Adrenergic activation of cardiac phospholipase D: role of α1-adrenoceptor subtypes

Objective: Adrenergic stimulation of the heart leads to activation of the phospholipase D signal transduction pathway with formation of the intracellular second messengers phosphatidic acid and diacylglycerol, which may play a role in the development of myocardial hypertrophy by activating mitogen-activated protein kinases and protein kinase C. So far, the adrenergic receptor subtypes mediating activation of cardiac phospholipase D are not known. Methods: We developed an assay for determination of phospholipase D activity in the isolated perfused rat heart. Utilizing the phospholipase D specific transphosphatidylation reaction the stable product phosphatidylethanol (PEtOH) is formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of phospholipase D activity and was determined by HPLC and evaporative light-scattering detection (PEtOH μg/mg myocardial protein). Results: Basal PEtOH formation in unstimulated hearts was 0.06±0.01 μg/mg. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent phospholipase D activation with a maximum formation of PEtOH (0.17±0.01 μg/mg) at 100 μmol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the α1-adrenoceptor antagonist prazosin and was unaffected by the β-adrenoceptor antagonist propranolol. Further characterisation of α1-adrenoceptor subtypes with selective α1-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (α1A-selective: 0.06±0.01 μg/mg) and by BMY 7378 (α1D-selective: 0.07±0.01 μg/mg). In contrast, the α1B-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14±0.01 μg/mg). Conclusion: Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by α1-adrenoceptors. Here, the α1A-adrenoceptor subtype, but not the α1B-adrenoceptor are coupled to activation of cardiac phospholipase D.