Enhancement of the RhoA/Rho kinase pathway is associated with stress-related erectile dysfunction in a restraint water immersion stress model

Abstract

Stress is a risk factor for erectile dysfunction (ED); however, the pathology of stress-induced ED remains unclear. Accordingly, in this study, we investigated the mechanisms of stress-induced ED using a rat model. Ten-week-old male Wistar/ST rats were maintained in a cage filled with water to a height of 2 cm (stress group) or a normal cage (control group). We found that water immersion stress significantly enhanced the contractile response to noradrenaline in the corpus cavernosum (CC) (p < 0.05). Moreover, stress significantly decreased erectile function, as assessed by changes in intracavernous pressure (p < 0.01). In addition, Rho kinase-1 (ROCK-1) protein expression was significantly upregulated under stress conditions (p < 0.05), and phosphorylated myosin light chain (phospho-MLC) levels, contribute to smooth muscle contraction, were also upregulated (p < 0.01). Treatment with fasudil hydrochloride, a Rho kinase inhibitor, for 5 days significantly improved erectile function (p < 0.01) and normalized ROCK-1 and phospho-MLC levels (p < 0.01). Thus, the RhoA/Rho kinase pathway may be associated with stress-induced ED via contraction of CC. Stress also decreased the smooth muscle/collagen ratio of CC (p < 0.01), and fasudil treatment did not alleviate these effects (p = 0.50). These findings suggested that penile fibrosis gradually progressed under stress conditions and that fibrosis may be independent of the RhoA/Rho kinase pathway, implying that longer exposure to stress may promote ED. We conclude that stress-induced ED was caused by contraction of CC mediated by the RhoA/Rho kinase pathway.

Keywords: RhoA/Rho kinase pathway; contraction; erectile dysfunction; rat; stress.

FIGURE 1

Experimental protocol

FIGURE 2

Cumulative dose responses to NA in isolated corpus cavernosum samples. The responses are relative to responses induced by 80 mM K⁺ Krebs solution. Curves were fitted to all the data by nonlinear regression. Data are reported as means ± standard deviations (n = 7/group). *p < 0.05 versus the control using Student's t‐tests. NA, noradrenaline

FIGURE 3

mRNA expression levels of sympathetic nervous system‐related genes in the rat corpus cavernosum. Target gene expression was quantified relative to the expression of β‐actin using the comparative cycle threshold method. Data are reported as means ± standard deviations (n = 6/group). *p < 0.05 using Student's t‐tests. TH: tyrosine hydroxylase, α_1AR, adrenaline α_1A receptor; α_1BR, adrenaline α_1B receptor; ROCK‐1, Rho kinase‐1; ROCK‐2, Rho kinase‐2

FIGURE 4

Representative ICP and AP results. Charts show ICP and AP during electrostimulation (bold bar) in each group of rats (a). The EFS period is shown by the thicker bar. ICP to MAP ratios in each group rats were measured (b). Data are reported as means ± standard deviations (n = 8/group). **p < 0.01 using Bonferroni's multiple t‐tests. ICP, intracavernous pressure; MAP, mean arterial pressure

FIGURE 5

Protein levels of ROCK‐1 (a) and phospho‐MLC (b) in rat corpus cavernosum. Quantification was performed by densitometry analysis, with normalization to β‐actin. Data are expressed as the ratio compared with the control. Data are reported as means ± standard deviations (n = 5–6/group). *p < 0.05, **p < 0.01 using Bonferroni's multiple t‐tests. ROCK‐1, Rho kinase‐1; phospho‐MLC, phosphorylated‐myosin light chain

FIGURE 6

Histological analysis of the rat corpus cavernosum. Representative rat penises stained with Masson's trichrome stain (a). SM is stained red and collagen is stained blue. SM/collagen ratios in the rat corpus cavernosum were evaluated (b). Data are reported as means ± standard deviations (n = 5–6/group). **p < 0.01 using Bonferroni's multiple t‐tests. SM, smooth muscle

FIGURE 7

mRNA expression levels of inflammatory cytokines in the rat corpus cavernosum. Target gene expression was quantified relative to the expression of β‐actin using the comparative cycle threshold method. Data are reported as means ± standard deviations (n = 5–6/group). *p < 0.05, **p < 0.01 using Bonferroni's multiple t‐tests. TNF‐α, tumor necrosis factor‐α