Effect of anandamide on nonadrenergic noncholinergic-mediated relaxation of rat corpus cavernosum

The purpose of this study was to investigate the effect of the endogenous cannabinoid anandamide on the nonadrenergic noncholinergic (NANC) relaxant responses to electrical field stimulation in isolated rat corpus cavernosum. The corporal strips were mounted under tension in a standard oxygenated organ bath with guanethidine sulfate (5 microM) and atropine (1 microM) (to produce adrenergic and cholinergic blockade). The strips were precontracted with phenylephrine hydrochloride (7.5 microM) and electrical field stimulation was applied at different frequencies to obtain NANC-mediated relaxation. The expression of CB1, CB2 and vanilloid receptor proteins within the rat corpus cavernosum was evaluated using western blot analysis. The results showed that the relaxant responses to electrical stimulation were significantly enhanced in the presence of anandamide at 1 and 3 microM. The potentiating effect of anandamide (1 microM) on relaxation responses was significantly attenuated by either the selective cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; 1 microM) or the vanilloid receptor antagonist capsazepine (3 microM), but not by the selective cannabinoid CB2 receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl) ethyl]-1H-indol-3-yl (4-methoxyphenyl)methanone (AM630; 1 microM). Neither of these antagonists had influence on relaxation responses. Indomethacin (20 microM) had no effect on NANC-mediated relaxation in the presence or absence of anandamide (1 microM). Preincubation with Nw-Nitro-L-Arginine Methyl Ester (L-NAME; 1 microM) significantly inhibited the relaxation responses in the presence or absence of 1 microM anandamide. Although at 30 nM, L-NAME did not cause a significant inhibition of relaxant responses individually, it significantly inhibited the potentiating effect of anandamide (1 microM) on relaxation responses. Anandamide (1 microM) had no influence on concentration-dependent relaxant responses to sodium nitroprusside (10 nM-1 mM), a nitric oxide (NO) donor. The western blotting of corporal tissues demonstrated the existence of both vanilloid and CB1 receptors in corporal strips. In conclusion, our results showed that anandamide has a potentiating effect on NANC-mediated relaxation of rat corpus cavernosum through both CB1 and vanilloid receptors and the NO-mediated component of the NANC relaxant responses to electrical stimulation is involved in this enhancement.     

Role of opioid and nitric oxide systems in the nonadrenergic noncholinergic-mediated relaxation of corpus cavernosum in bile duct-ligated rats

Changes in nonadrenergic noncholinergic (NANC)-mediated relaxation of the anococcygeus muscle have been demonstrated in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and nitric oxide (NO) overproduction. This study was therefore undertaken to investigate the effect of cholestasis on the NANC-mediated relaxation of corpus cavernosum in bile duct-ligated rats and to examine the possible roles of the opioid system and nitric oxide in the cholestasis-associated alterations of corpus relaxation. Bile duct-ligated and sham-operated rats were treated for 2 weeks with either normal saline, N (omega)-nitro L-arginine methylester (L-NAME) (3 mg/kg/day, i.p.) or naltrexone (20 mg/kg/day, i.p.). On the 14th day, the strips of corpus cavernosum were mounted under tension in a standard oxygenated organ bath with guanethidine sulfate (5 microM) and atropine sulfate (1 microM) (to produce adrenergic and cholinergic blockade). The strips were precontracted with phenylephrine hydrochloride (7.5 microM) and electrical field stimulation was applied at different frequencies to obtain NANC-mediated frequency-dependent relaxant responses. The results showed that the amplitudes of relaxation responses at each frequency in bile duct-ligated rats were greater than the responses of sham-operated animals. This increase in relaxation responses in bile duct-ligated rats was inhibited by chronic L-NAME administration for 2 weeks so it seemed that it might be due to the nitric oxide overproduction in cholestatic states. Chronic administration of naltrexone for 2 weeks to bile duct-ligated rats had the same inhibitory effect on the relaxation responses. Our results demonstrated that in cholestasis, there was an increase in NANC-mediated relaxation of corpus cavernosum and both opioid and nitric oxide systems were involved in this increase.     

