Nitric Oxide Synthetase Inhibitor Research Articles

Vasodilation in human subcutaneous arteries induced by neuropeptide Y is mediated by neuropeptide Y Y<sub>1</sub> receptors and is nitric oxide dependent

Neuropeptide Y (NPY) is known as a potent vasoconstrictor of peripheral blood vessels both in vivo and in vitro. There have been reports suggesting that NPY also has a dilatory effect. The aim of the present study was to elucidate whether NPY dilates small human subcutaneous arteries. Subcutaneous arteries, obtained from patients undergoing abdominal surgery, were mounted in in vitro tissue baths, and the vascular responses to NPY were investigated. The presence of mRNA encoding the human NPY Y1 receptor in endothelial cells from human umbilical veins was studied by the use of reverse transcriptase - polymerase chain reaction (RT-PCR). In arteries precontracted with the prostaglandin analogue U46619, NPY induced a concentration-dependent vasodilation (Emax 30 +/- 10% of the U46619-induced contraction), which was significantly inhibited by the NPY Y1 receptor antagonist BIBP3226 (1 microM), causing a rightward shift of the concentration-response curve, pEC50 7.1 +/- 0.3 vs. 7.7 +/- 0.3 for NPY alone. After pretreatment with the nitric oxide synthetase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) (10 microM), the dilation was abolished (Emax 6 +/- 5% of the U46619-induced contraction). mRNA encoding the human NPY Y1 receptor was detected in endothelial cells from human umbilical veins. It was concluded that NPY induces vasodilation in human subcutaneous arteries. The dilation is mediated via the NPY Y1 receptor and is dependent on nitric oxide.

P-393 - Influence of nitric oxide synthetase inhibitor on the cardiovascular response to clonidine adminis-tered intracerebroventricularly

P-393 - Influence of nitric oxide synthetase inhibitor on the cardiovascular response to clonidine adminis-tered intracerebroventricularly

Antiplatelet effect of amlodipine: A possible mechanism through a nitric oxide-mediated process

The effect of amlodipine, a novel calcium channel blocker of the dihydropyridine type, on rabbit platelet aggregation, and the possible antiaggregatory mechanisms of amlodipine, especially on the nitric oxide (NO) guanosine 3′,5′-cyclic monophosphate (cyclic GMP)-mediated pathway, were investigated. Other effects of amlodipine on thromboxane B 2 (TXB 2) formation in platelets also were examined. Amlodipine concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 μg/mL) or thrombin (0.1 U/mL) with an ic 50 range of 32–69 μM. Along with this inhibition, our results also demonstrated that in the presence of l-arginine (100 μM), amlodipine (50 μM) increased nitric oxide synthetase (NOS) activity (from the resting activity of 2.05 ± 0.36 to 7.11 ± 0.95 pmol/mg protein/min) and NO release (by 80%), accompanied by an elevation of the cyclic GMP level (from the resting platelet level of 1.27 ± 0.12 to 6.21 ± 0.55 pmol/10 9 platelets) induced by collagen (10 μg/mL). However, the antiaggregatory effect of amlodipine (50 μM) could be attenuated significantly by oxyhemoglobin (5 μM), a NO scavenger, or N G-nitro- l-arginine methyl ester (100 μM), a specific NOS inhibitor. In addition, the TXB 2 production in platelets induced by collagen or thrombin was concentration-dependently inhibited by amlodipine. Therefore, we propose that the antiaggregatory mechanisms of amlodipine might be mediated, in part, by a NO-cyclic GMP process accompanied by the inhibition of TXB 2 formation in platelets.

Acute management of migraine: triptans and beyond.

Migraine is a paroxysmal disorder characterized by attacks of headache, nausea, vomiting, photophobia and phonophobia, and malaise. This review summarizes new treatment options for the therapy of acute attacks. Sumatriptan was the first specific serotonin-1B/D agonist for the treatment of acute migraine attacks. Apart from the oral and subcutaneous formulation, it is also available as nasal spray and suppository. The other new migraine drugs zolmitriptan, naratriptan, rizatriptan and eletriptan differ in their pharmacological profiles, which translates into minor differences in efficacy, headache recurrence and side-effects. Importantly, in clinical practice individual patients may show a preference for one treatment over another. New drugs in migraine treatment include substance-P antagonists, nitric oxide synthetase inhibitors and calcitonin gene-related peptide antagonists.

