Non-adrenergic Non-cholinergic Relaxation Research Articles

Inhibitory NANC neurotransmission in choledocho–duodenal junction of rabbits—a possible role of PACAP

The pharmacological properties of non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission were investigated in the rabbit choledocho–duodenal junction (CDJ), using the microelectrode and tension recording methods. l-NAME (10 −4 M) and apamin (5×10 −6 M) suppressed NANC relaxation evoked by electrical field stimulation (EFS) in the presence of atropine and guanethidine (each 10 −6 M) to a similar extent (to about 40% of the initial control). However, combined application of l-NAME (10 −4 M) and apamin (5×10 −6 M) did not abolish it. EFS also evoked biphasic inhibitory junction potentials (IJPs) consisting of initial fast and slow sustained components in the presence of atropine and guanethidine (each 10 −6 M). Apamin (5×10 −8–5×10 −6 M) dose-dependently suppressed the initial fast component by about 70%. In contrast, l-NAME (10 −4 M) did not affect either the amplitude of IJP or the resting membrane potential. PACAP-38 (>10 −8 M) dose-dependently hyperpolarized the smooth muscle membrane of rabbit CDJ followed by a slow repolarization to the original level. After pretreatment with apamin (5×10 −7 M), PACAP-38 (10 −6 M) failed to evoke membrane hyperpolarization. During repolarization in the continued presence of PACAP-38, the amplitude of initial fast component of IJP was reduced to about 40–60% of control value, while that of the slow one was unaffected. A similar suppression of initial fast component of IJP (about 40% of the control value) also occurred after application of PACAP (6–38), a PACAP antagonist, or prolonged treatment with monoclonal antibodies to PACAP-27 or PACAP-38. Furthermore, the substantial part of residual fast IJP in the presence of PACAP (6–38) was suppressed by desensitization to α, β-methylene ATP (10 −3 M). These results indicate that in rabbit CDJ NANC relaxation consists mainly of apamin- and l-NAME-sensitive components, which occur in a membrane potential dependent (through membrane hyperpolarization) and independent fashion, respectively. It has further been suggested that PACAP, together with a smaller contribution of ATP, may be involved as the principal apamin-sensitive transmitter in NANC relaxation of this muscle.

Cross tolerance between nitroglycerin and neural relaxation of the rabbit sphincter of Oddi

We studied whether non-adrenergic, non-cholinergic (NANC) relaxation of the rabbit sphincter of Oddi was influenced by tolerance to nitroglycerin (NG)in vitro. Sphincter of Oddi (SO) muscle rings precontracted with EC50concentrations of cholecystokinin octapeptide (CCK8) were exposed to cumulative increases in NG concentrations and tested for relaxation by measurement of isometric tension. A separate group of six rings was subjected to a preceding exposure to 275 μmnitroglycerin over 60 min to inducein vitrotolerance to nitroglycerin. The rings (both tolerant and non-tolerant) were subjected to electrical field stimulation (FS: 50 V, 0.1 ms, 20 Hz, 3 and 10 stimuli). The rings were then preincubated with NANC solution: phentolamine, oxprenolol and atropine (all 1 μm) for 20 min and FS was applied again. FS was repeated after additional incubation withNG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthase (30 μm) and after a successive incubation with 3 mml-arginine (20 min). Maximum contractions produced by CCK8 in `tolerant' and ‘non-tolerant’ sphincters were 29.9±5.8 and 28.3±5.2 mN, respectively. The sensitivity to CCK8 also was not different between the two groups with EC50(−log M) values of 8.5±0.2 and 8.3±0.1, respectively. FS evoked twitchlike contraction followed by relaxation in the ampullary SO in both `tolerant' and `non-tolerant' preparations. Incubation in NANC solution resulted in monophasic relaxations in response to FS in non-tolerant sphincters but not in tolerant ones.l-NAME (30 μm) reversed NANC relaxation in non-tolerant muscle rings whereas it failed to modify NANC contractions in the tolerant preparations.l-arginine (3 mm) reversed the inhibitory effect ofl-NAME on NANC relaxation in the `non-tolerant' rings and it was without effect on FS-induced contractions in the `tolerant' SO. As measured by radioimmunoassay, tolerance to NG was without any significant effect on tissue content of both cyclic adenosine 3′:5′ monophosphate (cAMP) and cyclic guanosine 3′:5′ monophosphate (cGMP). FS significantly increased tissue cAMP and cGMP content in ‘non-tolerant’ preparations. FS failed to increase the level of either cyclic nucleotide in `tolerant' tissue. We conclude that NANC relaxation of the ampullary part of the rabbit SO is significantly impaired in the state of tolerance to NG`in vitro'.