Anandamide mediates hyperdynamic circulation in cirrhotic rats via CB(1) and VR(1) receptors

BACKGROUND AND PURPOSE: Hyperdynamic circulation and mesenteric hyperaemia are found in cirrhosis. To delineate the role of endocannabinoids in these changes, we examined the cardiovascular effects of anandamide, AM251 (CB(1) antagonist), AM630 (CB(2) antagonist) and capsazepine (VR1 antagonist), in a rat model of cirrhosis. EXPERIMENTAL APPROACH: Cirrhosis was induced by bile duct ligation. Controls underwent sham operation. Four weeks later, diameters of mesenteric arteriole and venule (intravital microscopy), arterial pressure, cardiac output, systemic vascular resistance and superior mesenteric artery (SMA) flow were measured after anandamide, AM251 (with or without anandamide), AM630 and capsazepine administration. CB(1), CB(2) and VR1 receptor expression in SMA was assessed by western blot and RT-PCR. KEY RESULTS: Anandamide increased mesenteric vessel diameter and flow, and cardiac output in cirrhotic rats, but did not affect controls. Anandamide induced a triphasic arterial pressure response in controls, but this pattern differed markedly in cirrhotic rats. Pre-administration of AM251 blocked the effects of anandamide. AM251 (without anandamide) increased arterial pressure and systemic vascular resistance, constricted mesenteric arterioles, decreased SMA flow and changed cardiac output in a time-dependent fashion in cirrhotic rats. Capsazepine decreased cardiac output and mesenteric arteriolar diameter and flow, and increased systemic vascular resistance in cirrhotic rats, but lacked effect in controls. Expression of CB(1) and VR1 receptor proteins were increased in cirrhotic rats. AM630 did not affect any cardiovascular parameter in either group. CONCLUSIONS AND IMPLICATIONS: These data suggest that endocannabinoids contribute to hyperdynamic circulation and mesenteric hyperaemia in cirrhosis, via CB(1)- and VR1-mediated mechanisms.     

Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus

The nitric oxide (NO) synthesis inhibitor NG-monomethyl L-arginine (L-NMMA) reduced NANC-mediated relaxations of isolated strips of the rat gastric fundus elicited by low frequencies or short periods of field stimulation, but D-NMMA had no effect. The inhibitory effect of L-NMMA on NANC-mediated relaxations was partially reversed by L-arginine but was not affected by D-arginine. A VIP antibody abolished the relaxant response to VIP and reduced the responses to stimulation. Residual responses to stimulation in the presence of VIP antibody were further reduced by L-NMMA. The tone of the fundus strip was slightly increased by L-NMMA and slightly reduced by L-arginine. The relaxation produced by VIP was slightly reduced by L-NMMA and enhanced by L-arginine. Relaxations produced by peptide histidine isoleucine, sodium nitroprusside or isoprenaline were not affected by L-NMMA or L-arginine. The results suggest that NO as well as VIP is involved in NANC-mediated relaxations of the rat gastric fundus.     

The effect of sildenafil on corpus cavernosal smooth muscle relaxation and cyclic GMP formation in the diabetic rabbit