Generation of reactive oxygen species, release of l-glutamate and activation of caspases are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture

Oxygen-induced cell death in embryonic neurons is a useful in vitro model of neuronal apoptosis to study the molecular mechanisms underlying the cell death induced by oxidative stress. In the present study, we examined the involvement of reactive oxygen species and glutamate in the high (50%) oxygen-induced death of cultured hippocampal neurons. During the course of cell death, increases in O 2 − and hydrogen peroxide (H 2O 2) levels were observed. On the other hand, superoxide dismutase (SOD), catalase and deferoxamine (DFX), which have inhibitory effects on the generation of O 2 −, H 2O 2 and hydroxyl radicals, respectively, protected the neurons. These results suggested that both O 2 − and H 2O 2 play important roles in this apoptosis. Antagonists of NMDA and AMPA/kinate (AMPA/KA) receptors and an inhibitor of glutamate release partially prevented the apoptosis, suggesting that exposure to high oxygen enhances glutamate release, which results in activation of NMDA receptor and AMPA/KA receptor. In addition, specific nitric oxide (NO) scavenger and NO synthetase inhibitors blocked the apoptosis, indicating that NO and/or peroxynitrite are involved in this mechanism of cell death. Caspase inhibitors also blocked the neuronal apoptosis. These results suggested that multiple effectors including generation of reactive oxygen species, release of l-glutamate and activation of caspases are activated during the death induced by high oxygen.

The role of nitro-reduction and nitric oxide in the toxicity of chloramphenicol.

Recent work on the toxicology of chloramphenicol suggests that its propensity to cause damage to the blood forming organs may be related to its potential for nitro-reduction and the subsequent production of nitric oxide. In this study both aerobic and anaerobic nitro-reduction of chloramphenicol by human foetal and neonatal liver results in the production of the amine derivative. However intermediates of the reaction nitroso- or glutathionesulphinamido-chloramphenicol could not be detected by hplc. Perfusion of chloramphenicol through isolated lobules of human placentae caused a decrease in blood pressure at a time which coincided with a peak of nitric oxide production. However, although the pressure drop could be reversed by an inhibitor of nitric oxide synthetase, the nitric oxide profile remained the same. These observations suggest that involvement of the para-nitro group of chloramphenicol could cause both hemotoxicity and hypotension in susceptible individuals.

Panax quinquefolium L. inhibits thrombin-induced endothelin release in vitro.

Endothelial cell damage is considered to be the initial step in the genesis of thrombosis and arteriosclerosis, the common precursors of cardiovascular disorders. In this study, we evaluated the protective effects of American ginseng or Panax quinquefolium L. extracts on endothelial cell injury, and investigated effects of ginseng extracts on thrombin-induced endothelin release using cultured human umbilical vein endothelial cells. We observed that when endothelial cells pretreated with 1, 10, and 100 micrograms/ml of Panax quinquefolium L. extracts were incubated for 4 and 24 hr with thrombin, the concentration of endothelin was significantly decreased in a concentration dependent, time related manner (at 4 hr, IC50 = 5.1 micrograms/ml; at 24 hr, IC50 = 6.2 micrograms/ml). We further evaluated the effects of NG-nitro-L-arginine (NLA), a nitric oxide (NO) synthetase inhibitor, on the activity of Panax quinquefolium L. extracts. Following pretreatment of cultured endothelial cells with NLA, the inhibition of thrombin-induced endothelin release by Panax quinquefolium L. was significantly reduced (P < 0.05). This result suggests that the pharmacological action of Panax quinquefolium L. is, at least partially, due to NO release. Our data demonstrate that American ginseng may play a therapeutic role in facilitating the hemodynamic balance of vascular endothelial cells.

Role of nitric oxide and cGMP in human septic serum-induced depression of cardiac myocyte contractility.

