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

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.

Naltrexone, an opioid receptor antagonist, attenuates liver fibrosis in bile duct ligated rats.

AIM: The aim of this study was to investigate the hypothesis that the opioid system is involved in the development of hepatic fibrosis. METHODS: The effect of naltrexone (an opioid receptor antagonist) on hepatic fibrosis in bile duct ligated (BDL) or sham rats was assessed by histology and hepatic hydroxyproline levels. Liver matrix metalloproteinase 2 (MMP-2) was measured by zymography, and alpha smooth muscle actin (alpha-SMA) and CD45 (leucocyte common antigen) by immunohistochemistry. The redox state of the liver was assessed by hepatic glutathione (GSH)/oxidised glutathione (GSSG) and S-nitrosothiol levels. Subtypes of opioid receptors in cultured hepatic stellate cells (HSCs) were characterised by reverse transcriptase-polymerase chain reaction, and the effects of selective delta opioid receptor agonists on cellular proliferation, tissue inhibitor of metalloproteinase 1 (TIMP-1), and procollagen I expression in HSCs determined. RESULTS: Naltrexone markedly attenuated the development of hepatic fibrosis as well as MMP-2 activity (p<0.01), and decreased the number of activated HSCs in BDL rats (p<0.05). The development of biliary cirrhosis altered the redox state with a decreased hepatic GSH/GSSG ratio and increased concentrations of hepatic S-nitrosothiols, which were partially or completely normalised by treatment with naltrexone, respectively. Activated rat HSCs exhibited expression of delta1 receptors, with increased procollagen I expression, and increased TIMP-1 expression in response to delta(1) and delta(2) agonists, respectively. CONCLUSIONS: This is the first study to demonstrate that administration of an opioid antagonist prevents the development of hepatic fibrosis in cirrhosis. Opioids can influence liver fibrogenesis directly via the effect on HSCs and regulation of the redox sensitive mechanisms in the liver.

Low plasma protein nitrotyrosine levels distinguish primary Raynaud's phenomenon from scleroderma.

OBJECTIVE: To investigate the hypothesis that increased formation of reactive nitrogen species may contribute to the vascular pathology that develops in patients with connective tissue disease such as scleroderma. PATIENTS AND METHODS: The level of protein-bound nitrotyrosine in plasma was measured by stable isotope dilution gas chromatography/negative ion chemical ionisation mass spectrometry in 11 patients with primary Raynaud's phenomenon, 37 with scleroderma, 13 with chronic renal impairment, and in 23 healthy controls. RESULTS: Plasma protein-bound nitrotyrosine was markedly decreased in patients with primary Raynaud's phenomenon (mean (SEM) 0.60 (0.06) ng/mg dry protein) compared with patients with scleroderma (1.78 (0.21) ng/mg protein), chronic renal impairment (1.42 (0.17) ng/mg protein) or healthy controls (1.63+/-0.15 ng/mg protein, ANOVA p<0.001). CONCLUSION: These data suggest that there is decreased nitration of plasma proteins, or increased degradation of nitrated proteins from the circulation of patients with primary but not secondary Raynaud's phenomenon.

Hydrogen sulphide and the hyperdynamic circulation in cirrhosis: a hypothesis.

Cirrhosis is associated with the development of a hyperdynamic circulation, which is secondary to the presence of systemic vasodilatation. Several mechanisms have been postulated to be involved in the development of systemic vasodilatation, including increased synthesis of nitric oxide, hyperglucagonaemia, increased carbon monoxide synthesis, and activation of K(ATP) channels in vascular smooth muscle cells in the systemic and splanchnic arterial circulation. Hydrogen sulphide (H2S) has recently been identified as a novel gaseous transmitter that induces vasodilatation through activation of K(ATP) channels in vascular smooth muscle cells. In this brief review, we comment on what is known about H2S, vascular and neurological function, and postulate its role in the pathogenesis of the vascular abnormalities in cirrhosis.

Inactivation of xanthine oxidoreductase is associated with increased joint swelling and nitrotyrosine formation in acute antigen-induced arthritis.