In vitro and in vivo effects of nitric oxide synthase inhibitors and nitric oxide inactivators on the South American opossum ileocolonic junction.

The potential role of nitrergic nerves in the regulation of the South American (SA) opossum ileocolonic junction (ICJ) function was investigated. In vitro, the effects of nitric oxide (NO) synthase inhibitors and NO inactivators on the non-adrenergic non-cholinergic (NANC) nerve-mediated relaxations of the circular muscle of the SA opossum ICJ were determined by employing isolated strips. Electrical field stimulation (0.2-8.0 Hz) caused frequency-dependent NANC relaxations. Nicotine and ATP also induced concentration dependent NANC relaxations that were abolished by tetrodotoxin (TTX). The relaxation response induced by NANC nerve activation was reduced in a dose dependent manner by NO synthase inhibitors while vasoactive intestinal peptide (VIP) and sodium nitroprusside (SNP) induced relaxations were uninfluenced by these drugs. In vivo, the NO synthase inhibitor, L-NAME, administered into the local artery caused a raise in intraluminal pressure of the ICJ in anaesthetized SA opossums in a L-arginine-preventable manner. Hydroquinone and pyrogallol, while being able to reduce, in a superoxide dimutase (SOD) reversible manner, the relaxations induced by exogenous NO failed to affect the NANC nerve-induced relaxations. Finally, neurones and nerve fibres in the myenteric plexus as well as varicose nerve fibres on the circular smooth layer were positive for NADPH-diaphorase activity. These findings indicate that nitrergic nerves inhibit ICJ circular smooth muscle in vitro and in vivo but cast doubts on the neuromediator being the NO radical.

No evidence for a significant non-nitrergic, hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus.

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli. 2. Phenylephrine (0.1-100 microM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 microM phenylephrine (EC26) was used to raise tone in subsequent experiments. 3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-NG-nitro-arginine (L-NOARG; 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01-5 microM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 microM). 4. Nifedipine (1 microM) reduced the contractile response to 0.2 microM phenylephrine by 38%; however, it had no effect on NANC relaxations. 5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.

Evidence for an esophageal origin of VIP-IR and NO synthase-IR nerves innervating the guinea pig trachealis: A retrograde neuronal tracing and immunohistochemical analysis

Nonadrenergic noncholinergic (NANC) relaxations of the guinea pig trachea are thought to be mediated by vasoactive intestinal peptide (VIP) and nitric oxide (NO). Physiological studies have indicated that the parasympathetic ganglion neurons mediating NANC relaxations of the guinea pig trachea but not the ganglion neurons mediating cholinergic contractions are in some way associated with the adjacent esophagus. In the present study, we attempted to locate precisely the noncholinergic parasympathetic ganglia innervating the trachealis. Two days after injection of the retrograde neuronal tracer DiI into the trachealis of organotypic cultures of the guinea pig trachea and esophagus, neurons within the myenteric plexus of the esophagus or closely associated with the outer striated longitudinal muscle layers of the esophagus were labeled. Subsequent immunohistochemical analyses revealed that a majority of the retrogradely labeled neurons possessed VIP immunoreactivity (IR) or NO synthase (NOS)-IR or had VIP-IR nerve fibers associated with their cell bodies. By contrast, no labeling of esophageal neurons was seen when the tissue between the trachea and esophagus had been disrupted by blunt dissection prior to tracer injection or when the cultures were treated with the axonal transport inhibitor colchicine. The results of these experiments provide the first direct evidence that VIP-IR and NOS-IR neurons intrinsic to the guinea pig esophagus project axons to the adjacent trachealis. Based on their location and phenotype and the results of our previous studies, it is likely that these neurons are the postganglionic parasympathetic neurons mediating NANC relaxations of the trachealis.

