Adrenergic activation of cardiac phospholipase D: role of alpha(1)-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 microg/mg myocardial protein).

Results: Basal PEtOH formation in unstimulated hearts was 0.06+/-0.01 microg/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 microg/mg) at 100 micromol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the alpha(1)-adrenoceptor antagonist prazosin and was unaffected by the beta-adrenoceptor antagonist propranolol. Further characterisation of alpha(1)-adrenoceptor subtypes with selective alpha(1)-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (alpha(1A)-selective: 0.06+/-0.01 microg/mg) and by BMY 7378 (alpha(1D)-selective: 0.07+/-0.01 microg/mg). In contrast, the alpha(1B)-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14+/-0.01 microg/mg).

Conclusion: Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by alpha(1)-adrenoceptors. Here, the alpha(1A)-adrenoceptor subtype, but not the alpha(1B)-adrenoceptor are coupled to activation of cardiac phospholipase D.