The role of nitric oxide in non-adrenergic non-cholinergic relaxation in the guinea-pig gastric fundus

The role of nitric oxide (NO) in non-adrenergic non-cholinergic (NANC) neurotransmission was studied on circular muscle strips of the dorsal part of the guinea-pig gastric fundus. In the presence of atropine and guanethidine, a low frequency of electrical stimulation (1≈10 Hz) induced frequency-dependent relaxation which were not affected by adrenergic and cholinergic blockage but abolished by tetrodotoxin. N^G-nitro-L-arginine (L-NNA), a stereospecific inhibitor of NO-biosynthesis, inhibited the relaxations induced by electrical stimulations but not the relaxations to exogenous nitric oxide. The effect of L-NNA was prevented by L-arginine, the precursor of the NO biosynthesis but not by its enantiomer, D-arginine. Exogenous administration of NO caused concentration-dependent relaxations which showed a similarity to those obtained with electrical stimulation. Hemoglobin, a NO scavenger, abolished the NO-induced relaxations and also markedly reduced those induced by electrical stimulation. The inhibitory effect of hemoglobin was similar to that of L-NNA. Application of ATP caused weak relaxations compared with those to electrical stimulation, which were unaffected by L-NNA. Exogenously applied vasoactive intestinal polypeptide (VIP) induced concentration-dependent relaxation which was not affected by L-NNA. These results suggest that NO is produced and released mainly as a neurotransmitter from enteric neurons during NANC relaxation induced by low frequencies and short trains of electrical stimulation and has a main role in NANC neurotransmission at relaxation induced by these electrical stimulations in the guinea-pig gastric fundus.     

Influence of alpha-chymotrypsin and trypsin on the non-adrenergic non-cholinergic relaxation in the rat gastric fundus.

Relaxations of the rat gastric fundus were induced by electrical stimulation of the non-adrenergic non-cholinergic (NANC) neurones, by vasoactive intestinal polypeptide (VIP), by noradrenaline and by isopropylnoradrenaline. The influence of alpha-chymotrypsin and trypsin thereupon was studied. alpha-Chymotrypsin 2 u ml-1, present for 30 min, antagonized completely the VIP-induced relaxation, but not the stimulation-induced relaxation; alpha-chymotrypsin 10 u ml-1 also partially antagonized the stimulation-induced relaxation. When alpha-chymotrypsin 2 u ml-1 was added after the relaxation had occurred, it antagonized completely the VIP-induced relaxation, but it also partially antagonized the stimulation-induced relaxation. The partial antagonism of the stimulation-induced relaxation was more pronounced with alpha-chymotrypsin 10 u ml-1. Trypsin, 10(-6) M and 3 X 10(-6) M, had effects on VIP- and stimulation-induced relaxations similar to those of alpha-chymotrypsin. The relaxations induced by noradrenaline and isopropylnoradrenaline were not influenced by alpha-chymotrypsin or trypsin, respectively. The results suggest that a peptide, possibly VIP, is involved in the NANC relaxation of the rat gastric fundus.     

Nitric oxide synthase activity and non-adrenergic non-cholinergic relaxation in the rat gastric fundus.

1. In the presence of atropine (1 microM) and guanethidine (5 microM), electrical field stimulation (EFS, 120 mA, 1 ms, 0.5-16.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM)-precontracted longitudinal muscle strips from the rat gastric fundus. 2. L-Citrulline concentrations were measured in the incubation medium of precontracted strips before and after EFS to investigate nitric-oxide (NO) synthase activity and its possible relation to non-adrenergic non-cholinergic (NANC) relaxation. 3. Basal NO synthase activity was reflected by the finding of prestimulation levels of L-citrulline of approximately 30 nM. These levels were unaffected by tetrodotoxin (3 microM) and NG-nitro-D-arginine methyl ester (D-NAME, 100 microM), slightly reduced by a calcium-free medium and halved by NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). 4. EFS evoked significant, frequency-dependent increases in bath levels of L-citrulline at all frequencies tested. The increases evoked by 16-Hz EFS were abolished by tetrodotoxin (3 microM), a calcium-free medium and L-NAME (100 microM) but not by D-NAME (100 microM). 5. L-NAME (0.1 microM-1.0 mM) produced significant reduction of 4-Hz EFS-induced L-citrulline production (100% inhibition at 10 microM), but had less marked effects on basal production (approximately 50% reduction at 100 microM) and 4-Hz EFS-induced NANC relaxation (approximately 50% reduction at 1 mM). 6. L-Arginine (1 mM), but not D-arginine (1 mM), increased basal L-citrulline levels and reversed the inhibitory effect of L-NAME (10 microM). 7. These findings represent clear biochemical evidence of both basal and EFS-stimulated NO synthase activity in the rat gastric fundus.     

Lead effects on non-adrenergic non-cholinergic relaxations in the rat gastric fundus.