Non-adrenergic, non-cholinergic neurons innervating the guinea-pig trachea are located in the oesophagus: evidence from retrograde neuronal tracing

The possibility that non-adrenergic, non-cholinergic (NANC) neurons innervating the guinea-pig trachea may be located within the oesophagus has been investigated using an in vitro retrograde tracing technique. The cervical trachea and oesophagus were excised from guinea-pigs and DiI was applied to a 5 mm region of the trachealis muscle. These preparations were maintained in organotypic culture for 3 days and processed for immunohistochemistry. A mean of 44 (4 neural cell bodies in the oesophageal myenteric plexus were found to be labelled by DiI. The vast majority of these neurons contained nitric oxide synthase, vasoactive intestinal polypeptide and neuropeptide Y. It is suggested that the population of neurons identified in this study are postganglionic parasympathetic neurons mediating NANC relaxation of the trachealis muscle in this species.

Extrinsic neural control of nitric oxide synthase expression in the myenteric plexus of rat jejunum.

1. The role of extrinsic innervation in the expression of nitric oxide synthase (NOS) in the myenteric plexus remains unknown. In this study, we investigated the effect of extrinsic denervation on NOS expression in rat jejunum. 2. NG-nitro-L-arginine methyl ester (L-NAME)-sensitive non-adrenergic, non-cholinergic (NANC) relaxations induced by transmural nerve stimulation were significantly increased in muscle strips obtained from rats treated with splanchnic ganglionectomy or 6-hydroxydopamine (6-OH-dopamine). Truncal vagotomy or treatment of the coeliac ganglia with capsaicin did not significantly affect NANC relaxations. 3. The number of NOS-immunopositive cells in the myenteric plexus was significantly increased in tissues obtained from rats treated with splanchnic ganglionectomy or 6-OH-dopamine. 4. Western blot analysis and Northern blot analysis showed a significant increase in the density of the immunoreactive NOS band and the NOS mRNA band, respectively, of the tissues obtained from rats treated with splanchnic ganglionectomy or 6-OH-dopamine. 5. Truncal vagotomy or treatment of the coeliac ganglia with capsaicin did not significantly affect the density of NOS band or NOS mRNA band. 6. It is concluded that neuronal NOS expression in the myenteric plexus is independent of vagus nerve and is negatively regulated by the splanchnic nerves in the rat small intestine.

Possible role of cAMP, cGMP and [Ca 2+] i during NANC relaxation in the cat airway smooth muscle

To investigate the possible role of cyclic nucleotides and [Ca 2+] i in non-adrenergic non-cholinergic (NANC) relaxations evoked by electrical field stimulation (EFS) in the cat airway, we studied the effects of specific phosphodiesterase (PDE) type IV or V inhibitors on the biphasic-NANC relaxations, the correlation between NANC relaxation and changes in intracellular levels of cAMP and cGMP ([cAMP] i and [cGMP] i), and measured changes in [Ca 2+] i during the NANC relaxation. EFS (repetitive stimuli at 20 Hz, 1 or 4 ms pulse duration, 50 V) applied to the bronchial smooth muscle during contraction induced by 5-HT (10 −5 M) in the presence of atropine (10 −6 M) and guanethidine (10 −6 M) elicited biphasic NANC relaxations. Zaprinast (>3×10 −7 M), a specific PDE type V inhibitor, preferentially enhanced the amplitude of the first component of the NANC relaxations. However, rolipram (>3×10 −7 M) enhanced both the first and second component of the NANC relaxation to a similar extent. In the trachea, EFS evoked monophasic NANC relaxation accompanied by a concomitant accumulation of [cAMP] i and [cGMP] i. Pretreatment with rolipram (3×10 −6 M) enhanced the accumulation of [cAMP] i and amplitude of NANC relaxation evoked by EFS. However, zaprinast did not affect the amplitude of NANC relaxation although it significantly increased the levels of [cGMP] i. N ω -Nitro- l-arginine methylester ( l-NAME; 10 −5 M) completely suppressed the accumulation of [cGMP] i but only partly suppressed the NANC relaxation evoked by EFS. In contrast, EFS significantly enhanced [cAMP] i in the presence of l-NAME. During NANC relaxation, time-dependent decrease in [Ca 2+] i occurred, which was partly suppressed by l-NAME. These results indicate that NANC relaxation is associated with concomitant accumulation in both [cAMP] i and [cGMP] i, and decrease in [Ca 2+] i. However, the timing of the action of [cGMP] i and [cAMP] i in NANC relaxations differs in the central and peripheral airway.

