Role of the nitric oxide pathway and the endocannabinoid system in neurogenic relaxation of corpus cavernosum from biliary cirrhotic rats

Abstract

Background and purpose: Relaxation of corpus cavernosum, which is mediated by nitric oxide (NO) released from non-adrenergic non-cholinergic (NANC) neurotransmission, is critical for inducing penile erection and can be affected by many pathophysiological conditions. However, the peripheral effect of liver cirrhosis on erectile function is as yet unknown. The aim of the present study was to investigate the effect of biliary cirrhosis on NANC-mediated relaxation of rat corpus cavernosum and the possible roles of endocannabinoid and nitric oxide systems in this model.

Experimental approach: Cirrhosis was induced by bile duct ligation. Controls underwent sham operation. Four weeks later, strips of corpus cavernosum were mounted in a standard organ bath and NANC-mediated relaxations were obtained by applying electrical field stimulation.

Key results: The NANC-mediated relaxation was enhanced in corporal strips from cirrhotic animals. Anandamide potentiated the relaxations in both groups. Either AM251 (CB(1) antagonist) or capsazepine (vanilloid VR(1) antagonist), but not AM630 (CB(2) antagonist), prevented the enhanced relaxations of cirrhotic strips. Either the non-selective NOS inhibitor L-NAME or the selective neuronal NOS inhibitor L-NPA inhibited relaxations in both groups, but cirrhotic groups were more resistant to the inhibitory effects of these agents. Relaxations to sodium nitroprusside (NO donor) were similar in tissues from the two groups.

Conclusions and implications: Cirrhosis potentiates the neurogenic relaxation of rat corpus cavernosum probably via the NO pathway and involving cannabinoid CB(1) and vanilloid VR(1) receptors.

Figure 1

Dose–response relationship of contractions induced by phenylephrine in isolated corpus cavernosum of sham-operated (SO) and cirrhotic (BDL) groups. Each group consisted of six rats.

Figure 2

Tracings of frequency-dependent relaxant responses to EFS in corporal strips precontracted with 7.5 μM phenylephrine (Phe) in the presence of guanethidine (5 μM) and atropine (1 μM). In comparison with sham-operated strips (a), relaxant responses to EFS were enhanced in corporal strips of biliary cirrhotic animals (b). Anandamide (AN, 1 μM) potentiated the NANC-mediated relaxations in both sham-operated (c) and cirrhotic (d) groups. EFS was applied at 2, 5, 10 and 15 Hz. EFS, electrical field stimulation; NANC, non-adrenergic non-cholinergic.

Figure 3

EFS-induced relaxation in corporal strips precontracted with phenylephrine (7.5 μM) in the presence of guanethidine (5 μM) and atropine (1 μM) from sham-operated group in the absence (SO) or presence of 1 μM anandamide (SO+AN) and cirrhotic group in the absence (BDL) or presence of 1 μM anandamide (BDL+AN). The frequency-dependent relaxations were significantly enhanced in cirrhotic group as compared with sham groups. Anandamide potentiated the relaxant responses to EFS in both sham and cirrhotic groups. Each group consisted of six rats (^*P<0.05, ^**P<0.001 and ^***P<0.001 compared with sham group without anandamide; ^#P<0.05 and ^##P<0.01 compared with cirrhotic group without anandamide). AN, anandamide; BDL, bile duct ligated; EFS, electrical field stimulation.

Figure 4

Effect of AM251 (10 μM) on frequency-dependent relaxations in sham-operated (SO) and cirrhotic (BDL) groups in the presence or absence of 1 μM anandamide (AN). Each group consisted of six rats (^*P<0.05 compared with cirrhotic group with anandamide; ^#P<0.05 compared with cirrhotic groups without anandamide, ns=nonsignificant difference). BDL, bile duct ligated.

Figure 5

Effect of 10 μM capsazepine (CZ) on frequency-dependent relaxations in sham-operated (SO) and cirrhotic (BDL) groups in the presence or absence of 1 μM anandamide (AN). Each group consisted of six rats (^*P<0.05, ^**P<0.01 compared with cirrhotic groups with anandamide; ^#P<0.05 compared with cirrhotic groups without anandamide; ns=nonsignificant difference). BDL, bile duct ligated.

Figure 6

L-NAME inhibited the relaxant responses to 10-Hz stimulation in precontracted rat corpus cavernosum in a concentration-dependent manner. In the presence of L-NAME (30 nM), the potentiating activity of anandamide (AN) on relaxant responses to EFS was significantly inhibited in sham rats (SO). L-NAME (30 nM) alone did not cause a significant attenuation of NANC-mediated relaxation in other groups (a; ^*P<0.05, ^**P<0.01, and ^***P<0.001 compared with corresponding groups without L-NAME). There was no significant difference in NANC-mediated relaxations between cirrhotic groups without anandamide (BDL) and cirrhotic groups with anandamide (BDL+AN) in the presence of different doses of L-NAME (b). Each groups consisted of six rats (b; ^*P<0.05, ^**P<0.01, and ^***P<0.001 compared with corresponding sham groups without anandamide; ^#P<0.05 compared with corresponding cirrhotic groups without anandamide; ns=nonsignificant difference). BDL, bile duct ligated; L-NAME, N^ω-nitro-L-arginine methyl ester; NANC, non-adrenergic non-cholinergic.

Figure 7

Effect of L-NPA on the relaxant responses to 10-Hz stimulation in precontracted corpus cavernosum from sham-operated (SO) and cirrhotic (BDL) rats in the presence or absence of anandamide (AN, 1 μM). In the presence of L-NPA (0.1 μM), the potentiating activity of anandamide on relaxant responses to EFS was significantly inhibited in sham rats. L-NPA (0.1 μM) alone did not cause a significant attenuation of NANC-mediated relaxation in other groups (a; ^*P<0.05, ^**P<0.01, and ^***P<0.001 compared with corresponding groups without L-NPA). There is no significant difference in NANC-mediated relaxations between cirrhotic groups without anandamide (BDL) and cirrhotic groups with anandamide (BDL+AN) in the presence of different doses (1–100 μM) of L-NPA (b). Each groups consisted of six rats (b; ^*P<0.05, ^**P<0.01, and ^***P<0.001 compared with corresponding sham groups without anandamide; ^#P<0.05 compared with corresponding cirrhotic groups without anandamide; ns=nonsignificant difference). BDL, bile duct ligated; L-NPA, N^ω-propyl-L-Arginine.

Figure 8

Concentration-dependent relaxation in response to sodium nitroprusside in precontracted rat corporal smooth muscles of sham-operated (SO) and cirrhotic (BDL) groups. Each group consisted of six rats. BDL, bile duct ligated.

Figure 9

Western blot of neuronal NO synthase (nNOS) enzyme, cannabinoid CB₁ and vanilloid VR₁ receptor protein in corporal tissue strips from two representative sham-operated (SO) and biliary cirrhotic (BDL) rats.