Sildenafil, a type V phosphodiesterase inhibitor, enhances smooth muscle relaxation in normal human and rabbit corpus cavernosum. We investigated the in vitro effects of sildenafil on non-adrenergic, non-cholinergic and nitric oxide (NO)-mediated cavernosal smooth muscle relaxation in diabetic rabbits, since alterations in this pathway are recognised in diabetic erectile dysfunction. Diabetes mellitus was induced in male New Zealand White rabbits with alloxan. Cavernosal strips from age-matched control, 3- and 6-month diabetic animals were mounted in organ baths. Relaxation responses to electrical field stimulation (1-20 Hz) or sodium nitroprusside (10(-8)-10(-4) M) were assessed in the absence and presence of sildenafil (10(-8) and 10(-7) M). The effect of sildenafil on cGMP formation by the corpus cavernosum was also assessed following stimulation with sodium nitroprusside, A23187 and acetylcholine. Sodium nitroprusside-stimulated relaxations were significantly (P<0.03) impaired in the corpus cavernosum from both diabetic groups, (IC(50)=4.6 x 10(-6) M following 3 months of diabetes mellitus and 4.0 x 10(-6) M following 6 months of diabetes mellitus; compared to 7.5 x 10(-7) M for pooled age-matched controls). Sildenafil (10(-7) M) significantly enhanced sodium nitroprusside-stimulated relaxation in control (P<0.05) and diabetic groups (P<0.03). Electrical field stimulation-mediated relaxations of the corpus cavernosum were significantly impaired after 6-month diabetes mellitus and enhanced by sildenafil (10(-8) M). cGMP formation by the diabetic corpus cavernosum was impaired significantly, but restored towards normal by sildenafil. We suggest that the impairment of NO-mediated relaxation of the corpus cavernosum reflect, at least in part, a defect in guanylyl cyclase activity. These findings support the use of sildenafil as an effective, orally administered, treatment for diabetic erectile dysfunction.     

Ginsenosides-induced nitric oxide-mediated relaxation of the rabbit corpus cavernosum.

1. Ginsenosides, the active ingredients extracted from Panax ginseng, have been shown to promote nitric oxide (NO) release in bovine aortic endothelial cells. Since the endothelial cells and the perivascular nerves in penile corpus cavernosum contain NO synthase and an NO-like substance has been shown to be released from these cells which relaxes corpus cavernosum, the possibility that ginsenosides may relax corpus cavernosum by releasing endogenous NO was examined. 2. With an in vitro tissue superfusion technique, ginsenosides (250, 500 and 750 micrograms ml-1) relaxed corpus cavernosum, concentration-dependently. 3. Using an in vitro tissue bath technique, acetylcholine (ACh)-induced relaxations were increased in the presence of ginsenosides (250 micrograms ml-1). 4. Ginsenosides at 100 micrograms ml-1 significantly enhanced the tetrodotoxin (TTX)-sensitive relaxation of corpus cavernosum elicited by transmural nerve stimulation. 5. The ginsenosides-induced, ACh-induced and ginsenosides-enhanced transmural nerve stimulation-elicited relaxations were significantly attenuated by NG-nitro-L-arginine (100 microM) and oxyhaemoglobin (oxyHb; 5-10 microM), and were enhanced by superoxide dismutase (SOD; 50 u ml-1). 6. The relaxations and their attenuation by NG-nitro-L-arginine and TTX were associated with increase and decrease in tissue cyclic GMP levels, respectively. 7. It is concluded that ginsenosides may release NO from endothelial cells, and enhance NO release from endothelial cells elicited by other vasoactive substances and from perivascular nitrergic nerves in the corpus cavernosum. These endothelial and neurogenic effects of ginsenosides in inducing relaxation of the corpus cavernosum may account for the aphrodisiac effect of Panax ginseng.     

Different responses to angiotensin-(1-7) in young, aged and diabetic rabbit corpus cavernosum.