Decrease in Participation of Nitric Oxide in Nonadrenergic, Noncholinergic Relaxation of Rat Intestine With Age

Participation of nitric oxide in the electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation in various intestinal regions was studied in 2- to 50-week-old Wistar rats. In the jejunum of 2-week-old rats, the extent of the nitric oxide-mediated component of the relaxation of longitudinal muscle was approximately 60–70%, whereas the component was 40–50% in 4-week-old rats and was absent in 8- and 50-week-old rats. Thus, nitric oxide seems to be the most important mediator at young ages but its significance is lost with age. The same tendency as that in the jejunum was also shown in longitudinal muscle of the ileum, proximal and distal colon, and rectum. The tendency was also shown in the circular muscle of the rectum. Sensitivity of the longitudinal muscle of the jejunum and proximal colon to exogenously added nitric oxide was high in younger rats. Immunoreactive structures for nitric oxide synthase were observed in the circular muscle layer of the rectum. The population of the structures was denser in 4-week-old than that in 50-week-old. The results suggest that NANC relaxation in every region of the intestine at 2-week-old is almost solely mediated by nitric oxide, and its significance as an inhibitory mediator gradually or rapidly decreases with age.

Endothelium-dependent sensory NANC vasodilatation: involvement of ATP, CGRP and a possible NO store.

1 Non-adrenergic non-cholinergic (NANC) vasodilator nerves regulate tone in certain vascular beds. We have investigated the mechanisms of the NANC dilator response in the isolated small mesenteric artery of the rabbit by use of the tension myograph. 2 Small second or third order (150-300 microm in diameter) arteries of the rabbit mesenteric bed were mounted in a Mulvany tension myograph. Responses to electrical field stimulation (EFS) and exogenous vasodilators were investigated. 3 EFS (0.5-16 Hz, 10 V, 0.3 ms for 5 s), in the presence of guanethidine (5 microM) and atropine (1 microM) produced frequency-dependent relaxation of small arteries. Pretreatment with tetrodotoxin (1 microM) abolished the relaxation and desensitization with capsaicin (10 microM) strongly inhibited the relaxation. 4 Pretreatment with a P2Y-purinoceptor antagonist, basilen blue (3 microM) or a human calcitonin gene-related peptide (hCGRP) receptor antagonist, hCGRP8-37 (1 microM) suppressed the NANC relaxation by approximately 40-60 % in each case and combined pretreatment almost abolished the relaxation. 5 The EFS-induced relaxation was suppressed by endothelium-removal, pretreatment with the soluble guanylyl cyclase inhibitor ODQ (1 microM) and the NO scavenger oxyhaemoglobin (OxyHb; 20 microM) but not by NO synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME; 300 microM) or NG-nitro-L-arginine (L-NOARG; 300 microM). Combined pretreatment with ODQ and CGRP8-37 almost abolished the relaxation. 6 A P2Y-purinoceptor agonist, 2-methylthio ATP, produced endothelium-dependent relaxation which was inhibited by L-NAME and ODQ (1 microM), whilst hCGRP produced endothelium-independent and ODQ-insensitive relaxation. 7 Ultraviolet light (320 nm, 5 shots over 20 s) produced relaxation that was blocked by both OxyHb and ODQ but not by NG-monomethyl-L-arginine (L-NMMA, 300 microM). 8 The present study suggests that EFS-induced NANC relaxation of the mesenteric small artery of the rabbit is mediated mainly by capsaicin-sensitive sensory C-fibres and that both ATP and CGRP are involved. The action of ATP released by EFS appears to be endothelium-dependent and involve activation of soluble guanylyl cyclase, but is resistant to inhibitors of NO synthase. The response to CGRP is endothelium-independent. These results show that ATP and CGRP account fully for the NANC relaxation of this vessel type and that the endothelium is involved in NANC-induced relaxation. The endothelium-dependent part of the response is consistent with the release of NO, either from NO synthase, incompletely inhibited by the NO synthase inhibitors, or by some preformed stores.

