α1-Adrenoceptor-mediated depletion of phosphatidylinositol 4, 5-bisphosphate inhibits activation of volume-regulated anion channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE Volume-regulated anion channels (VRACs) play an important role in cell-volume regulation. alpha(1)-Adrenoceptor stimulation by phenylephrine (PE) suppressed the hypotonic activation of VRAC current in mouse ventricular cells and regulatory volume decrease (RVD) was also absent in PE-treated cells. We examined whether the effects of alpha(1)-adrenoceptor stimuli on VRAC current were modulated by phosphatidylinositol signalling. EXPERIMENTAL APPROACH Whole-cell patch-clamp method was used to record the hypotonicity-induced VRAC current in mouse ventricular cells. RVD was analyzed by videomicroscopic measurement of cell images. KEY RESULTS The attenuation of VRAC current by PE was suppressed by alpha(1A)-adrenoceptor antagonists (prazosin and WB-4101), anti-G(q) protein antibody and a specific phosphoinositide-specific phospholipase C (PLC) inhibitor (U-73122), but not by antagonists for alpha(1B)-, alpha(1D)- or beta-adrenoceptor, or protein kinase C inhibitors. The inhibition of VRAC by PE was antagonized by intracellular excess phosphatidylinositol 4,5-bisphosphate (PIP2), while intracellular anti-PIP2 antibody (PIP2 Ab) inhibited the activation of VRAC currents. When cells were loaded with phosphatidylinositol 3,4,5-trisphosphate (PIP3) with or without PIP2 Ab, PE little affected the VRAC current. Extracellular m-3M3FBS (an activator of PLC) suppressed VRAC in the absence of PE, and this effect was reversed by intracellular excess PIP2. CONCLUSIONS AND IMPLICATIONS Our results indicate that the stimulation of alpha(1A)-adrenoceptors by PE inhibited the activation of cardiac VRAC current via PIP3 depletion brought about by PLC-dependent reduction of membrane PIP2 level.