OBJECTIVE: Arthritis is associated with increased articular formation of nitrotyrosine, which may contribute to injury. Nitrotyrosine is formed by nitration of tyrosine by reactive nitrogen species such as peroxynitrite, the formation of which may be enhanced by xanthine oxidoreductase (XOR), since it can generate nitric oxide from nitrite/nitrate, and superoxide during xanthine metabolism. We hypothesized that inactivation of XOR would protect against antigen-induced arthritis (AIA) and decrease nitrotyrosine formation. METHODS: AIA was induced with methylated bovine serum albumin (mBSA) in three groups of Wistar rats: animals fed on (1) tungsten-enriched chow (0.7 g/kg) (TG), which inactivates XOR, (2) standard chow (SG), and (3) rats treated with allopurinol (50 mg/kg/day; p.o.) (AG). Nitrotyrosine in patella-synovium was quantified by mass spectrometry three weeks after intra-articular (i.a.) antigen injection. RESULTS: Treatment with tungsten, but not allopurinol, suppressed plasma and articular XOR activity at < or = 0.9% of normal levels. XOR inactivation was associated with increased knee swelling 24-48 hrs post i.a. mBSA, compared with controls (mean increase +/- SEM of knee diameter from baseline of 3.3 +/- 0.5, 2.0 +/- 0.3 and 1.9 +/- 0.2 mm in TG, SG and AG (n = 14 each group), respectively; p < 0.05, TG vs SG, ANOVA). Mean ratio of articular nitrotyrosine-tyrosine (+/- SEM) was increased in the XOR-inactivated group, compared with controls: 12.3 +/- 0.7, 9.6 +/- 0.8 and 10.4 +/- 0.5 pg/microg in TG, SG and AG, respectively; p < 0.05, TG vs SG. CONCLUSION: Contrary to expectation, XOR inactivation was associated with increased joint swelling and articular tyrosine nitration in acute AIA, suggesting a novel, protective role for XOR in inflammatory arthritis.

Enhancement of indomethacin-induced gastric damage by mouth breathing in rabbits.

Previous studies have reported that mouth breathing is associated with respiratory acidosis. Regarding to the reports that renal elimination of weak acids such as indomethacin is pH dependent, this study was carried out to evaluate the role of mouth breathing on plasma level of indomethacin and indomethacin-induced gastric damage in rabbits. Mouth breathing was induced by surgical ligation of nostrils under general anesthesia. One day after the operation, arterial blood samples were collected for acid-base balance analysis and indomethacin was administered intraperitoneally in a single dose of 40mg/kg. The animals were killed 4h after indomethacin administration and blood samples were collected for spectrofluorometric determination of indomethacin in plasma. The results showed that indomethacin induces more severe gastric damage in nose obstructed rabbits compared with sham and unoperated (UNOP) animals. Acid-base analysis revealed a respiratory acidosis in nose obstructed rabbits and indomethacin level of plasma was significantly higher in nose obstructed animals in comparison with control rabbits. The study shows that mouth breathing can increase the potentiation of indomethacin-induced gastric mucosal damage that may be due to higher level of indomethacin in plasma of nose obstructed animals.

The role of the interaction between endogenous opioids and nitric oxide in the pathophysiology of ethanol-induced gastric damage in cholestatic rats.

Interaction between endogenous opioids and nitric oxide (NO) has been shown in different biological models and pharmacological evidence suggest that opioids can induce NO release in endothelium as well as in neural cells. Cholestasis is associated with NO overproduction. The reason for increased NO synthesis is not clearly known but it can potentiate development of gastric mucosal damage in cholestatic subjects. Based on increased plasma levels of endogenous opioids and existence of NO overproduction in cholestasis, the present experiments were performed to investigate the role of interaction between endogenous opioids and NO in generation of ethanol-induced gastric damage in cholestatic rats. Cholestasis was induced by surgical ligation of bile duct and sham-operated rats served as controls. The animals received either 20 mg/kg of naltrexone or saline for 6 days and then were fasted and received L-arginine (200 mg/kg), NG-nitro-L-arginine methylester (L-NAME; 2, 5 and 10 mg/kg) or saline. The ethanol-induced gastric mucosal damage was significantly more severe in cholestatic rats than in sham-operated animals (115 +/- 12 mm2 vs. 72 +/- 11 mm2, P < 0.05). L-NAME significantly enhanced the development of gastric mucosal lesions in sham-operated rats. But in cholestatic animals, L-NAME decreased and L-arginine enhanced the severity of gastric damage. Pretreatment of animals with naltrexone decreased severity of gastric mucosal damage in cholestatic rats. Concurrent administration of naltrexone with L-arginine was protective against ethanol-induced gastric damage in both normal and cholestatic groups. Administration of naltrexone with L-NAME had the same effect in cholestatic and control rats and increased severity of gastric damage. Plasma levels of NO2- + NO3- were significantly higher in cholestatic rats than control animals (72 +/- 6 microM vs. 39 +/- 3 microM, P < 0.05). Pretreatment of animals with naltrexone significantly reduced plasma levels of NO2- + NO3- in cholestatic animals, but not in control rats (33 +/- 6 microM vs. 32 +/- 4 microM). The protective effect of L-NAME against gastric damage in cholestatic rats can be explained by inhibition of NO overproduction and it seems that interaction between opioids and NO may have an important role in generation of NO overproduction and gastric complications in cholestatic rats.

