Involvement of beta 3-adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function

Abstract

The beta(3)-adrenoreceptor plays a major role in lipolysis but the role and distribution of beta(3)-receptors in other specific sites have not been extensively studied. beta(3)-adrenergic receptors are present not only in adipose tissue but also in human gall bladder, colon, prostate, and skeletal muscle. Recently, beta(3)-adrenergic receptor stimulation was shown to elicit vasorelaxation of rat aorta through the NO-cGMP signal transduction pathway. Here we show that beta(3)-receptors are present in human corpus cavernosum and are localized mainly in smooth muscle cells. After activation by a selective beta(3)-adrenergic receptor agonist, BRL 37344, there was a cGMP-dependent but NO-independent vasorelaxation that was selectively blocked by a specific beta(3)-receptor antagonist. In addition, we report that the human corpus cavernosum exhibits basal beta(3)-receptor-mediated vasorelaxant tone and that beta(3)-receptor activity is linked to inhibition of the RhoA/Rho-kinase pathway. These observations indicate that beta(3)-receptors may play a physiological role in mediating penile erection and, therefore, could represent a therapeutic target for treatment of erectile dysfunction.

Figures

Figure 1

Relaxant effects of BRL 37344 on HCC strips. (a) BRL 37344 (BRL) relaxed HCC strips in the presence (+ end) and absence (− end) of endothelium. (b) l-NAME (100 μM) did not inhibit BRL 37344-induced vasorelaxation of HCC. (c) 1-H-[1,2,4]oxadiazolo [4,3a]quinoxalin-1-one (10 μM) did not inhibit BRL 37344-induced vasorelaxation. (d) Methylene blue (MB; 10 μM) inhibited BRL 37344-induced vasorelaxation. Data were analyzed by using ANOVA. **, P < 0.01, significantly different from corresponding control values. The data represent the mean ± SEM from eight separate experiments (eight strips per experiment).

Figure 2

Influence of BRL 37344 on cGMP levels in HCC. Refer to Materials and Methods for experimental details. Addition of IBMX caused a 3-fold increase in cGMP levels (τ, P < 0.05 vs. basal). Addition of BRL 37344 (BRL; 1 and 10 μM) caused a significant increase in cGMP (**, P < 0.01 vs. IBMX) that was not inhibited by l-NAME. In the same assay, ACh caused an increase in cGMP (P < 0.01 vs. IBMX) that was significantly inhibited by l-NAME (#, P < 0.01 vs. ACh). Experiments were from four different HCC strips and each sample was run in triplicate. The results are expressed as mean ± SEM, and data were compared by ANOVA followed by Bonferroni post hoc test.

Figure 3

Immunohistochemical localization of β3-adrenergic receptors in HCC. (Upper) The red/brown color is indicative of the presence of β3-adrenergic receptors in the tissue. Positive areas are indicated by the dotted lines (A, C, and E) and by the arrows (B, D, and F). The positive staining is absent in the stromal component, e.g., the area outside the dotted line in A, C, and E. The positive reaction is homogeneously diffuse both in smooth muscle cells that transversally define the vascular lumen and longitudinally define the intimal cushions (A and B). The positive reaction is also evident in the sections where the smooth muscle cells are associated with the venous wall (F). There is also a positive reaction in the smooth muscle trabecular components that are interconnected with the more external layers of the venous wall (C). (Lower) Two examples of Western blots for β3-adrenergic receptors in HCC performed with 30 μg of protein and two different Abs (a, from Alpha Diagnostic International; b, from Santa Cruz Biotechnology).

Figure 4

Influence of the β3-adrenergic receptor antagonist SR 59230A on vasorelaxation and smooth muscle tone in HCC. (a) SR 59230A virtually abolished BRL 37344-induced vasorelaxation. (b) SR 59230A alone caused a concentration-dependent contraction. (c) SR 59230A inhibited the contractile effects of 1 μM PE. In these experiments, SR 59230A was added to organ baths 15 min before addition of PE. The data in a represent four separate experiments. The data in b and c represent the mean ± SEM of duplicate determinations from four to five separate experiments. Data are expressed as mean ± SEM. In b, * (P < 0.05) and ** (P < 0.01) signify that values were significantly different from smooth muscle tone in the absence of added SR 59230A. In c, * (P < 0.05) and ** (P < 0.01) signify that values were significantly different from PE-induced contractions in the absence of added SR 59230A. Data were analyzed by ANOVA followed by Bonferroni post hoc test.

Figure 5

Influence of hET-1 on vasorelaxant responses to BRL 37344 (BRL), ACh, and SNP. (a) Precontraction of HCC strips with 10 nM hET-1 resulted in the abolition of the smooth muscle relaxant action of BRL. The smooth muscle relaxant actions of ACh and SNP were maintained. The relaxant response to ACh was slightly reduced (see b) but that of SNP was not affected by hET-1 (data not shown). (b) HCC strips were precontracted with either PE (1 μM) or hET-1 (10 nM). The smooth muscle relaxant effect of ACh in HCC strips precontracted with hET-1 was significantly reduced when compared with HCC strips precontracted with PE. All strips were tested at the end of experiments for their capacity to relax in response to added SNP to be certain that the smooth muscle in HCC was functionally intact. Data are expressed as mean ± SEM of duplicate determinations from four to five separate experiments. In a, *** (P < 0.001) signifies that values for BRL in the presence of hET-1 were significantly different from values for BRL in the presence of PE (data not shown). In b, *** (P < 0.001) signifies that values for ACh in the presence of hET-1 (endothelin) were significantly different from values for ACh in the presence of PE. Data were analyzed by ANOVA followed by Bonferroni post hoc test.