NO-mediated Relaxation Research Articles

Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications.

1. To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO-mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2. NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg-1 protein min-1): urethra (33 +/- 3.3), detrusor (13.1 +/- 1.1) and ureter (1.5 +/- 0.2). No activity was detected in the particulate fraction of any region. 3. NOS activity was dependent on Ca(2+)-calmodulin and required exogenously added NADPH and tetrahydrobyoptein (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4. NOS activity was inhibited to a greater extent by NG-nitro-L-arginine (L-NOARG) and its methyl ester (L-NAME) than by NG-monomethyl-L-arginine (L-NMMA), while 7-nitroindazole (7-NI) was a weak inhibitor and L-cannavine had no effect. 5. Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin-sensitive manner, suggesting feedback inhibition of NOS by NO. 6. EFS induced prominent NO-mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L-NOARG = L-NAME > 7-NI > L-NMMA. 7. In conclusion, we have demonstrated the presence of NO-synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO-mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely.

Inhibition by SK&F96365 of NO‐mediated relaxation induced by Ca2+ ‐ATPase inhibitors in rat thoracic aorta

1. We investigated the effect of SK&F96365, a putative inhibitor of receptor-operated Ca2+ entry, on the endothelium-dependent, NO-mediated relaxation and cyclic GMP formation induced by Ca2(+)-ATPase inhibitors in rat thoracic aorta. 2. SK&F96365 inhibited cyclopiazonic acid or thapsigargin-induced relaxation and cyclic GMP formation mediated by a constitutive NO synthase, which is known to be activated by the Ca2+ that enters into the endothelial cells via plasma membrane Ca2+ channels subsequent to depletion of stored Ca2+ by Ca2(+)-ATPase inhibitors. 3. SK&F96365 also inhibited relaxation and cyclic GMP formation induced by acetylcholine, without affecting those induced by nitroprusside and A23187. 4. Ni2+ attenuated relaxation and cyclic GMP formation induced by cyclopiazonic acid and acetylcholine. 5. In contrast, the voltage-dependent Ca2+ channel blocker, nifedipine, did not affect the relaxation caused by Ca2(+)-ATPase inhibitors. 6. These results suggest that endothelium-dependent, NO-mediated relaxation of the arteries induced by Ca2(+)-ATPase inhibitors is triggered by the Ca2+ that enters into endothelial cells via receptor-operated channels (SK&F96365-sensitive channels) subsequent to depletion of stored Ca2+ as a result of inhibition of the Ca2(+)-ATPase (Ca2+ pump) of the stores.

Inhibition of the contraction of the ductus arteriosus to oxygen by 1-aminobenzotriazole, a mechanism-based inactivator of cytochrome P450.

1. We have proposed that contractile tension of the ductus arteriosus is sustained by a cytochrome P450-linked mechanism acting as a limiting step in the synthesis of endothelin-1 (ET-1). In the present study, we have used the isolated ductus from near-term foetal lambs and guinea-pigs to investigate the effect on both muscle tone and ET-1 formation of 1-aminobenzotriazole (ABT), a suicide substrate for mono-oxygenase reactions. 2. ABT relaxed the lamb ductus at rest (2.5% O2) and during the oxygen contraction (15 to 95% O2). The effect was seen at 40 microM, and at 0.8 mM active tone was almost completely abolished. ABT (1 mM) also reversed the oxygen contraction in the guinea-pig ductus. 3. In the lamb ductus, the ABT response was not affected by removal of the endothelium or by treatment with 2.8 microM indomethacin (at 2.5% O2) and the ensuing contraction. 4. At both low and high concentration, ABT relaxed marginally, or not at all, the potassium-contracted (55 mM) ductus from either species. 5. ET-1 release from either the intact or endothelium-denuded lamb ductus tended to decrease in the presence of ABT (1 mM), whilst during the same treatment cyclic GMP content of the tissue remained unchanged. 6. We conclude that ABT relaxation is due to suppression of a contractile mechanism and not to activation of prostaglandin- and NO-mediated relaxing mechanisms. This contractile mechanism has a cytochrome P450-based mono-oxygenase reaction as a key component.