Role of nitric oxide in hypodipsia of rats with obstructive cholestasis.

Cholestasis is associated with the overproduction of nitric oxide (NO), and NO acts as an inhibitory mechanism when thirst is stimulated by water deprivation or by angiotensin II. Due to the presence of hypodipsia in the cholestatic condition, we have compared the rate of water intake between bile duct-ligated (cholestatic) and sham-operated rats. We have evaluated the effect of NO synthesis inhibition by N(G)-nitro-L-arginine (L-NNA, 10 mg kg(-1)/day) on the rate of water intake in cholestatic rats. The results showed that plasma alkaline phosphatase activity (a marker of liver damage) increased after bile-duct ligation, and that its elevation was partially (but significantly) prevented by treatment with L-arginine. A two-week bile-duct obstruction induced a significant decrease in the rate of water intake compared with sham-operated animals (35.87 +/- 1.45 vs 42.37 +/- 1.99 mL/day, P < 0.05). This effect was corrected by the daily administration of L-NNA. Surprisingly, L-arginine (200 mg kg(-1)/day) showed similar activity as L-NNA in cholestatic rats and increased water intake, but not in control animals. Systemic NO synthesis inhibition corrected the decrease in water intake observed in cholestatic rats. This suggests an important role for NO in the pathophysiology of hypodipsia in cholestatic subjects. The effect of chronic L-arginine administration observed in cholestatic rats but not seen in the control rats could be explained theoretically by the amelioration of cholestasis-induced liver damage by chronic L-arginine administration in bile duct-ligated rats.

The role of nitric oxide in bradycardia of rats with obstructive cholestasis.

Nitric oxide (NO) has an important role in controlling heart rate and contributes to the cholinergic antagonism of the positive chronotropic response to adrenergic stimulation. Based on evidence of NO overproduction in cholestasis and also on the existence of bradycardia in cholestatic subjects, this study aimed to evaluate the chronotropic effect of epinephrine in isolated atria of cholestatic rats and determine whether alterations in epinephrine-induced chronotropic responses of cholestatic rats are corrected after systemic inhibition of NO synthase (NOS) with N(G)-nitro-L-arginine (L-NNA). Male Sprague-Dawley rats were used. Cholestasis was induced by surgical ligation of the bile duct under general anesthesia and sham-operated animals were considered as control. The animals were divided into three groups, which received either L-arginine (200 mg/kg/day), L-NNA (10 mg/kg/day) or saline. One week after the operation, a lead II ECG was recorded from the animals, then spontaneously beating atria were isolated and chronotropic responses to epinephrine were evaluated in a standard oxygenated organ bath. The results showed that plasma gamma-glutamyl transpeptidase and alanine aminotransferase activity was increased by bile-duct ligation, and that L-aginine treatment partially, but significantly, prevented the elevation of these markers of liver damage. The results showed that heart rate of cholestatic animals was significantly less than that of sham-operated control rats in vivo and this bradycardia was corrected with daily administration of L-NNA. The basal spontaneous beating rate of atria in cholestatic animals was not significantly different from that of sham-operated rats in vitro. Meanwhile, cholestasis induced a significant decrease in chronotropic effect of epinephrine. These effects were corrected by daily administration of L-NNA. Surprisingly L-arginine was as effective as L-NNA and increased the chronotropic effect of epinephrine in cholestatic rats but not in sham-operated animals. Systemic NOS inhibition corrected the decreased chronotropic response to adrenergic stimulation in cholestatic rats, and suggests an important role for NO in the pathophysiology of heart rate complications in cholestatic subjects. The opposite effect of chronic L-arginine administration in cholestasis and in control rats could be explained theoretically by an amelioration of cholestasis-induced liver damage by chronic L-arginine administration in bile duct-ligated rats.

