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).


CONCLUSIONS
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.
Mier
Kenneth
K
Medizinische Klinik II, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, Germany.
Kemken
Dorit
D
Katus
Hugo A
HA
Richardt
Gert
G
Kurz
Thomas
T
D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D006332:Cardiomegaly / Q000378:metabolism*; D003000:Clonidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004146:Dioxanes / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D009638:Norepinephrine / Q000494:pharmacology; D010477:Perfusion; D010739:Phospholipase D / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011224:Prazosin / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction; D013268:Stimulation, Chemical
D000317:Adrenergic alpha-Antagonists; D000319:Adrenergic beta-Antagonists; D004146:Dioxanes; D010879:Piperazines; D018340:Receptors, Adrenergic, alpha-1; C051731:chlorethylclonidine; D011433:Propranolol; C010654:(2-(2',6'-dimethoxy)phenoxyethylamino)methylbenzo-1,4-dioxane; D010739:Phospholipase D; C053428:BMY 7378; D003000:Clonidine; D009638:Norepinephrine; D011224:Prazosin