We evaluated the ability of angiotensin-(1-7) [Ang-(1-7)] to produce relaxation of the corpus cavernosum of New Zealand White rabbits. The reactivity of corpus cavernosal strips isolated from young rabbits (8-10 months old) was assessed in organ-bath chambers. Cumulative concentration response curves for Ang-(1-7), angiotensin II (Ang II), carbachol and sodium nitroprusside (SNP) were established. Ang-(1-7) (10(-12) to 10(-5)M) produced a concentration-dependent relaxation of the corpus cavernosal strips with a pD(2) value of 9.8+/-0.3. Ang-(1-7)-induced maximal relaxant response was reduced by 48+/-2%, 57+/-3% and 76+/-2% in the presence of A-779 (10(-6)M), a selective Ang-(1-7) receptor (AT(1-7)) antagonist, nitro-l-arginine methyl ester (l-NAME) (10(-4)M), an inhibitor of nitric oxide (NO) synthase, or iberiotoxin (5 x 10(-8)M), an inhibitor of calcium-activated potassium (BK) channels, respectively. In contrast, Ang II-induced contraction was increased in the presence of A-779. Carbachol-, SNP- and Ang-(1-7)-induced relaxations were significantly reduced whereas Ang-II induced contraction was significantly increased in the cavernosum strips from older (18-24 months old) and diabetic rabbits compared to the young. Pre-incubation of the cavernosum strips obtained from young, older or diabetic rabbits with Ang-(1-7) resulted in a significant attenuation of Ang II-induced contraction. In conclusion, these results demonstrate that Ang-(1-7) can produce nitric oxide-dependent relaxation of the corpus cavernosum through activation of AT(1-7) and BK channels. Older and diabetic animals showed impaired Ang-(1-7)-mediated relaxation suggesting that aging and diabetes related erectile dysfunction (ED) may be partly due to decreased Ang-(1-7)-mediated relaxation of the corpus cavernosum. Acute pre-incubation with Ang-(1-7) was effective in attenuating Ang II-induced contraction of rabbit corpus cavernosum suggesting that the possible role of Ang-(1-7) in treatment of ED should be investigated.     

The nonadrenergic noncholinergic-mediated relaxation of corpus cavernosum was impaired in chronic lithium-treated rats: improvement with l-arginine

One-third of lithium-treated men complain from sexual dysfunction, although the exact mechanisms of which are not yet known. In this study we investigated the effect of chronic lithium (LiCl, 600 mg/l for 30 days) administration on the neurogenic relaxation of isolated rat corpus cavernosum. The corporal strips were precontracted with phenylephrine and electrical field stimulation (EFS) was applied to obtain relaxation. Relaxation to EFS was significantly (P<0.001) impaired in LiCl-treated rats. The nitric oxide (NO) synthase inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME; 100 microM) inhibited the relaxation to EFS in both LiCl-treated and control rats. The NO precursor l-arginine, at per se noneffective concentration (0.1 mM), significantly (P<0.001) enhanced the EFS-induced relaxation of LiCl-treated corporal strips. The relaxation responses to the NO donor sodium nitroprusside were similar between two groups. These data demonstrate that chronic lithium treatment could impair the NO-mediated neurogenic relaxation of rat corpus cavernosum which could be prevented by l-arginine.     

Enzyme activities of the nitric oxide-cGMP pathway in corpus cavernosum isolated from middle-aged rats

Cyclic guanosine-3',5'-monophosphate (cGMP)-mediated mechanisms play an important role in vasodilation and blood pressure regulation. We investigated basal activity of the nitric oxide (NO)-cGMP signal transduction pathway in corpus cavernosum from both middle-aged and young rats, and the electrical field stimulation-induced relaxation in the organ was also evaluated. In middle-aged rats, nitric oxide synthase (NOS) and cGMP-phosphodiesterase activities were significantly decreased; however, guanylate cyclase activity was similar. cGMP concentration, a secondary messenger of NO, remained almost the same level as compared with young rats. These results suggest that decrease in cGMP-phosphodiesterase activity is likely to account for the maintenance of cGMP concentration. In isolated corpus cavernosum from middle-aged rats, electrical field stimulation-induced relaxation was partially impaired. These results suggest that downregulation of the NOS and cGMP-phosphodiesterase activities are early events in the pathogenesis of erectile dysfunction.     

The anandamide transport inhibitor AM404 activates vanilloid receptors

The possibility that the anandamide transport inhibitor N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM404), structurally similar to the vanilloid receptor agonists anandamide and capsaicin, may also activate vanilloid receptors and cause vasodilation was examined. AM404 evoked concentration-dependent relaxations in segments of rat isolated hepatic artery contracted with phenylephrine. Relaxations were abolished in preparations pre-treated with capsaicin. The calcitonin-gene related peptide (CGRP) receptor antagonist CGRP-(8-37) also abolished relaxations. The vanilloid receptor antagonist capsazepine inhibited vasodilation by AM404 and blocked AM404-induced currents in patch-clamp experiments on Xenopus oocytes expressing the vanilloid subtype 1 receptor (VR1). In conclusion, AM404 activates native and cloned vanilloid receptors.     

