No abstract

No abstract

The alpha1-adrenoceptor-G protein-phosphoinositide-specific phospholipase C (PLC) signal transduction pathway is assumed to play an important role in the regulation of contractile force and in the pathophysiology of myocardial hypertrophy. In the present study, the components of this pathway were investigated in left ventricles of hearts from hypertensive transgenic rats overexpressing the mouse renin gene [TG(mREN2)27] in comparison to age- and weight-matched Sprague-Dawley control rats. Contractile force was assessed in isolated electrically driven left ventricular papillary muscle strips. Alpha1-adrenoceptor density was measured by radioligand binding using [3H]prazosin, steady state levels of alpha q/11, and G protein beta-subunits by Western blotting. PLC activity was determined by a cell-free assay using exogenous phospholipid vesicles containing [3H]phosphatidylinositol (4,5)-bisphosphate as a substrate. Alpha1-adrenoceptor density was significantly increased (by 80%) in transgenic rats compared with control rats, while the positive inotropic response to the alpha1-adrenoceptor agonist phenylephrine was significantly reduced, suggesting a postreceptor defect in TG(mREN2)27. The expression of alpha q and alpha11 was verified by reverse transcription-polymerase chain reaction, and alpha q/11 steady state protein levels were shown to be similar in transgenic and control rats. Western blotting using a beta-common antibody revealed two bands at approximately 35 and 36 kD. The quantities of both were similar in TG(mREN2)27 compared with those in control rats. In contrast, PLC activity was significantly reduced (by 32%) in transgenic rats. In conclusion, our findings are consistent with a desensitization of the alpha1-adrenergic signal transduction pathway at the level of the effector.

1 Phosphoinositide-speci®c phospholipase C (PLC) is involved in the regulation of many cellular functions. In the myocardium, PLC-generated second messengers play a role in the regulation of contractile function and in the pathophysiology of myocardial hypertrophy. 2 In the present study, the eect of mastoparan, a tetradecapeptide which is capable of activating heterotrimeric G proteins by mimicking the action of an activated receptor, on membrane-bound human myocardial PLC, was investigated in a cell-free assay with exogenous phospholipids as a substrate. 3 Mastoparan stimulated human myocardial PLC approximately two fold with a half-maximal eect at approximately 2 mM and a maximal eect at 10 mM. The peptide did not alter the dependence of PLC on free calcium ions. In order to exclude non-speci®c eects of mastoparan due to its amphiphilic properties, dierent mastoparan derivatives were used as positive and negative controls. Mas17, an inactive mastoparan analogue with phsyical properties very similar to mastoparan, did not induce substantial PLC stimulation in human myocardial membranes. In contrast, Mas7, the most active mastoparan derivative known, caused a more pronounced PLC activation compared with the mother compound indicating that the eect was sequence-speci®c. Human myocardial PLC stimulation was pertussis toxin-insensitive and could not be abolished by addition of excess a-subunits from puri®ed retinal transducin or by excess GDP or GDPbS. In order to investigate whether mastoparan stimulated PLC via pertussis toxin-insensitive a q , a deletion mutant of PLCb 2 de®cient of the site of interaction with a q -subunits was expressed in COS-1 cells. Both wild-type and mutant PLCb 2 were similarly sensitive to stimulation by mastoparan. 4 It is concluded that mastoparan stimulates human myocardial PLC by a mechanism distinct from heterotrimeric G proteins.