Previous studies have demonstrated the existence of a circulating myocardial depressant substance during human septic shock. We have recently identified this substance as a synergistic combination of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). This study utilized an in vitro cardiac myocyte assay to evaluate the potential mechanistic role of nitric oxide (NO) and cGMP in depression of myocyte contractility induced by TNF-alpha, IL-1beta, TNF-alpha + IL-1beta (at low concentrations), and human septic shock serum (HSS). TNF-alpha, IL-1beta, TNF-alpha + IL-1beta, and each of 5 sera from patients with acute septic shock caused depression of both maximum extent and peak velocity of cardiac myocyte shortening and an increase in intracellular cGMP concentration during 30 min of exposure (minimum P < 0.01). NO synthetase (NOS) and guanylate cyclase inhibitors such as N-methyl-L-arginine (L-NMA) and methylene blue prevented these effects; an excess of L-arginine with L-NMA restored them (minimum P < 0.01). In contrast, D-arginine failed to reestablish cytokine-induced myocyte depression and cGMP accumulation prevented by L-NMA. Exposure of myocytes to TNF-alpha, IL-1beta, or TNF-alpha + IL-1beta produced a concentration-dependent increase in intracellular cGMP that paralleled the depression of cardiac myocyte contractility (minimum P < 0.001). In addition, TNF-alpha, IL-1beta, TNF-alpha + IL-1beta, or HSS application to cardiac myocytes resulted in increased NO gas generation, which was inhibited by L-NMA (minimum P < 0.01). Furthermore, unstimulated cardiac myocytes were shown to harbor constitutive but not inducible NOS activity. These data suggest that the sequential generation of NO by a constitutive NOS and cGMP by guanylate cyclase represents an important mechanism of cardiac myocyte depression by TNF-alpha, IL-1beta, TNF-alpha + IL-1beta, and the myocardial depressant substance(s) of septic shock.

Nerve Growth Factor Treatment Prevents the Increase in Superoxide Produced by Epidermal Growth Factor in PC12 Cells

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21(ras) construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140(trk) receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140(trk) (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140(trk) receptors that lack binding sites for Shc and phospholipase Cgamma. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, L-nitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.

Adjuvant effect of grifolan on antibody production in mice.

The effects of grifolan (GRN), a gel-forming (1-->6)-branched (1-->3)-beta-D-glucan, on antibody production were examined. Sera from mice that were injected with GRN and trinitrophenyl ovalbumin (TNP-OVA) intraperitoneally showed a significantly increased level of anti-TNP IgG. However, injection of TNP-OVA alone showed a lower antibody level. Two hundred fifty microg of GRN and 10 microg of TNP-OVA gave the maximum production of anti-TNP antibody. Enhanced antibody production was also observed in the culture supernatant of splenocyte obtained from GRN-administered mice. The culture supernatant contained a significant amount of nitric oxide (NO) in the case of GRN-administered mice. To observe the effect of NO on the antibody production induced by GRN, N-monomethyl arginine (NMMA), an inhibitor of NO synthetase, was added to the splenocyte cultures. The antibody level of supernatants containing NMMA was higher than that of control supernatants. These results suggest that GRN can enhance antibody production and that NO induced by stimulation with GRN concomitantly with antibody production is a negative factor on the adjuvant activity. Inhibition of NO may increase the adjuvant effect of GRN.

Direct evidence for the role of nitric oxide on the glutamate-induced neuronal death in cultured cortical neurons

It has been reported that glutamate-induced neurotoxicity is related to an increase in nitric oxide (NO) concentration. An NO-sensitive electrode has been developed to measure NO concentration directly. Using this electrode, we examined NO concentration and neuronal survival after glutamate application in rat cultured cortical neurons. We also examined the effects of NMDA receptor antagonists, MK-801 and ketamine, and the NO synthetase inhibitor, l-NMMA on NO production and neuronal death. After 7 days in culture, application of glutamate (1 mM) or l-arginine (0.3 mM) to the cultured medium increased NO concentration, and decreased the number of anti-microtubule-associated protein 2 positive neurons. Both pretreatment with MK-801 (300 μm) and ketamine (300 μm) prevented glutamate-, but not l-arginine-induced increase in NO concentration and neuronal death. l-NMMA prevented both glutamate- and l-arginine-induced NO production and neuronal death. The nitric oxide donor, S-nitroso- N-acetyl- d, l-penicillamine (SNAP) also caused neuronal death, and MK-801, ketamine and l-NMMA did not prevent SNAP-induced toxicity. We have demonstrated excitatory amino acid-induced changes of NO concentration and the parallel relationship between changes of NO concentration and neuronal death. In conclusion, an increase in NO concentration does induce neuronal death, and the inhibition of the production of NO prevents glutamate-induced neuronal death.