EFFECT OF SUPEROXIDE DISMUTASE INHIBITOR ON NITRIC OXIDE-INDUCED AIRWAY RELAXATION AND INTRACELLULAR Ca2+ TRANSIENTS. ▴ 2307

Endogenous production of superoxide anion (SA) is known to affect a variety of signal transduction pathways in smooth muscle. In this study, we investigated the effect of diethyldithiocarbamate (DETCA), an inhibitor of Zn, Cu-superoxide dismutase (SOD), on nitric oxide (NO)-mediated relaxation and intracellular Ca2- ([Ca2+]i) transients in porcine tracheal smooth muscle (TSM) cells. In carbachol (CH)-contracted TSM strips, DETCA (10 mM) inhibited relaxations mediated by cGMP and induced by NO released from intrinsic nerves as well as by the NO donor, sodium nitroprusside (SNP), but not that mediated by cAMP and induced by isoproterenol. In CH-contracted tissues, exposure to DETCA resulted in increased lucigenin-dependent chemiluminescence. DETCA did not inhibit the elevation of cGMP in response to SNP. In single, freshly isolated TSM cells loaded with fura-2/AM, exposure to 100 nM acetylcholine (ACh), which acts through the inositol 1,4,5-trisphosphate (IP3) receptor, resulted in elevation of [Ca2+]i. DETCA attenuated this Ca2+ response, but not to caffeine, an inducer of Ca2+ release through the ryanodine receptor. These results suggest that SA attenuates NO-mediated relaxations at a step distal to the generation of cGMP, and decreases ACh-induced elevation of [Ca2+]i by inhibiting IP3-induced Ca2+ release selectively. Supported by Univ. of Minnesota, Mayo Foundation, and NIH grant HL51736-02C. MSK was recipient of an Abbott Senior Fellowship.

A role for dietary copper in nitric oxide-mediated vasodilation.

This study was designed to investigate the role of dietary copper in nitric oxide-mediated arteriolar dilation. Male weanling Sprague-Dawley rats were fed a purified diet that was either copper-adequate (6.0 micrograms Cu per g diet) or copper-deficient (0.3 microgram Cu per g diet) for a period of 4 weeks. Each rat was anesthetized with pentobarbital and its cremaster muscle was positioned in a Krebs'-filled bath to which graded concentrations of vasoactive agents were added. In the first series, responses to norepinephrine (NE 10(-9)-10(-6) M) and acetylcholine (ACH 10(-7)-10(-4) M) were compared in third-order arterioles. Second, the dilator response to 10(-5) M ACH in the absence and presence of 240 U/ml Cu, Zn-superoxide dismutase (SOD) was determined. Third, arteriolar dilation was determined in response to NO-independent stimulation of soluble guanylate cyclase with hydrogen peroxide (10(-7)-10(-5) M) and to dibutyryl cGMP (10(-6)-10(-4) M), dibutyryl cAMP (10(-6)-10(-4) M), and papaverine (10(-4) M). The arteriole constrictor response to NE and the dilator response to hydrogen peroxide, dibutyryl cGMP and cAMP, and papaverine were not different between the dietary groups. Copper deficiency attenuated the ACH-induced dilation, but the response was restored in the presence of SOD. The inactivation of cytosolic Cu, Zn-SOD by restriction of dietary copper results in the depression of NO-mediated vascular smooth-muscle relaxation probably by interaction of NO with superoxide.

Cyclic GMP relaxes the internal anal sphincter in Hirschsprung's disease

In Hirschsprung's disease (HD), the aganglionic colon and internal anal sphincter (IAS) fail to relax. Aganglionic colon of HD patients relaxes in response to exogenous nitric oxide (NO), whereas the IAS from HD patients does not relax. The authors hypothesized that the failure of IAS relaxation is caused by a local deficiency of cyclic guanosine monophosphate (cGMP), the final metabolite in NO-mediated smooth muscle relaxation. To test this hypothesis, the authors measured the isometric tension of smooth muscle strips taken from the IAS and aganglionic colon of patients with HD before and after exposure to cGMP and compared this with ganglionic colon and IAS from normal controls. In HD patients both the IAS and aganglionic colon relaxed in response to cGMP ( P < .05). The amount of relaxation observed in both the aganglionic colon and IAS was comparable to that measured in the normal controls. The observation that exogenous cGMP relaxes the IAS, whereas exogenous NO does not, suggests that mechanisms for relaxation may be different than those in the aganglionic colon and may explain persistent IAS dysfunction after resection of aganglionic colon. The defect of the IAS in HD may be the inability of the NO cGMP pathway to induce smooth muscle cell relaxation rather than a defect in the smooth muscle cell.