Possible role of nitric oxide in NANC relaxation of the airway and in airway hyperreactivity in airway disease

In the lung, there is increasing evidence that endogenous nitric oxide (NO) plays multiple roles in physiological control of airway functions, immune responses and host defense against infection, and it is also implicated in inflammatory disease of the airways. As elsewhere, it is generally accepted that NO derived from constitutive NO synthase (cNOS) is involved in physiological regulation of airway function, whereas NO derived from inducible NO synthase (iNOS) is involved in immune responses and inflammatory diseases of the airway. In this mini review, first we describe evidence showing the possible roles of NO and NO-related compounds in nonadrenergic noncholinergic (NANC) relaxation and inhibitory action on excitatory neuro-effector transmission, thereby indicating that NO derived from neuronal cNOS plays a role to provide "double braking" in bronchoconstrictions. Secondly, we will discuss the possible involvement of epithelial damage due to excessive NO production through iNOS in the genesis of airway hyperreactivity and airway inflammation.

Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide.

Neuronal nitric oxide synthase (nNOS) generates NO in neurons, and heme-oxygenase-2 (HO-2) synthesizes carbon monoxide (CO). We have evaluated the roles of NO and CO in intestinal neurotransmission using mice with targeted deletions of nNOS or HO-2. Immunohistochemical analysis demonstrated colocalization of nNOS and HO-2 in myenteric ganglia. Nonadrenergic noncholinergic relaxation and cyclic guanosine 3',5' monophosphate elevations evoked by electrical field stimulation were diminished markedly in both nNOSDelta/Delta and HO-2(Delta)/Delta mice. In wild-type mice, NOS inhibitors and HO inhibitors partially inhibited nonadrenergic noncholinergic relaxation. In nNOSDelta/Delta animals, NOS inhibitors selectively lost their efficacy, and HO inhibitors were inactive in HO-2(Delta)/Delta animals.

Impaired expression of nitric oxide synthase in the gastric myenteric plexus of spontaneously diabetic rats

BACKGROUND & AIMS: The mechanism responsible for defective gastric accommodation in diabetes is unknown. The aim of this study was to investigate if this abnormality is due to a defective nitric oxide pathway secondary to impaired nitric oxide synthase (NOS) expression in the gastric myenteric plexus.METHODS: To test this hypothesis, we studied nonadrenergic, noncholinergic (NANC) relaxation, NOS activity, NADPH diaphorase histochemistry, NOS immunohistochemistry, NOS immunoblotting, and NOS messenger RNA expression in the gastric myenteric plexus of spontaneously diabetic biobreeding/Worcester (BB/W) rats. Age-matched nondiabetic Wistar rats were used as controls.RESULTS: Gastric neuromuscular preparations from control rats showed a frequency-dependent NANC relaxation in response to transmural stimulation. This relaxation was markedly antagonized by N(G)-nitro-L- arginine-methyl ester, indicating mediation by the neuronal release of NO. The NANC relaxation in gastric muscle preparations obtained from diabetic BB/W rats was significantly impaired. The number of NOS- immunoreactive cells in the gastric myenteric plexus and the NOS activity were significantly reduced in diabetic BB/W rats, suggesting that NOS synthesis is impaired in diabetes. Northern blot analysis showed that the density of NOS messenger RNA bands at 9.5 kilobases was significantly reduced in the gastric tissues of diabetic BB/W rats.CONCLUSIONS: These results indicate that gastric relaxation in diabetics is hampered mainly by impaired NOS expression in the gastric myenteric plexus.(Gastroenterology 1997 Nov;113(5):1535-44)

Influence of endotoxin on contractility in the rat gastric fundus

The influence of in vivo treatment with E. coli lipopolysaccharide endotoxin on the contractility of the rat gastric fundus was studied. Four h after lipopolysaccharide treatment (20 mg/kg i.p.), the contractile responses to prostaglandin F 2 α in longitudinal muscle strips from the gastric fundus were not different from those in control animals, while the well-known decreased response to noradrenaline in rings of the thoracic aorta was confirmed. Incubation of the tissues with l-arginine did not depress the response to prostaglandin F 2 α in fundus strips of lipopolysaccharide-treated rats. Twelve h after lipopolysaccharide treatment (6.7 mg/kg i.p.), the prostaglandin F 2 α -induced contractions were consistently depressed. The impairment of the prostaglandin F 2 α -induced responses by lipopolysaccharide treatment was not reversed by the nitric oxide synthase inhibitors N G-nitro- l-arginine ( l-NNA, 10 −4 M), N G-nitro- l-arginine methyl ester ( l-NAME, 3×10 −4 M), aminoguanidine (10 −4 M) and l- N 6-1-iminoethyl-lysine ( l-NIL, 10 −4 M) nor by the cyclooxygenase inhibitor indomethacin (10 −5 M). The impairment was prevented by pretreating the animals with dexamethasone (5 mg/kg i.p.), which had no effect per se on the contractile response to prostaglandin F 2 α . Lipopolysaccharide treatment did not influence the contractile responses to KCl and serotonin. The nonadrenergic noncholinergic relaxant responses to transmural electrical stimulation were not influenced 4 h after lipopolysaccharide treatment but were moderately reduced after 12 h. The results illustrate that the selective impairment of prostaglandin F 2 α -induced contractions in the rat gastric fundus by lipopolysaccharide treatment is not mediated via generation of nitric oxide; downregulation of the prostaglandin F 2 α -receptor by lipopolysaccharide treatment might be involved.