N(G)-nitro-L-arginine methylester is protective against ethanol-induced gastric damage in cholestatic rats.

In this study the effect of nitric oxide (NO) synthesis inhibition on ethanol-induced gastric damage was evaluated in bile duct-ligated, sham-operated and unoperated rats. The animals were injected intraperitoneally with saline, L-arginine (200 mg/kg) or N(G)-nitro-L-arginine methylester (L-NAME) in doses of 5, 15 and 30 mg/kg, 30 min before ethanol administration. The animals were killed 1 h after ethanol administration and their stomachs were removed for measurement of gastric mucosal damage. The results showed that L-NAME significantly enhanced the development of gastric mucosal lesions in sham-operated and unoperated rats, while in bile duct-ligated animals, L-NAME decreased and L-arginine enhanced the potentiation of ethanol-induced gastric mucosal damage. The plasma level of nitrite and nitrate was also measured and was significantly higher in bile duct-ligated rats than in control groups. The results suggest that inhibition of NO synthase with L-NAME has different effects on ethanol-induced gastric damage in cholestatic groups and in normal rats and that these effects can be explained by overproduction of NO in bile duct-ligated animals.

Naloxone is protective against indomethacin-induced gastric damage in cholestatic rats.

We compared indomethacin-induced gastric damage in three groups of rats-bile duct-ligated, sham-operated, and unoperated-and evaluated the role of the opioid system by blocking the effects of endogenous opioids with naloxone. Indomethacin was administered orally in a dose-dependent manner at 10, 30, and 45 mg/ kg. Naloxone was administered intraperitoneally in several doses of 0.5 and 1 mg/kg, starting 30 min before indomethacin (10mg/kg) administration and continued every 30 min. The animals were killed 4h after indomethacin administration. Indomethacin induced more severe gastric damage in bile duct-ligated rats than in sham and unoperated animals, and administration of naloxone (1 mg/kg) every 30 min inhibited the potentiation of indomethacin-induced gastric damage in bile duct-ligated rats, but not in the control groups (sham-operated and unoperated rats). Plasma indomethacin level was also measured, by fluorometry, but showed no significant difference between the groups. Endogenous opioids have been reported to accumulate in plasma of cholestatic animals, and, considering the results of this study, we suggest the opioid system plays an important pathophysiologic role in the pathogenesis of peptic ulcers in cholestatic subjects.

Synthesis and pharmacological activity of thebaine-derived mu-opioid receptor agonists.

Thebaine-derived mu-opioid agonists were synthesized through the reaction of thebaine with N-aryl maleimide and tested for opioid activity. Morphine was used as reference compound. Our results show that an attachment of aryl succinimide group to thebaine produced series of compounds with mu-opioid agonist activity. The most active compound in smooth muscle preparation was compound 6 with an IC50 ratio of delta/mu = 248.69 and was as potent as morphine with ED50 value 26.65 mg kg-1 i.p. in hot-plate test and showed good antinociceptive activity.

Enhancement of aspirin-induced gastric damage by cholestasis in rats.

In this study the severity of aspirin-induced gastric mucosal damage was investigated in rats with obstructive cholestasis. Cholestasis was induced by ligation and resection of the bile duct under general anesthesia. Two weeks after operation, the rats were fasted for 24 hours. Aspirin was administered orally in doses of 0, 128, 192, 266 and 335 mg/kg, and the animals were killed four hours after dosing. The dose of 266 mg/kg was chosen for a study of the time-dependency; other groups of animals were killed at time intervals of one, three, five, seven and nine hours after aspirin administration. The results showed that aspirin induces more severe gastric damage in bile duct resected rats compared with sham-operated and control animals. Salicylate levels of serums were also measured but there was no significant difference in serum salicylate levels between bile duct resected, sham-operated and control rats. It can be concluded that cholestasis can potentiate aspirin-induced gastric damage in rats.