α1-adrenoceptor-mediated breakdown of phosphatidylinositol 4,5-bisphosphate inhibits pinacidil-activated ATP-sensitive K+ currents in rat ventricular myocytes

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates ATP-sensitive K+ (KATP) channel activity. Because phospholipase C (PLC) hydrolyzes membrane-bound PIP2, which in turn may potentially decrease KATP channel activity, we investigated the effects of the α1-adrenoceptor-Gq-PLC signal transduction axis on pinacidil-activated KATP channel activity in adult rat and neonatal mouse ventricular myocytes. The α1-adrenoceptor agonist methoxamine (MTX) reversibly inhibited the pinacidil-activated KATP current in a concentration-dependent manner (IC50 20.9±6.6 μmol/L). This inhibition did not occur when the specific α1-adrenoceptor antagonist, prazosin, was present. An involvement of G proteins is suggested by the ability of GDPβS to prevent this response. Blockade of PLC by U-73122 (2 μmol/L) or neomycin (2 mmol/L) attenuated the MTX-induced inhibition of KATP channel activity. In contrast, the MTX response was unaffected by protein kinase C inhibition or stimulation by H-7 (100 μmol/L) or phorbol 12,13-didecanoate. The MTX-induced inhibition became irreversible in the presence of wortmannin (20 μmol/L), an inhibitor of phosphatidylinositol-4 kinase, which is expected to prevent membrane PIP2 replenishment. In excised inside-out patch membranes, pinacidil induced a significantly rightward shift of ATP sensitivity of the channel. This phenomenon was reversed by pretreatment of myocytes with MTX. Direct visualization of PIP2 subcellular distribution using a PLCδ pleckstrin homology domain-green fluorescent protein fusion constructs revealed reversible translocation of green fluorescent protein fluorescence from the membrane to the cytosol after α1-adrenoceptor stimulation. Our data demonstrate that α1-adrenoceptor stimulation reduces the membrane PIP2 level, which in turn inhibits pinacidil-activated KATP channels.