Time-dependent reduction of acetylcholine-induced relaxation in corpus cavernosum of cholestatic rats: role of nitric oxide and cyclooxygenase pathway

The endothelium-dependent relaxation of corpus cavernosum smooth muscle and the roles of nitric oxide (NO) and arachidonic acid products of cyclooxygenase were investigated in non-operated, SHAM-operated, and bile duct-ligated rats. We further investigated the time-dependent alterations of corpus cavernosum relaxation in 2-, 7-, and 14-day bile duct-ligated animals. Acetylcholine produced concentration-dependent relaxation in phenylephrine-precontracted strips of corpus cavernosum. A significant reduction in the acetylcholine-induced relaxation was observed 2 days after bile duct ligation, and a greater reduction was observed on subsequent days. Incubation with 20 microM indomethacin reduced the acetylcholine-induced relaxation of the corpus cavernosum of unoperated rats while it had no effect in the corpus cavernosum of bile duct-ligated rats. Chronic treatment with Nomega-Nitro-L-Arginine Methyl Ester (L-NAME, 3 mg/kg/day, intraperitoneally) reduced the relaxation responses in the unoperated group while it had no effect in the bile duct-ligated group. These results show that acetylcholine-induced corporal relaxation is impaired in cholestatic rats, and this may be related to deficient nitric oxide production by the endothelium. The involvement of prostaglandins in this impairment seems unlikely.     

Mechanisms of anandamide-induced vasorelaxation in rat isolated coronary arteries

1. The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2. This relaxation was endothelium-independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 microM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3. Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 microM), but unaffected by AM 251 (1 microM) and AM 630 (1 microM), more selective antagonists of cannabinoid CB(1) and CB(2) receptors respectively. Palmitoylethanolamide, a selective CB(2) receptor agonist, did not relax precontracted coronary arteries. 4. Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 microM) or blockade of vanilloid VR1 receptors with capsazepine (5 microM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5. Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)). Gap junction inhibition with 18alpha-glycyrrhetinic acid (100 microM) did not affect anandamide relaxations. 6. This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB(1) or CB(2) receptors, but may involve activation of BK(Ca). Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.     

Mechanisms underlying endothelium-dependent, nitric oxide/prostacyclin-independent, acetylcholine-induced relaxation in canine corpus cavernosum

The mechanisms underlying endothelium-dependent and nitric oxide (NO)/prostacyclin-independent, acetylcholine-induced relaxation in isolated canine corpus cavernosum were investigated. In isolated canine corpus cavernous strips treated with indomethacin (10(-6) M) and N(G)-nitro-L-arginine (10(-4) M), acetylcholine produced relaxations in a concentration-dependent manner. The relaxations were not affected by treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide, sodium salt (carboxy PTIO, 3 x 10(-4) M), glibenclamide (10(-6) M), iberiotoxin (10(-7) M) or charybdotoxin (10(-7) M), but were abolished or reversed to contractions by treatment with apamin (10(-8) M) or scyllatoxin (10(-8) M). Levcromakalim (10(-7)-10(-6) M) induced a concentration-dependent relaxation which was abolished by treatment with glibenclamide (10(-6) M), but was not affected by treatment with apamin (10(-8) M) or scyllatoxin (10(-8) M). These findings indicate that endothelial cells of canine corpus cavernosum have an ability to produce a relaxing substance(s) other than NO or prostacyclin in response to acetylcholine. The substance(s) may open solely small conductance Ca2+-dependent K+ channels.     

