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

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 alpha(1)-adrenoceptor-G(q)-PLC signal transduction axis on pinacidil-activated K-ATP channel activity in adult rat and neonatal mouse ventricular myocytes. The alpha(1)-adrenoceptor agonist methoxamine (MTX) reversibly inhibited the pinacidil-activated K-ATP current in a concentration-dependent manner (IC50 20.9+/-6.6 mumol/L). This inhibition did not occur when the specific alpha(1)-adrenoceptor antagonist, prazosin, was present. An involvement of G proteins is suggested by the ability of GDPbetaS to prevent this response. Blockade of PLC by U-73122 (2 mumol/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 mumol/L) or phorbol 12,13-didecanoate. The MTX-induced inhibition became irreversible in the presence of wortmannin (20 mumol/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 PLCdelta pleckstrin homology domain-green fluorescent protein fusion constructs revealed reversible translocation of green fluorescent protein fluorescence from the membrane to the cytosol after alpha(1)-adrenoceptor stimulation. Our data demonstrate that alpha(1)-adrenoceptor stimulation reduces the membrane PIP2 level, which in turn inhibits pinacidil-activated K-ATP channels.