Reduction of Surgery-induced Peritoneal Adhesions by Methylene Blue

Background: The possible involvement of inflammatory mediators such as nitric oxide (NO), and reports of protective effects of antioxidants, led us to test the effectiveness of methylene blue and NO synthesis inhibitor in reducing adhesion formation. Methods: Generation of adhesions in rats, by scraping the anterior uterine horn wall, was followed by intraperitoneal administration of saline, methylene blue, or Nα-t-BOC-ω-nitro-L-arginine. Additional rats received identical treatments, but without the serosal damage. Two weeks later, formation of adhesions was quantitatively graded. Results: Adhesions were found in <5% of the rats with the sham surgery, regardless of treatment. In the experimental group, >95% of the rats treated with saline or NO synthetase inhibitor had severe adhesions, in contrast to 5% of the methylene blue treated rats. Severity of adhesion was lower in the methylene blue group ( P <0.001). Conclusions: Methylene blue was very effective in preventing formation of peritoneal adhesions. Its activity is probably through inhibition of free-radical generation and not of nitric oxide action.

Nitric Oxide and Parasympathetic Vascular and Secretory Control of the Dog Nasal Mucosa

Nasal vascular and secretory responses to local intra-arterial injection of acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) and to electrical stimulation of the nasal parasympathetic nerve fibres were recorded in dogs anaesthetized with pentobarbital. The influence of pretreatment with atropine and propranolol and the nitric oxide synthetase (NOS) inhibitor N

Impaired insulin but normal pentagastrin effect on gastric acid secretion in diabetic rats: a role for nitric oxide

Significant changes in gastrointestinal function, decreased gastric secretion and motility in particular, are often observed in patients with chronic diabetes. The mechanisms leading to those remain unclear. In these studies we evaluated the gastric acid secretory response to insulin and pentagastrin in normal Wistar and streptozotocin diabetic rats. We also sought to determine the role of nitric oxide (NO) in this process. The animals were anesthetized with sodium pentobarbital. Warm saline was perfused through a polyethylene tube placed in the oesophagus and collected from the duodenum at 10 min intervals. Following a 50 min equilibration period, a bolus intra-jugular infusion of insulin (4.0 U/kg), 2-deoxyglucose (200 mg/kg) or pentagastrin 4.0 ( μg/kg) was started and samples of the gastrointestinal perfusate were collected for an additional 80 min. Insulin-stimulated acid secretion peaked 60 min after bolus infusion in normal animals; a response that was significantly decreased in the diabetic rats. Similarly, 2-deoxyglucose-induced glucopenia increased gastric acid secretion to a lower extent in diabetic versus normal rats. The stimulatory response to pentagastrin was prompt and essentially equal in normal and diabetic animals. However, when hypoglycemia was prevented by glucose infusion, insulin did not stimulate gastric acid secretion in normal rats. Further, glucose infusion in these animals actually enhanced the secretory response to pentagastrin. Nitro- l-arginine methyl ester (L-NAME 20 mg/kg i.v.), an inhibitor of NO synthetase, also prevented the secretory response to insulin but not to pentagastrin. Preinfusion of arginine (100 mg/kg i.v.) in diabetic rats restored the gastric secretory response to insulin toward that of normal animals. We conclude that the gastric acid secretory response to insulin, but not to pentagastrin, is decreased in diabetic animals, that this response may operate through a NO mediated mechanism possibly set in motion by central nervous system glucopenia and that this NO-mediated mechanism is attenuated in diabetes.

