Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+in arterial smooth muscle cells and elicits vasocontraction

Abstract

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P2] is a newly identified phosphoinositide that modulates intracellular Ca2+by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca2+, we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca2+stores PI(3,5)P2would increase intracellular Ca2+in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P2was present in the mouse aorta and that exogenously applied PI(3,5)P2readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P2elevated intracellular Ca2+, and it also contracted aortic rings. Both the rise in intracellular Ca2+and the contraction caused by PI(3,5)P2were prevented by antagonizing RyRs, while the majority of the PI(3,5)P2response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca2+stores with thapsigargin or caffeine and/or ryanodine blunted the Ca2+response and greatly attenuated the contraction elicited by PI(3,5)P2. The removal of extracellular Ca2+or addition of verapamil to inhibit voltage-dependent Ca2+channels reduced but did not eliminate the Ca2+or contractile responses to PI(3,5)P2. We also found that PI(3,5)P2depolarized aortic smooth muscle cells and that LaCl3inhibited those aspects of the PI(3,5)P2response attributable to extracellular Ca2+. Thus, full and sustained aortic contractions to PI(3,5)P2required the release of SR Ca2+, probably via the activation of RyR, and also extracellular Ca2+entry via voltage-dependent Ca2+channels.