Effects of calcium antagonists on the nitrergic nerve function in canine corpus cavernosum

Effects of calcium antagonists on nitrergic nerve function were examined in the isolated canine corpus cavernosum. In the cavernous strips precontracted with phenylephrine, transmural electrical stimulation elicited frequency-dependent (2 - 5 Hz) relaxations that were abolished by N(G)-nitro-L-arginine (10(-5) M), a nitric oxide (NO) synthase inhibitor; 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10(-6) M), a soluble guanylate cyclase inhibitor; and tetrodotoxin (3 x 10(-7) M). The relaxations were not affected by treatment with nifedipine or nicardipine (10(-8) - 10(-6) M), L-type specific calcium channel inhibitors, but were significantly inhibited by amlodipine or cilnidipine, inhibitors of L- plus N-type calcium channels, in a concentration-related manner (10(-7) - 10(-6) M). All of the inhibitors used did not affect the relaxations induced by exogenous NO (acidifed NaNO2). These findings suggest that N-type, but not L-type, calcium channels are responsible for increasing cytosolic free calcium, a prerequisite for the synthesis of NO, in the nitrergic dilator nerves innervating the corpus cavernosum.     

Vasorelaxant activities of the putative endocannabinoid virodhamine in rat isolated small mesenteric artery

Virodhamine is a recently identified novel endocannabinoid. Cannabinoids may evoke vasorelaxation through novel receptors in the vasculature and/or through release of vasodilator peptides from sensory nerve endings. Virodhamine induced endothelium-dependent relaxation in the rat isolated small mesenteric artery mounted in a myograph and precontracted with methoxamine. Desensitization of vanilloid receptors by capsaicin did not affect relaxation responses to virodhamine. The CB(1) receptor antagonist SR 141716A (3 microM), but not the more CB(1)-selective blocker AM 251 (1 microM), attenuated the response, while two CB(2) receptor antagonists, SR 144528 (1 microM) and AM 630 (10 microM), had no effect. The novel antagonist for the putative endothelial 'abnormal-cannabidiol receptor', O-1918 (30 microM), inhibited virodhamine relaxations. Hence virodhamine may activate this novel receptor, which might also recognize SR 141716A. Inhibition of nitric oxide synthase (L-NAME 300 microM) did not affect relaxation to virodhamine but the responses were markedly reduced when tone was induced with 60 mM KCl, suggesting a role for the activation of K(+) channels. The Ca(2+)-activated K(+) channel (K(Ca)) blockers, apamin (50 nM) and charybdotoxin (50 nM), inhibited virodhamine vasorelaxation. Combination of these blockers with SR 141716A (3 microM) caused no further inhibition. It was concluded that virodhamine relaxes the rat small mesenteric artery by endothelium-dependent activation of K(Ca), perhaps via the putative abnormal-cannabidiol receptor.     

Pharmacological characterization of rabbit corpus cavernosum relaxation mediated by the tissue kallikrein-kinin system.

1. The roles of the tissue kallikrein-kinin system and nitric oxide (NO) release in Phoneutria nigriventer venom-induced relaxations of rabbit corpus cavernosum (RbCC) smooth muscle have been investigated by use of a bioassay cascade. 2. Phoneutria nigriventer venom (10-30 micrograms), porcine pancreatic kallikrein (100 mu), rabbit urinary kallikrein (10 mu), bradykinin (BK, 0.3-3 nmol), acetylcholine (ACh, 0.3-30 nmol) and glyceryl trinitrate (GTN, 0.5-10 nmol) caused relaxations of the RbCC strips. Captopril (1 microM) substantially potentiated Phoneutria nigriventer venom- and BK-induced RbCC relaxations without affecting those elicited by GTN. 3. The bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3,Thi5,D- Tic7,Oic8]-BK, 50 nM), aprotinin (10 micrograms ml-1) and the tissue kallikrein inhibitor, Pro-Phe-Aph-Ser-Val- Gln-NH2 (KIZD-06, 1.3 microM) significantly inhibited Phoneutria nigriventer venom-induced RbCC relaxations, without affecting those provoked by GTN and ACh. The B1 receptor antagonist, [Leu9]des Arg10BK (0.5 microM) and soybean trypsin inhibitor (SBTI, 10 micrograms ml-1) had no effect on Phoneutria nigriventer venom-induced RbCC relaxations. 4. The relaxations induced by Phoneutria nigriventer venom, porcine pancreas kallikrein, BK and ACh were significantly inhibited by N omega-nitro-L-arginine methyl ester (L-NAME, 10 microM) but not by D-NAME (10 microM). L-NAME did not affect GTN-induced relaxations. L-Arginine (300 microM), but not D-arginine (300 microM), significantly reversed the inhibitory effect of L-NAME. 5. Our results indicate that Phoneutria nigriventer venom activates the tissue kallikrein-kininogen-kinin system in RbCC strips leading to NO release and suggest a functional role for this system in penile erection.     