Characterisation of Endothelin Induced Relaxation in Guinea-pig Airways: Evidence for Dilatory ETA-and ETB-receptors

Receptors involved in endothelin induced relaxation were characterized on isolated circular segments of guinea-pig trachea. The motor responses to endothelin-1 (ET-1), ET-2 and ET-3 as well as the effects of the ETB-receptor agonist, BQ 3020, and the ETA-receptor antagonist, FR 139317, were tested. The responses obtained were analysed in relation to the nitric oxide (NO) synthase inhibition by NG-monomethyl L-arginine (L-NMMA). In submaximally precontracted tracheal segments ET-1 and ET-2 induced concentration-dependent dilatations. ET-3 induced a biphasic response in precontracted segments, at low concentrations a small contraction followed by a dilatation at higher concentrations. FR 139317 blocked the low concentration induced dilatation by ET-1/ET-2, unmasking a contractile response. The dilatation induced by higher ET-1/ET-2 concentrations was unaffected by FR 139317 which also did not affect the dilatory response of ET-3. These results indicate the presence of two dilatory receptors. One of these was of ETAtype, since it was antagonized by FR 139317 while the other receptor probably was of the ETBtype, since all three endothelins in the moderate concentration range caused a uniform dilatation which was unaffected by FR 139317. Furthermore, BQ 3020 induced potent relaxation of the precontracted segments. In resting tracheal segments all three endothelins caused an identical contraction, suggesting a contractile ETB-receptor. However, BQ 3020 along with another ETB-agonist, IRL 1620, failed to induce contraction which indicates the possibility of two different subtypes of the ETB-receptor, one involved in dilatation and the other in contraction. Experiments with the nitric oxide synthetase inhibitor L-NMMA on precontracted segments showed a similar change as when FR 139317 was used. This suggests that the dilatory ETA-receptor depends on nitric oxice (NO) for mediating the dilation. On the other hand, the dilatory ETB-receptor response was unaffected by L-NMMA which suggests a mechanism unrelated to NO.

Anaplasma marginale: effect of the treatment of cattle with an interferon gamma-neutralizing monoclonal antibody or the nitric oxide synthetase inhibitor aminoguanidine on the course of infection.

Cattle undergoing initial infection with the rickettsia Anaplasma marginale were treated with either a monoclonal antibody (MoAb) with neutralizing activity for bovine interferon gamma (IFN-gamma) or aminoguanidine (AG), a specific inhibitor of the inducible form of nitric oxide synthetase (iNOS). Plasma levels of MoAb and AG were measured over the time of administration. The course of A. marginale infection was not altered in the MoAb-treated cattle relative to untreated controls. In cattle treated with AG however, A. marginale infection was significantly ameliorated, as judged by lower parasite levels and decreased anaemia in these cattle relative to the controls. The implications of these findings in relation to the basis for immunity against this economically important haemoparasite are discussed.

Physiology and biochemistry of endothelial function in children with chronic renal failure

Premature atherosclerosis is a major cause of morbidity and mortality in chronic renal failure (CRF). Endothelial dysfunction is a key early event in atherogenesis. The aim of this study was to assess the effect of CRF on endothelial function using physiological and biochemical measures. To focus on the effect of CRF itself, 23 children (matched with 23 controls for age and vessel diameter) were selected because they were normotensive, had normal total cholesterol (TC) levels, and were not on vasoactive drugs. Their mean (range) age was 12.0 (7.8 to 17.0) years; GFR 17.5 (8.8 to 34.5) ml/min/1.73 m2. The physiology of endothelial function in the brachial artery was assessed using high resolution ultrasound by measuring its diameter at rest, during reactive hyperemia (endothelium dependent dilation) and after sublingual glyceryl trinitrate (GTN; endothelium independent dilation). Nitric oxide (NO) metabolites and endogenous NO synthetase (eNOS) inhibitors were measured as an assessment of endothelial metabolism. Brachial artery dilation to flow [FMD, mean (SEM)%] was reduced in CRF to 4.9 (0.6) and controls 8.6 (0.6), P < 0.0001. In contrast, the response to GTN was similar in both groups: CRF 25.1 (1.6), controls 23.3 (1.2), P = 0.31. There was no difference in TC, low density lipoprotein (LDL) or high density lipoprotein (HDL) between the patients and the controls. Triglycerides (TG) were higher in the patients but within the normal range. Antibodies against oxidized LDL (ox-LDL) were high in CRF. Endogenous NOS inhibitors were high in CRF, and intermediate NO metabolites were low. There was no correlation between FMD of the brachial artery and lipid subfractions, or with NO metabolites or eNOS inhibitors. Endothelium dependent dilation of the brachial artery is impaired in children with CRF who do not have co-existing risk factors for atherosclerosis. This may represent early evidence of atherogenic vascular disease.

