RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: a mechanism for diabetes-associated erectile dysfunction

Abstract

Significant impairment in endothelial-derived nitric oxide is present in the diabetic corpus cavernosum. RhoA/Rho-kinase may suppress endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that RhoA/Rho-kinase contributes to diabetes-related erectile dysfunction and down-regulation of eNOS in the streptozotocin (STZ)-diabetic rat penis. Colocalization of Rho-kinase and eNOS protein was present in the endothelium of the corpus cavernosum. RhoA/Rho-kinase protein abundance and MYPT-1 phosphorylation at Thr-696 were elevated in the STZ-diabetic rat penis. In addition, eNOS protein expression, cavernosal constitutive NOS activity, and cGMP levels were reduced in the STZ-diabetic penis. To assess the functional role of RhoA/Rho-kinase in the penis, we evaluated the effects of an adeno-associated virus encoding the dominant-negative RhoA mutant (AAVTCMV19NRhoA) on RhoA/Rho-kinase and eNOS and erectile function in vivo in the STZ-diabetic rat. STZ-diabetic rats transfected with AAVCMVT19NRhoA had a reduction in RhoA/Rho-kinase and MYPT-1 phosphorylation at a time when cavernosal eNOS protein, constitutive NOS activity, and cGMP levels were restored to levels found in the control rats. There was a significant decrease in erectile response to cavernosal nerve stimulation in the STZ-diabetic rat. AAVT19NRhoA gene transfer improved erectile responses in the STZ-diabetic rat to values similar to control. These data demonstrate a previously undescribed mechanism for the down-regulation of penile eNOS in diabetes mediated by activation of the RhoA/Rho-kinase pathway. Importantly, these data imply that inhibition of RhoA/Rho-kinase improves eNOS protein content and activity thus restoring erectile function in diabetes.

Figures

Fig. 1.

Colocalization of Rho-kinase and eNOS in the rat corpus cavernosum. Confocal microscopic images of Rho-kinase (a and d; green), eNOS (b and e; red), and an overlay image of Rho-kinase and eNOS(c and f; yellow) in control rat corpus cavernosum demonstrating that Rho-kinase and eNOS are coexpressed in the endothelium (white arrow) are shown. Photomicrograph is representative of three experiments.

Fig. 2.

In vivo erectile responses to Y-27632 in control and STZ-diabetic rats. Shown is a bar graph demonstrating the increase in ICP in control and STZ-diabetic rats in response to intracavernous injection of the Rho-kinase inhibitor Y-27632 (3-30 nmol). n, number of experiments; *, P < 0.05, response significantly different to control.

Fig. 3.

Effect of diabetes on corporal RhoA, Rho-kinase, and MYPT-1 protein expression. (a) Western blot analysis demonstrating corporal expression of RhoA, Rho-kinase, and GAPDH protein in control (lanes 1 and 2) and 7 days after gene transfer of AAVCMVβgal (lanes 3-5) or AAVCMVT19NRhoA (lanes 6-8) in STZ-diabetic (DM) rats. (b) Densitometry analysis of RhoA and Rho-kinase protein. (c) Western blot analysis demonstrating corporal expression of Phos-Thr-696 MYPT-1 in control (lanes 1 and 2) and 7 days after gene transfer of AAVCMVβgal (lanes 3 and 4) or AAVCMVT19NRhoA (lanes 5 and 6) in STZ-diabetic rats. (d) Densitometry analysis of Phos-Thr-696 MYPT-1. *, P < 0.05 when compared to control; **, P < 0.05 when compared to STZ-diabetic tissue transfected with AAVCMVβgal.

Fig. 4.

Effect of diabetes on corporal eNOS and nNOS protein expression. (a) Western blot analysis demonstrating corporal expression of eNOS, nNOS, and GAPDH protein in control (lanes 1 and 2) and 7 days after gene transfer of AAVCMVβgal (lanes 3 and 4) or AAVCMVT19NRhoA (lanes 5 and 6) in STZ-diabetic rats. (b) Densitometry analysis of eNOS and nNOS protein. *, P < 0.05 when compared to control; **, P < 0.05 when compared to STZ-diabetic tissue transfected with AAVCMVβgal.

Fig. 5.

Effect of diabetes on corporal NOS activity and cGMP levels. (a) NOS activity as measured by calcium-dependent conversion of l-arginine to l-citrulline. (b) cGMP levels in control and STZ-diabetic rats corporal tissue after intracavernosal administration of vehicle, AAVCMVβgal, and AAVT19NRhoA. n, number of tissue samples; *, P < 0.05 when compared to control; **, P < 0.05 when compared to STZ-diabetic tissue treated with vehicle or AAVCMVβgal.

Fig. 6.

Influence of in vivo gene transfer of the dominant-negative RhoA mutant on neurogenic-mediated erectile responses. (a) Representative ICP tracing showing responses to CNS at the 5-V setting for 1 min in control (Left) and STZ-diabetic (DM) rats transfected with AAVCMVβgal (Center) or AAVCMVT19NRhoA (Right). (b) Bar graph depicting the voltage-dependent erectile response (ICP/MAP) and total ICP (area under the erectile curve in mmHg/sec) in response to CNS in control and STZ-diabetic rats transfected with AAVCMVβgal or AAVCMVT19NRhoA. In vivo experiments were conducted 7 days after transfection with adeno-associated viruses. n, number of experiments; *, P < 0.05, response significantly different compared to control; **, P < 0.05, response significantly different compared to STZ-diabetic rats transfected with AAVCMVβgal.