Constitutive Nitric Oxide Synthase Is Expressed and Nitric Oxide-Mediated Relaxation Is Preserved in Retrieved Human Aortocoronary Vein Grafts

Although little is known about the endothelial cell function of human saphenous vein coronary artery bypass grafts, there is evidence to suggest that receptor-activated, endothelial-dependent relaxation mediated by nitric oxide is impaired. This study examines the expression and function of endothelial cell constitutive nitric oxide synthase (cNOS) of aortocoronary vein bypass grafts and human saphenous veins obtained from 10 patients undergoing repeat coronary artery bypass grafting for recurrent ischemic symptoms. Following precontraction with norepinephrine (10 -5 M), responses to acetylcholine (receptor-mediated, endothelium-dependent), calcium ionophore (A23187; receptor-independent, endothelium-dependent), and sodium nitroprusside (endothelium-independent) were assessed. Following total RNA extraction using phenol/guanidinium isothiocyanate from specimens of human saphenous vein and vein graft, a quantitative RNase Protection Assay (RPA) was performed using a cRNA riboprobe corresponding to a fragment of the human endothelial cell cNOS gene. Histologically, the vein grafts showed both intimal hyperplasia development and focal atherosclerosis formation compared to the saphenous veins. Scanning electron microscopy of the saphenous veins and the vein grafts showed an intact endothelium. Precontracted vein grafts did not relax in response to acetylcholine; in contrast, the saphenous vein relaxed in a dose-dependent manner to reach a maximal relaxation of 19 ± 4% precontracted tension. Saphenous veins and vein grafts relaxed in response to A23187 with maximal relaxation of 92 ± 5 and 73 ± 13%, respectively. Both vessels relaxed in a dose dependent manner to sodium nitroprusside. RPA normalized to β-actin showed similar levels of expression of endothelial cell cNOS equivalent to 1 pg of sense RNA in both the saphenous vein and vein graft. These data demonstrate that the endothelium of coronary artery vein grafts shows both expression and function of endothelial cell cNOS. However, the impaired NO-mediated relaxation of aortocoronary vein grafts in response to acetylcholine appears to be due to altered receptor activation of the endothelial cNOS system.

In the presence of atropine, cisapride induces a no-mediated gastric relaxation: [formula omitted], L.F. Helsen, A.L. Meulemans. Dept. of Gastrointestinal Pharmacology, Janssen Research Foundation, Beerse, Belgium

In the presence of atropine, cisapride induces a no-mediated gastric relaxation: [formula omitted], L.F. Helsen, A.L. Meulemans. Dept. of Gastrointestinal Pharmacology, Janssen Research Foundation, Beerse, Belgium

Effects of ethanol and other aliphatic alcohols on NO-mediated relaxations in rat anococcygeus muscles and gastric fundus strips.