Chronic administration of fluoxetine impairs neurogenic and endothelium-dependent relaxation of the rabbit corpus cavernosum smooth muscle

Antidepressants, including selective serotonin reuptake inhibitors (SSRIs), cause erectile dysfunction; however, the mechanism by which they cause erectile function is unclear. We investigated the reactivity of the corpus cavernosum after chronic fluoxetine treatment in rabbits. Twelve rabbits were randomly divided into two groups: control (n = 6) or 20 mg/kg/day of fluoxetine delivered i.p. (n = 6). The reactivity of the corpus cavernosum tissue from the fluoxetine-treated and control groups was studied in organ chambers after 21 days of fluoxetine injection. In the fluoxetine-treated group, endothelium-dependent relaxation of the corpus cavernosum in response to acetylcholine was significantly decreased compared to the control group. However, the sensitivity (i.e., pD₂) of the fluoxetine-treated cavernosal tissue strips to acetylcholine was not changed with respect to controls. Electrical field stimulation (EFS)-induced neurogenic relaxation was also significantly reduced in the fluoxetine-treated group. Relaxation in response to the nitric oxide (NO) donor sodium nitroprusside was similar between the cavernosal tissues from the two groups. There was also no change in agonist potency between the two groups. Additionally, chronic fluoxetine treatment had no effect on KCl-induced contractile responses. When tissue contraction was produced with phenylephrine to study relaxation in response to various stimuli, the tension induced was similar between the fluoxetine-treated and control groups. This study suggests that chronic fluoxetine treatment causes significant functional changes to the penile erectile tissue of rabbits, and these changes may contribute to the development of impotence.     

Involvement of nitric oxide in the non-adrenergic non-cholinergic neurotransmission of horse deep penile arteries: role of charybdotoxin-sensitive K(+)-channels.

