Beta 3-adrenoceptor in rat aorta: molecular and biochemical characterization and signalling pathway.

1. We have previously demonstrated that beta(3)-adrenoceptor (beta(3)-AR) stimulation induces endothelium-dependent vasorelaxation in rat aorta through the activation of an endothelial NO synthase associated with an increase in intracellular cGMP. The aim of the present study was to localise beta(3)-AR to confirm our functional study and to complete the signalling pathway of beta(3)-AR in rat aorta. 2. By RT-PCR, we have detected beta(3)-AR transcripts both in aorta and in freshly isolated endothelial cells. The absence of markers for adipsin or hormone-sensitive lipase in endothelial cells excluded the presence of beta(3)-AR from adipocytes. The localization of beta(3)-AR in aortic endothelial cells was confirmed by immunohistochemistry using a rat beta(3)-AR antibody. 3. To identify the G protein linked to beta(3)-AR, experiments were performed in rat pre-treated with PTX (10 microg kg(-1)), a G(i/0) protein inhibitor. The blockage of G(i/0) protein by PTX was confirmed by the reduction of vasorelaxation induced by UK 14304, a selective alpha(2)-AR agonist. The cumulative concentration-response curve for SR 58611A, a beta(3)-AR agonist, was not significantly modified on aorta rings from PTX pre-treated rats. 4. At the same level of contraction, the relaxations induced by 10 microM SR 58611A were significantly reduced in 30 mM-KCl pre-constricted rings (E(max)=16.7+/-8.4%, n=5), in comparison to phenylephrine (0.3 microM) pre-constricted rings (E(max)=49.11+/-11.0%, n=5, P<0.05). In addition, iberotoxin (0.1 microM), glibenclamide (1 microM) and 4-aminopyridine (1 mM), selective potassium channels blockers of K(Ca), K(ATP), and K(v) respectively, decreased the SR 58611A-mediated relaxation. 5. We conclude that beta(3)-AR is preferentially expressed in rat aortic endothelial cells. Beta(3)-AR-mediated aortic relaxation is independent of G(i/0) proteins stimulation, but results from the activation of several potassium channels, K(Ca), K(ATP), and K(v).