α 1 -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⁺ (K_ATP) channel activity. Because phospholipase C (PLC) hydrolyzes membrane-bound PIP₂, which in turn may potentially decrease K_ATP channel activity, we investigated the effects of the α₁-adrenoceptor-G_q-PLC signal transduction axis on pinacidil-activated K_ATP channel activity in adult rat and neonatal mouse ventricular myocytes. The α₁-adrenoceptor agonist methoxamine (MTX) reversibly inhibited the pinacidil-activated K_ATP 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 K_ATP 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 K_ATP channels.