Nonadrenergic–noncholinergic relaxations of isolated circular muscle from South American opossum esophagogastric junction: Is nitric oxide the inhibitory mediator?

Nonadrenergic–noncholinergic (NANC) inhibitory nerves are responsible for most of the nerve induced relaxations of gastrointestinal muscle. It has recently been proposed that NANC nerves may release nitric oxide (NO) or a related compound derived from l-arginine. We have recently shown that the South American (SA) opossum is another suitable model to elucidate the mechanism involved in these NANC relaxations. In the present study the effect of NO synthase inhibitors as well as NO inactivators on the NANC-nerve induced relaxations of the circular muscle of the esophagogastric junction (EGJ) of the SA opossum was investigated. It was observed that the NO synthase inhibitors, l-NOARG and l-NAME, caused a concentration-dependent reduction of NANC-nerve induced relaxations which was reversed by l- but not d-arginine. The NO-donors sodium nitroprusside and hydroxilamine as well as NO caused concentration-dependent relaxations of the EGJ circular muscle. In the myenteric plexus of this region, NADPH-diaphorase positive neurons and nerve fibers were observed while in the circular muscle layer only numerous positive fibers were found. The NO inactivators, hydroquinone, pyrogallol and carboxy-PTIO, reduced NO-induced relaxations but failed to affect NANC nerve- and sodium nitroprusside-induced relaxations. Taken together, these findings indicate that NANC nerve induced relaxation of the SA opossum EGJ circular muscle is dependent on neural NO synthase activity and suggest that the neurotransmitter being released is a superoxide resistant molecule, which is unlikely to be the NO radical, or that the activity of NO synthase is required for the release of the actual neurotransmitter rather than for synthesizing the neuromediator.

Evidence for muscarinic M4 receptors mediating nonadrenergic noncholinergic relaxations in rabbit anococcygeus muscle.

The aim of the present study was to characterize the muscarinic receptor subtype mediating nonadrenergic noncholinergic (NANC) relaxations in the rabbit anococcygeus muscle (RAM) by the use of muscarinic receptor agonists and a battery of key muscarinic antagonists. In addition, experiments were carried out to identify the NANC neurotransmitter(s) involved in the inhibitory NANC neurotransmission. In preparations with histamine-raised tone, the nonselective muscarinic agonists (pD2 values) (+)-muscarine (5.23), cis-dioxolane (5.16), oxotremorine M (4.95) and carbachol (4.06) produced concentration-dependent relaxations corresponding to 72.6-85.0% of the histamine-induced precontraction. In contrast, the subtype-preferring (M1/M4 over M2 and M3 receptors) agonists 4-(3-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343), (S)-4-(dimethylamino)-1-methyl-2-butynyl-N-(3-chlorophenyl)carbamate methobromide [(S)-BN 228], (R)- and (S)-N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide [(R)- and (S)-BM 5] showed no or rather low [(S)-BN 228] muscarinic activity. The low potencies, together with the ineffectiveness of some agonists, indicated a low effective receptor reserve associated with the relaxant response. No contractile responses to (+)-muscarine were observed neither in unstimulated nor in precontracted preparations suggesting that the existence of an excitatory postjunctional muscarinic receptor may be excluded. The nicotinic antagonist hexamethonium had no influence on relaxant responses to (+)-muscarine, but abolished relaxations elicited by (-)-nicotine. This demonstrates that the RAM contains also nicotinic acetylcholine receptors (AchRs) mediating inhibitory NANC responses. Relaxations induced by the stimulation of muscarinic and nicotinic AchRs as well as by electrical field stimulation (EFS) were completely abolished by tetrodotoxin and were also sensitive to the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG), indicating that NO plays an important role as an inhibitory NANC transmitter in RAM. All muscarinic antagonists investigated did not influence the histamine-induced precontraction, but shifted the concentration-response curve of (+)-muscarine to the right in a parallel fashion. Schild analysis yielded regression lines of unit slope, indicating competitive antagonism. The following rank order of antagonist potencies (pA2 values) was found: 11-([4-[4-(diethylamino)-butyl]-1-piperidinyl]-acetyl-5,11-dihydro-6H-py rido (2,3-b) (1,4)-benzodiazepine-6-one hydrochloride (AQ-RA 741; 8.08) = himbacine (8.03) > or = tripitramine (7.69) > or = p-fluorohexahydro-sila-difenidol (p-F-HHSiD; 7.48) > or = methoctramine (7.30) > or = pirenzepine (7.18) > or = guanylpirenzepine (6.24). A comparison of the pA2 values determined in the RAM with published data from binding studies at muscarinic M1-M4 and m5 receptors suggests that the functional muscarinic receptor mediating NANC relaxation in the RAM is of the M4 subtype. Taken together, the results obtained in the present study provide convincing evidence that relaxant responses elicited by muscarinic stimuli in RAM are nitrergic in nature and mediated by muscarinic M4 receptors located somadendritically on NANC neurons. Thus, the isolated RAM may serve as a functional muscarinic M4 receptor model.