Estrogen-induced increases in coronary blood flow are antagonized by inhibitors of nitric oxide synthesis

Objective: Estrogen receptors have been found in coronary arterial endothelial and vascular smooth muscle cells. Therefore the present study was designed to determine if estradiol-17β can increase coronary blood flow and if so whether the changes are mediated by nitric oxide. Study design: Five oophorectomized non-pregnant sheep were chronically instrumented to measure blood pressure, heart rate, cardiac output, left circumflex coronary blood flow and central venous pressure. Animals received estradiol-17β (1.0 μg/kg) and cardiovascular responses were followed for 135 min. Results: Estradiol-17β (1.0 μg/kg) increased left circumflex (coronary) blood flow 28±3%, cardiac output 15±1% and heart rate by 13±3% Coronary vascular resistance decreased 23±5%, systemic vascular resistance decreased by 12±2% while blood pressure did not change significantly. Administration of the nitric oxide synthetase inhibitor l-nitroarginine methylester ( l-NAME), had no effect on basal coronary blood flow but completely reversed estradiol-17β induced increases in coronary blood flow. Conclusion: These results demonstrate that estrogen increases coronary blood flow in the non-pregnant sheep and that l-NAME, an inhibitor of nitric oxide, is able to reverse the estrogen induced flow changes.

Measurement of Upregulation of Inducible Nitric Oxide Synthase in the Experimental Autoimmune Encephalomyelitis Model Using a Positron Emitting Radiopharmaceutical

Excess nitric oxide has been implicated in the pathogenosis of experimental autoimmune encephalomyelitis (EAE) which is an animal model for multiple sclerosis. Positron emission tomography (PET) is an imaging technique that has shown utility for studying enzyme systemsin vivo.A positron-labeled inducible nitric oxide synthetase (iNOS) inhibitor has been studied in EAE-affected mice as well as controls. Greater uptake of the radiolabeled inhibitor was observed in the spinal cord of the affected mice than of control mice. Increased uptake was also observed in other organs not previously implicated in this experimental model. The increased uptake of the radiopharmaceutical in this model suggests that this tracer may have the potential for measuring increased levels of iNOS in humans by PET.

Evidence of P2Y-purinoceptor mediated bladder neck smooth muscle post-contractile relaxation in the male mini-pig

The functional role of non-adrenergic non-cholinergic (NANC) nerves in the autonomic control of the male mini-pig bladder neck was investigated in the present study. Electrical stimulation of muscle strips from male mini-pig bladder neck showed biphasic response with initial phasic contraction followed by post-contractile relaxation. Electrical stimulation in the presence of four autonomic blockers (atropine 10−6 M, propanolol 10−6 M, phentolamine 10−6 M and guanethidine 10−6 M) showed suppression of 68±15% of the contractile response (P<0.05, n=8) but no significant change in the relaxation response. Alpha-chymotrypsin 2 U/ml, l-NG-monomethyl-l-arginine acetate (a nitric oxide synthetase inhibitor) 10−4 M, 8-phenylthlophylline (a P1-purinoceptor antagonist) 10−6 M, and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphoric acid tetrasodium salt (a P2Y-purinoceptor antagonist) 3×10−5 M did not alter the NANC response significantly. On the other hand, reactive blue-2 (a P2Y-purinoceptor antagonist) 3×10−5 significantly reduced the relaxation by 79±9%. The result suggested that the P2Y-purinoceptor is involved in the electrically induced NANC post-contractile relaxation of the mini-pig bladder neck smooth muscle.