1. In anococcygeus muscles, ethanol (20-500 mM) slightly increased the tone and inhibited relaxations elicited by nitrergic nerve stimulation (0.5-5 Hz) in a concentration-dependent manner. 2. Other aliphatic alcohols decreased the tone but had inhibitory effects similar to ethanol on stimulation-induced relaxations, the EC50 (mM) values being: methanol 280, ethanol 80, propan-1-ol 20, propan-2-ol 55, propan 1,2-diol 135, butan-1-ol 120, butan-2-ol 15 and pentan-1-ol 3. 3. Relaxations induced by sodium nitroprusside (SNP, 10 nM) were inhibited by ethanol (20-500 mM) in a concentration-dependent manner and by propan-2-ol (100 mM). Relaxations induced by NO (1 microM) were inhibited by high concentrations of ethanol (200-300 mM) and by propan-2-ol (100 mM). 4. In gastric fundus strips, ethanol (60-200 mM) did not affect the resting tone but inhibited NO-mediated relaxations elicited by low frequency (1 Hz) field stimulation and reduced the initial relaxation by high frequency field stimulation (10 Hz) and by SNP (50 nM). The relaxant action of isoprenaline (10 nM) was not reduced although it was slightly slower in onset. Other aliphatic alcohols tested decreased the tone and inhibited relaxations elicited by field stimulation. 5. Acetaldehyde (1-10 mM) inhibited relaxations elicited by field stimulation and SNP in both the rat anococcygeus muscles and gastric fundus strips. The tone of gastric fundus strips was decreased by acetaldehyde but it was transiently increased in anococcygeus muscles. 6. The dehydrogenase inhibitors, 4-methylpyrazole (100 MicroM) and disulfiram (100 MicroM) did not significantly affect the inhibition by ethanol of nitrergic nerve stimulation-induced relaxations in anococcygeus muscles, which suggests that ethanol acts directly.7. The inhibition of NO-mediated relaxations by alcohols is attributed to sequestration of NO to form nitroso-alcohols.

Regional differences in nitric oxide-mediated relaxation of the rabbit sphincter of Oddi

We studied the role of the l-arginine-nitric oxide (NO) pathway in non-adrenergic, non-cholinergic (NANC) relaxation of the rabbit sphincter of Oddi by recording changes in isometric tension in response to electrical field stimulation in two series of experiments. In a first set of experiments, biliary sphincters of Oddi removed from New Zealand white rabbits were placed horizontally in an organ bath containing oxygenized, buffered (pH 7.4) Krebs solution. Contractile responses of the whole sphincter to field stimulation were determined. In the second set of experiments, sphincter of Oddi was divided into two parts and the effects of field stimulation wewe studied separately on areas close to the duodenal papilla (area I) and areas close to the common bile duct (area II). In the whole sphincter of Oddi, field stimulation induced an initial twitch-like contraction followed by relaxation proportional to the number of stimuli (3 and 10 stimuli at 20 Hz, 50 V, 0.1 ms). The magnitude of the contractile responses was considerably reduced by 1 μM atropine, phentolamine and oxprenolol (NANC solution). Field stimulation produced dose-dependent contractions of both segments of sphincter of Oddi in response to the same protocol as used with whole sphincter of Oddi. However, preincubation with NANC solution produced monophasic relaxation in response to field stimulation in area I, whereas area II preparations such as the whole sphincter of Oddi respondend with contractions followed by minimal relaxations. Field stimulation failed to induce either contractions or relaxations in the presence of 1 μM tetrodotoxin. 30 μM N G - nitro- l- arginine methyl ester abolished NANC relaxation in area I, but did not influence significantly contractile responses of area II, or the whole sphincter of Oddi. l-Arginine but not d-arginine (both 3 mM) reversed the inhibitory effect of N G - nitro- l- arginine on NANC relaxation in area I. We conclude that (i) the l-arginine-NO pathways is involved in NANC relaxation of sphincter of Oddi segment close to the duodenal papilla and (ii) that there are regional differences in NO-mediated relaxation of the sphincter of Oddi in rabbits.

Chronic treatment with the anabolic steroid, nandrolone, inhibits vasodilator responses in rabbit aorta

The effect of chronic treatment of rabbits for 4, 8 and 12 weeks with the anabolic steroid, nandrolone, on vasodilator responses was studied in segments of different arteries. The treatment abolished endothelium-dependent relaxation caused by acetylcholine and the Ca 2+-ionophore, A23187, in thoracic aorta, and reduced endothelium-independent relaxations induced by exogenous nitric oxide (NO) or sodium nitroprusside. The inhibitor of NO synthesis, N G-monomethyl- l-arginine, abolished vasodilator responses to acetylcholine and A23187. In contrast, relaxation induced by acetylcholine, NO or sodium nitroprusside in mesenteric and femoral arteries was unaltered by nandrolone treatment. Bioassay experiments using donor segments and endothelium-denuded bioassay rings from thoracic aorta show that acetylcholine, applied either through control or treated (12 weeks) donor segments, produced similar relaxation in bioassay rings from control rabbits, but this relaxation was markedly reduced in rings from treated rabbits. The increases of cyclic GMP levels induced by acetylcholine or sodium nitroprusside in segments from thoracic aorta were abolished by nandrolone treatment. These results suggest that the treatment with nandrolene reduces NO-mediated relaxation only in thoracic aorta by inhibition of guanylate cyclase, endothelial NO production and vasodilator machinery being unaltered.