Evidence for a role for nitric oxide in relation of the frog oesophageal body to electrical field stimulation.

1. Electrical field stimulation (EFS) (1-10 Hz, 30 V, 2 ms) of frog oesophageal body strips resulted in frequency-dependent non-adrenergic, non-cholinergic (NANC) relaxations. 2. Tetrodotoxin (TTX) (10(-6)-10(-5) M) had no effect on EFS evoked relaxations with a 2 ms pulse width. At a pulse width of 0.5 ms only the responses to the highest frequency (10 Hz) were significantly inhibited by TTX at 10(-5) M. Relaxation at 2 ms pulse width were unaffected by omega-conotoxin (10(-6) M), nifedipine (10(-6) M) or cobalt (5 x 10(-4) M). 3. NG-nitro-L-arginine (L-NOARG) (10(-6)-10(-4) M), a nitric oxide synthase (NOS) inhibitor, caused a concentration-dependent inhibition of the EFS-induced NANC relaxant responses. The inhibitory effect of L-NOARG was both prevented and reversed by L-arginine but not D-arginine (5 x 10(-3) M). 4. The phosphodiester type V inhibitor (PDE V), SK&F 96231 (10(-7)-10(-4) M), caused a concentration-dependent potentiation of both the percentage relaxation and the duration of the relaxant responses to EFS. 5. ODQ (10(-7)-10(-5) M), a guanylate cyclase inhibitor, produced a concentration-dependent inhibition of EFS-evoked NANC relaxations. 6. Oxyhaemoglobin (10(-6) M), which binds nitric oxide (NO), inhibited NANC relaxations to EFS. 7. The NO donor sodium nitroprusside (SNP) (10(-8)-10(-4) M) produced a concentration-dependent inhibition of evoked tone. L-NOARG (10(-4) M) had no effect on the SNP evoked relaxations. Preincubation with oxyhaemoglobin (10(-6) M) caused a reduction in the SNP (10(-6)-10(-5) M) induced relaxations. 8. These results suggest NO is the relaxant transmitter of the frog oesophageal body and the source of NO may be non-neuronal.

Sodium azide induces relaxation of the canine gastric body by activating a guanylate cyclase-dependent pathway.

In order to study the inhibitory mechanism by which sodium azide eliminates smooth muscle contraction in vivo and in vitro, gastrointestinal motility was monitored via chronically implanted force transducers in the stomach and duodenum of conscious dogs. Circular smooth muscle strips with myenteric plexus from the canine gastric body were used for in vitro measurement of isometric tension. In conscious dogs, sodium azide (50 micrograms kg-1, i.v.) abolished both the spontaneously occurring phase III contractions and postprandial motility. Exogenous motilin (100 ng kg-1)- and bethanechol (50 micrograms kg-1)-induced contractions were also abolished by sodium azide. In vitro, sodium azide and electrical field stimulation (EFS) caused a concentration- or frequency-dependent nonadrenergic noncholinergic relaxation in the gastric body strips. The relaxation induced by EFS, but not sodium azide, was abolished by tetrodotoxin. NG-nitro-L-arginine and oxyhaemoglobin failed to attenuate the relaxant effect of sodium azide, but strongly inhibited EFS-induced relaxation. Methylene blue inhibited both sodium azide- and EFS-induced relaxation. cGMP concentrations in muscle strips were markedly increased by sodium azide. These findings indicate that sodium azide induces relaxation in the canine gastric body through a direct action on smooth muscle, by activating a guanylate cyclase-dependent pathway; endogenous NO synthesis does not participate in this inhibitory mechanism.