1. The involvement of nitric oxide (NO) and the signal transduction mechanisms mediating neurogenic relaxations were investigated in deep intracavernous penile arteries with an internal lumen diameter of 600-900 microns, isolated from the corpus cavernosum of young horses. 2. The presence of nitric oxide synthase (NOS)-positive nerves was examined in cross and longitudinal sections of isolated penile arteries processed for NADPH-diaphorase (NADPH-d) histochemistry. NADPH-d-positive nerve fibres were observed in the adventitia-media junction of deep penile arteries and in relation to the trabecular smooth muscle. 3. Electrical field stimulation (EFS) evoked frequency-dependent relaxations of both endothelium-intact and denuded arterial preparations treated with guanethidine (10(-5) M) and atropine (10(-7) M), and contracted with 10(-6) M phenylephrine. These EFS-induced relaxations were tetrodotoxin-sensitive indicating their non-adrenergic non-cholinergic (NANC) neurogenic origin. 4. EFS-evoked relaxations were abolished at the lowest frequency (0.5-2 Hz) and attenuated at higher frequencies (4-32 Hz) by the NOS inhibitor, NG-nitro-L-arginine (L-NOARG, 3 x 10(-3) M). This inhibitory effect was antagonized by the NO precursor, L-arginine (3 x 10(-3) M). NG-nitro-D-arginine (10(-4) M) did not affect the relaxations to EFS. 5. Incubation with either the NO scavenger, oxyhaemoglobin (10(-5) M), or methylene blue (10(-5) M), an inhibitor of guanylate cyclase activation by NO, caused significant inhibitions of the EFS-evoked relaxations, and while oxyhaemoglobin abolished the relaxations to exogenously added NO (acidified sodium nitrite, 10(-6) - 10(-3) M), there still persisted a relaxation to NO of 24.4 +/- 5.1% (n = 6) in the presence of methylene blue. 6. Glibenclamide (3 x 10(-6) M), an inhibitor of ATP-activated K(+)-channels, did not alter the relaxations to either EFS-stimulation or NO, while the blocker of Ca(2+)-activated K(+)-channels, charybdotoxin (3 x 10(-8) M), caused a significant inhibition of both the electrically-induced relaxations and the relaxations to exogenously added NO. Furthermore, charybdotoxin blocked relaxations induced by the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8 Br-cyclic GMP). 7. These results suggest that relaxations of horse deep penile arteries induced by NANC nerve stimulation involve mainly NO or a NO-like substance from nitrergic nerves. NO would stimulate the accumulation of cyclic GMP followed by increases in the open probability of Ca(2+)-activated K(+)-channels and hyperpolarization leading to relaxation of horse penile arteries.     

Role of endogenous hydrogen sulfide in neurogenic relaxation of rat corpus cavernosum

Relaxation of corpus cavernosum during penile erection is mediated by a non-adrenergic non-cholinergic (NANC) neurotransmission and by the endothelium via the release of nitric oxide. Hydrogen sulfide (H(2)S) is an endogenous gaseous mediator which is a potent vasodilator and a neurotransmitter. This study was initiated to characterize the role of H(2)S in NANC neurogenic transmission in rat corpus cavernosum. The expression of H(2)S producing enzymes was assessed using RT-PCR as well as Western blotting and showed the expression of cystathionine γ-lyase (CSE) in rat corporal tissue. Homogenates from rat corpus cavernosum convert l-cysteine to H(2)S and this was partially inhibited by a CSE inhibitor, propargylglycine. Electrical stimulation of corporal tissue strips caused NANC relaxation. This neurogenic relaxation was significantly enhanced by inhibition of CSE by propargylglycine indicating that endogenously produced H(2)S may have a negative regulatory role in neurogenic relaxation of rat corpus cavernosum. To investigate this further we used physiologically relevant concentrations of exogenous NaHS, and showed that nanomolar concentrations could inhibit corporal relaxation induced by a nitroxyl (HNO) donor (Angeli's salt) but not with nitrosonium (NO(+)) or NO donors. This suggests that an interaction between endogenously produced H(2)S and nitroxyl (HNO) might be involved in erectile function.     

枸橼酸西地非尔对兔海绵体组织作用及其机制的离体研究

目的　研究枸橼酸西地非尔对兔海绵体组织舒张功能的影响及其作用机制。方法　 37℃温孵兔海绵体组织(RCC) ,用放射免疫方法监测在硝普钠存在下 ,西地非尔10、10 0、10 0 0nmol·L-1对cGMP水平的影响 ;采用离体器官水浴技术 ,将RCC用苯丙肾上腺素 (PE)收缩 ,观察西地非尔对NO供体硝普钠 (SNP)及促内皮依赖性NO释放剂氯化乙酰甲基胆碱介导RCC舒张作用的影响。结果　SNP可提高RCC中cGMP水平 ,西地非尔 10、10 0、10 0 0nmol·L-1可剂量依赖性使之升至更高水平。西地非尔使SNP、氯化乙酰甲基胆碱 (MCH)作用IC50 显著减小。结论　以上结果表明枸橼酸西地非尔通过NO/cGMP呈剂量依赖性增强MCH和SNP对RCC的舒张作用 ,对动物阴茎海绵体组织舒张功能有显著性改善作用 ,从而大大提高动物性功能。