P-332 - Requirement of extracellular Ca2+ in cyclopiazonic acid-induced, NO-mediated relaxation of rat thoracic aorta.

P-332 - Requirement of extracellular Ca2+ in cyclopiazonic acid-induced, NO-mediated relaxation of rat thoracic aorta.

Angiotensin II: nitric oxide interaction and the distribution of blood flow

Nitric oxide (NO) contributes to the regulation of regional blood flow. Inhibition of NO synthesis increases blood pressure and vascular resistance. Using radioactive microspheres and the substrate antagonist N omega-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) to block NO synthesis, we tested the hypothesis that there is a significant interaction between the vasodilator NO and the vasoconstrictor angiotensin II, which regulates regional hemodynamics. Further, we investigated the influence of anesthesia on this interaction. L-NAME increased blood pressure, decreased cardiac output, and increased total peripheral resistance in both anesthetized and conscious rats. In anesthetized rats, L-NAME decreased blood flow to visceral organs (i.e. kidney, intestine, and lung) but had little effect on blood flow to the brain, heart, or hindlimb. Treating anesthetized rats with the angiotensin II receptor antagonist losartan (10 mg/kg) attenuated the decrease in cardiac output and the increase in total peripheral resistance without affecting the pressor response to L-NAME. Losartan also attenuated the visceral hemodynamic responses to L-NAME. In conscious rats, L-NAME decreased blood flow to all organ beds. Treating these rats with losartan only marginally attenuated the increase in total peripheral resistance to L-NAME without significantly affecting the pressor response or the decrease in cardiac output. Losartan had no effect on the regional hemodynamic responses to L-NAME. These data suggest that NO-mediated vascular relaxation is an important regulator of total peripheral and organ vascular resistance. (ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthase-immunoreactive nerve fibers in dog cerebral and peripheral arteries

Nitric oxide synthase (NOS)-immunoreactive fibers innervating the dog arterial wall were histochemically determined by the use of NOS antiserum. NOS-immunoreactive fibers were consistently found in every arterial wall examined. In a whole-mount preparation, NOS-positive fibers were detectable in the small pial artery having a diameter of about 100 μm as well as the proximal middle cerebral artery. Further detailed analyses in thin cryostat sections indicated that in middle cerebral, basilar, temporal, mesenteric and femoral arteries, fine NOS-positive fibers were detected in outer zones of the media in addition to many thicker fibers in the adventitia. However, in the coronary artery, many thick fibers ere situated in the adventitia, and fine NOS-positive fibers were not the media. Injection of ethanol to the pterygopalatine ganglion markedly decreased or abolished the NOS immunoreactivity in nerve cells and fibers and abolished the innervation of NOS-positive fibers in the wall of middle cerebral artery of the ipsilateral side. Together with findings in our previous publications concerning the functional role of nitroxidergic nerve in the control of arterial tone, we conclude that perivascular nerves containing NOS are crucial in eliciting the neurally induced, NO-mediated arterial relaxation.

Effect of nitric oxide-donor, linsidomine chlorhydrate, in treatment of human erectile dysfunction caused by venous leakage

Recent experimental work has demonstrated that nitric oxide (NO) is the neurotransmitter responsible for cavernous smooth muscle relaxation. We studied the effect of a direct NO-donor, linsidomine chlorhydrate (SIN-1), in 30 patients with venous leakage confirmed by dynamic pharmacocavernosography and pharmacocavernosometry that was refractory to prostaglandin E 1 (PGE 1) under the assumption that the more physiologic approach might give better results. In all 30 patients, response to SIN-1 was no better, and in 22 cases it was less than the response to PGE1. No systemic or local side effects of SIN-1 were observed. SIN-1 is not superior to PGE1 in the treatment of erectile dysfunction caused by venous leakage, and failure of NO-mediated smooth muscle relaxation does not play a part in the entity, “venous leakage”.

