4,5)P4) and thereby regulates cellular transformation. To investigate the role Ins(1,4,5)P3-mediated Ca2+ oscillations play in cellular transformation, we studied Ins(1,4,5)P3-mediated Ca2+ responses in cells underexpressing the 43 kDa 5-phosphatase. Chronic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fold increase in the resting Ins(1,4,5)P3 concentration and a 4.1-fold increase in basal intracellular Ca2+. The increased Ins(1,4,5)P3 levels resulted in partial emptying (40%) of the Ins(1,4,5)P3-sensitive Ca2+ store, however, store-operated Ca2+ influx remained unchanged. In addition, Ins(1,4,5)P3 receptors were chronically down-regulated in unstimulated cells, as shown by a 53% reduction in [3H]Ins(1,4,5)P3 binding to microsomal receptor sites. Agonist stimulation with endothelin-1 resulted in the rapid rise and fall of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels, with no significant differences in the rates of hydrolysis of these second messengers in antisense-or vector-transfected cells. These studies indicate, in contrast to its predicted action, the 43 kDa 5-phosphatase does not metabolise Ins(1,4,5)P3 and Ins(1,3,4,5)P4 post agonist stimulation. Cells with decreased 43 kDa 5-phosphatase activity exhibited spontaneous Ca2+ oscillations in the absence of any agonist stimulation, and increased sensitivity and amplitude of intracellular Ca2+ responses to both high and low dose endothelin-1 stimulation. We conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1,4,5)P3-induced Ca2+ spiking, both in the unstimulated cell and following agonist stimulation. We propose the enhanced Ca2+ oscillations may mediate cellular transformation in cells underexpressing the 43 kDa 5-phosphatase.