Involvement of vasoactive intestinal polypeptide in nicotine-induced relaxation of the rat gastric fundus.

1. Nicotine-induced relaxation and release of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-like immunoreactivity (LI) were measured in longitudinal muscle strips from the rat gastric fundus. 2. Under non-cholinergic conditions (0.3 microM atropine), nicotine (3-300 microM) produced concentration-dependent relaxations of the 5-hydroxytryptamine (3 microM)-precontracted strips. Under non-adrenergic non-cholinergic (NANC) conditions (0.3 microM atropine + 1 microM phentolamine + 1 microM nadolol), relaxations induced by sub-maximal nicotine concentrations (10 and 30 microM) were significantly smaller, while that produced by the highest concentration used (300 microM) was similar to that seen under non-cholinergic conditions. 3. Re-exposure to the same nicotine concentration 1 h later induced smaller relaxations, indicating desensitization. The reductions seen in the second responses were proportional to the concentration used. 4. Under non-cholinergic conditions, the relaxant response to 30 microM nicotine was abolished by hexamethonium (100 microM) and significantly reduced by tetrodotoxin (TTX, 3 microM). The TTX-resistant component was not observed under NANC conditions. 5. NANC relaxation induced by 30 microM nicotine was significantly reduced by a specific anti-VIP serum (approximately 35% less than that seen with normal rabbit serum). 6. Nicotine (30-300 microM) caused significant, concentration-dependent increases in the outflow of VIP- and PHI-LI from the strips; these effects were also diminished with re-exposure. The increases in both types of immunoreactivity evoked by nicotine (300 microM) were abolished by hexamethonium (300 microM), TTX (3 microM) and a calcium-free medium. 7. These findings indicate that VIP and possibly PHI are involved in NANC relaxation of the rat gastric fundus induced by nicotine.

Prejunctional alpha sub 2-Adrenoceptors Inhibit Nitrergic Neurotransmission in Horse Penile Resistance Arteries

Purpose To study the influence of alpha-adrenergic stimuli on non-adrenergic non-cholinergic (NANC) neurogenic relaxation in isolated horse penile resistance arteries. Materials and Methods Deep intracavernous penile arteries with an internal lumen diameter of 200-500 micro m., isolated from the corpus cavernosum of young horses, were mounted in microvascular myographs for isometric tension recording and electrical field stimulation (EFS) of autonomic nerve terminals. Results In the presence of guanethidine (10 sup -5 M) and atropine (10 sup -7 M) tone of the arteries was raised by the thromboxane analogue, U46619. EFS (1, 4 and 32 Hz) induced frequency-dependent relaxations, which were abolished in the presence of tetrodotoxin, while N G-nitro-L-arginine (L-NOARG, 10 sup -4 M) abolished the relaxations to EFS at 1 Hz, and significantly reduced the relaxations at 4 Hz and 32 Hz by 82.5 +/- 10.2% and 52.9 +/- 4.7%, respectively (n = 6). EFS induced relaxations of a similar magnitude in penile arteries contracted with U46619 or the alpha 1-adrenoceptor agonist, phenylephrine, while the alpha 2-adrenoceptor agonist, BHT920 (10 sup -6 M), produced an inhibitory effect on the EFS-evoked relaxations which was inversely related to the stimulus frequency (1, 4 and 32 Hz). BHT920 had no effect on the relaxations induced by exogenous nitric oxide (NO), added as acidified sodium nitrite (10 sup -6-10 sup -3 M). The inhibitory effect of BHT920 on NANC relaxations was reversed by 10 sup -7 M rauwolscine. Conclusion These results suggest that the release of a NANC neurotransmitter primarily thought to be NO is inhibited by stimulation of prejunctional alpha 2-adrenoceptors in horse penile resistance arteries.