Nitric oxide synthase in pig lower urinary tract: immunohistochemistry, NADPH diaphorase histochemistry and functional effects.

1. The distribution and colocalization of nitric oxide synthase (NOS)-like immunoreactivity and NADPH diaphorase activity in the pig lower urinary tract were investigated by immunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS-immunoreactivity/NADPH diaphorase staining with smooth muscle responses involving the L-arginine/nitric oxide (NO) pathway. 2. NOS-immunoreactivity and NADPH diaphorase activity were expressed in nerve trunks and fine nerve fibres in and/or around muscular bundles in the detrusor, trigone and urethra. Thin nerve fibres that dispersed within the muscle bundles were mainly found in the urethral/trigonal area, whereas such fibres were less common in the detrusor. 3. Almost all neuronal structures that were NOS-immunolabeled were also stained for NADPH diaphorase. In contrast, the urothelium, which was intensively stained by the NADPH diaphorase technique, remained unstained by immunohistochemistry. 4. Electrical field stimulation of pig isolated trigonal and urethral preparations induced relaxations, which were inhibited by tetrodotoxin (1 microM) and NG-nitro-L-arginine (L-NOARG, 10 microM). 5. L-Arginine (1 mM), but not D-arginine, inhibited (25-30%) electrically evoked detrusor contractions. This inhibition was reversed by L-NOARG (0.1 mM). L-Arginine did not inhibit detrusor contractions in the presence of scopolamine (1 microM) and had no direct smooth muscle effects per se. 6. Acetylcholine (1 nM-10 microM) caused concentration-dependent relaxations of noradrenaline-induced contractions in pig vesical arteries. Removal of the endothelium practically abolished the acetylcholine-induced relaxation. Pretreatment with L-NOARG (0.1 mM and 0.3 mM) caused a rightward shift of the concentration-response curves to acetylcholine, but the maximal relaxation obtained was significantly reduced (to 65 +/- 12%; n = 6; P < 0.05) only at 0.3 mM L-NOARG. 7. In vessel segments contracted with K+ (60 mM), acetylcholine induced concentration-dependent relaxations. When the vessels were incubated with 0.3 mM L-NOARG and then contracted with K+ (60 mM) all relaxant responses to acetylcholine were abolished. 8. The presence of NO synthesizing enzyme in nerve fibres and the pharmacological evidence for NO-mediated relaxation of the trigone and urethra suggest that NO or a NO-related substance may have a role in inhibitory neurotransmission in these regions. In the detrusor, the presence of NO-synthesizing enzyme in nerves can be demonstrated, but its functional importance is unclear. NO, as well as other endothelium-derived factors seem to be involved in the endothelium-dependent acetylcholine-induced relaxation of pig vesical arteries.

Nitric oxide mediates the neural nonadrenergic, noncholinergic relaxation of pig tracheal smooth muscle.

In pig tracheal smooth muscle, the isometric tension responses to electrical field stimulation (EFS) were studied after raising the tone with carbamylcholine chloride (carbachol). EFS induced frequency-dependent relaxations that were nonadrenergic, noncholinergic (NANC) in nature. Addition of NG-nitro-L-arginine (L-NOArg), an inhibitor of nitric oxide (NO) synthesis from L-arginine, resulted in concentration-dependent inhibition of the relaxation response to EFS. Pretreatment of the tissues with L-arginine (1 mM) prevented the inhibitory effect of L-NOArg on the EFS-induced relaxations at the frequencies studied. However, in the presence of D-arginine, EFS-induced relaxations were inhibited by L-NOArg. L-Arginine, D-arginine, and L-NOArg had no significant effects on basal tone of the muscle. In the presence of L-NOArg, vasoactive intestinal polypeptide (3 x 10(-7) M), the nicotinic agonist dimethylphenyl piperazinium bromide (100 microM), and isoproterenol (1 microM) relaxed carbachol-induced tone. The concentration-dependent selective inhibition of neural relaxation by L-NOArg and its reversal by L-arginine in a stereospecific manner are consistent with NO-mediated NANC relaxation of pig tracheal smooth muscle.