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

Abstract

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).

Figure 1

Detection by RT–PCR of β₃-AR and adipsin gene transcripts in white adipose tissue, heart, thoracic aorta and freshly isolated aortic endothelial cells. Amplified cDNA fragments were separed on 2% agarose gels and visualized by staining with ethidium bromide. Sizes of the PCR products are indicated on the left and right of the panels. M: 100 pb ladder (Biolabs, New England); NRT: none reverse transcription; RT: reverse transcription; β₃-AR: β₃-adrenoceptors; WAT: white adipose tissue; Thor. Aorta: thoracic aorta; Endo. Cell: endothelial cells.

Figure 2

Comparison of von Willebrand factor (A) and β₃-AR expression in rat thoracic aorta (B). Adjacent 10 μm thick sections were incubated with either von Willebrand antibody (vWf Ab; A) or rat β₃-AR antibody (rβ₃-AR Ab; B) revealed by peroxydase-conjugated second antiserum. Black arrowhead shows endothelium intensively stained with vWf and rβ₃-AR Ab (A–B). Same staining after pre-absorption of rβ₃-AR Ab with the blocking peptide (rβ₃-AR Ab +B pept.; C). Micrographs X100.

Figure 3

Inhibition of α₂-AR induced relaxation, in thoracic aorta rings from PTX pre-treated rats. The rats were injected i.v. with saline (sham) or PTX 10 μg kg⁻¹ (pre-treated) 3 days before experiments. Typical recordings of relaxant effects of UK 14304 for concentrations ranging from 0.01 to 1 μM in aortic rings from sham operated rats (A) and PTX pre-treated rats (B). (C) Concentration-response curves for UK 14304 in aortic rings from sham operated rats (n=7) and PTX pre-treated rats (n=8). Results are expressed as the percentage of relaxation from the steady-state contraction level induced by phenylephrine. Each point is the mean of n experiments, and the vertical lines show the s.e. of the mean. When no error bar is shown, the error is smaller than the symbol. *P<0.05 and **P<0.01 indicate significant differences from control. Both curves are significantly different (two-way ANOVA).

Figure 4

Effects of PTX pre-treatment on β₃-AR-induced relaxation in rat aorta. The rats were injected i.v. with saline (sham) or PTX 10 μg kg⁻¹ (pre-treated) 3 days before experiments. Typical recordings of relaxant effects induced by SR 58611A for concentrations ranging from 0.1 to 30 μM on aortic rings from sham operated rats (A) and PTX pre-treated rats (B). (C) Concentration-response curves for SR 58611A in aortic rings from sham operated rats (n=7) and PTX pre-treated rats (n=8). The mean curves resulting from subtraction of the spontaneous relaxation of control vessels pre-treated or not with PTX are shown. Results are expressed as the percentage of relaxation from the steady-state contraction level induced by phenylephrine. Each point is the mean of n experiments, and the vertical lines show the s.e. of the mean. When no error bar is shown, the error is smaller than the symbol. *P<0.05 and **P<0.01 indicate significant differences from control.

Figure 5

Inhibition of β₃-AR-induced relaxation in rat aorta by high extra-cellular potassium concentration. Typical recordings of SR 58611A for concentrations ranging from 10 to 30 μM on rings pre-constricted with either phenylephrine (PE) (A) or KCl (B). The recordings are representative in each case of five experiments.

Figure 6

Inhibition of β₃-adrenergic induced relaxation in rat aorta by potassium channel blockers. The mean curves resulting from subtraction of the spontaneous relaxation of control vessel pre-treated or not with potassium channels blockers are shown. (A) Concentration-response curves for SR 58611A in the absence (n=9) and presence of 0.1 μM iberiotoxin (n=8) in rat thoracic aortic rings constricted with phenylephrine. (B) Concentration response curves for SR 58611A in the absence (n=21) and presence of 1 μM glibenclamide (n=6) or 100 μM tolbutamide (n=6) in rat thoracic aortic rings constricted with phenylephrine. (C) Concentration response curves for SR 58611A in the absence (n=21) and presence of 1 mM 4-aminopyridine (n=7) in rat thoracic aortic rings constricted with phenylephrine. Results are expressed as the percentage of relaxation from the steady-state contraction level induced by phenylephrine. Each point is the mean of n experiments, and the vertical lines show the s.e. of the mean. When no error bar is shown, the error is smaller than the symbol. *P<0.05 and **P<0.01 indicate significant differences from control.