The effect of lead exposure on non-adrenergic non-cholinergic (NANC) relaxations in rat gastric fundus was evaluated in this work. Wistar rats were divided into four groups: The control group received tap water and the three other received 0.008% of lead acetate in their drinking water for 15, 30 and 120 days. NANC relaxations induced by electrical field stimulation (0.5-8 Hz, 1 ms, 60 V) of gastric fundus strips was inhibited in all groups treated with lead. The strips from groups, control and 120 days of lead treatment (LEAD 120), were incubated with L-NOARG (100 microM). The presence of this blocker did not produce any additional inhibition. Sodium nitroprusside (10(-10)-10(-6) M) and 8-Br-GMPc (3 x 10(-8)-3 x 10(-4) M) produced dose-dependent relaxations in strips of both groups control and LEAD 120, however, in the LEAD 120, the potencies were significantly reduced from 7.32 +/- 0.05 to 6.40 +/- 0.09 (n = 5) and 4.26 +/- 0.06 to 3.69 +/- 0.05 (n = 5), respectively. Our data suggest that the chronic exposure to lead inhibits NANC relaxations probably by modulating NO release from NANC nerves and/or by interacting with intracellular transducer mechanisms in rat gastric fundus.     

Effect of 3-isobutyl-1-methylxanthine and zaprinast on non-adrenergic non-cholinergic relaxation in the rat gastric fundus

Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) have been proposed as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in the rat gastric fundus. The smooth muscle relaxant actions of VIP and NO are mediated by cAMP and cGMP, respectively; therefore the effect of inhibitors of phosphodiesterases responsible for cyclic nucleotide breakdown on relaxation induced by VIP, NO and electrical field stimulation was investigated. The non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), the cGMP-specific phosphodiesterase inhibitor, zaprinast, the adenylate cyclase activator, forskolin, and the cyclic nucleotide analog, 8-bromo cGMP, produced concentration-dependent relaxation of rat gastric fundus strips precontracted by PGF₂. IBMX potentiated isoprenaline-induced relaxation but not relaxation induced by sodium nitroprusside, VIP, NO or electrical field stimulation. Zaprinast potentiated the relaxation induced by sodium nitroprusside, while having no influence on relaxation due to any other stimulus. The combination of both phosphodiesterase inhibitors did not significantly affect the electrically induced relaxation. It is concluded that both cAMP and cGMP mediate relaxation in the rat gastric fundus. Further research is needed to investigate the role of the cyclic nucleotides during NANC relaxation of this tissue.     

Influence of age on the signal transduction pathway of non-adrenergic non-cholinergic neurotransmitters in the rat gastric fundus.

1. The influence of aging on the relaxant response and the change in cyclic nucleotide content induced by vasoactive intestinal polypeptide (VIP), nitric oxide (NO), electrical field stimulation of the non-adrenergic non-cholinergic neurones and substances acting at different levels of the cyclic AMP and cyclic GMP transduction pathways was studied in longitudinal muscle strips of the rat gastric fundus. 2. The relaxant responses to VIP, sustained electrical stimulation, forskolin and 3-isobutyl-1-methylxanthine were reduced with age, while the responses to dibutyryl cyclic AMP were not. The increase in cyclic AMP content induced by sustained electrical stimulation and forskolin was lower in old rats. 3. The relaxant responses to NO and to short train electrical stimulation were similar in the three age groups. The inhibitory effect of NG-nitro-L-arginine methyl ester on relaxations induced by short train electrical stimulation was more pronounced in old rats. The relaxant responses to sodium nitroprusside (SNP), 8-bromo-cyclic GMP and zaprinast were reduced with age. SNP induced a similar elevation of the cyclic GMP content in the three age groups. 4. These results suggest that aging differentially affects the cyclic AMP and cyclic GMP pathway for relaxation by VIP and NO in the rat gastric fundus, as the defect seems to occur at the level of the adenylate cyclase and cyclic GMP-dependent protein kinase respectively.     

Study of NO and VIP as non-adrenergic non-cholinergic neurotransmitters in the pig gastric fundus.

1. The contribution of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) to non-adrenergic non-cholinergic (NANC) relaxations in the pig gastric fundus was investigated. 2. Circular and longitudinal muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine; tone was raised with 5-hydroxytryptamine. Electrical field stimulation with 10 s trains at 5 min intervals induced responses were abolished by tetrodotoxin. 3. The short-lasting as well as the sustained electrically induced NANC relaxations were significantly reduced by NG-nitro-L-arginine methyl ester (L-NAME). Pretreatment with L-arginine but not D-arginine, prevented the inhibitory effect of L-NAME except for sustained relaxations in the longitudinal muscle strips. 4. Sodium nitroprusside, forskolin, zaprinast and 3-isobutyl-l-methylxanthine induced concentration-dependent relaxations. Exogenous NO mimicked the short-lasting electrically induced relaxations, while endogenous VIP evoked sustained relaxations. The responses to exogenous NO and VIP were not influenced by tetrodotoxin and L-NAME. 5. alpha-Chymotrypsin abolished the responses to exogenous VIP but only moderately reduced NANC relaxations induced by continuous electrical stimulation. Zaprinast potentiated the relaxant responses to sodium nitroprusside and increased the duration of the NANC relaxations induced by electrical stimulation with 10 s trains in circular muscle strips but not in longitudinal muscle strips. 6. The cyclic GMP and cyclic AMP response to electrical stimulation, NO and VIP was measured in circular muscle strips. Short-lasting as well as sustained electrical field stimulation induced an approximately 1.5 fold increase in cyclic GMP content, while NO induced nearly a 40 fold increase. An increase in cyclic AMP content was obtained only with sustained electrical field stimulation. 7. Immunocytochemistry for NO synthase (NOS) revealed immunoreactive neuronal cell bodies in the submucous and myenteric plexuses and nerve fibres in both the circular and longitudinal muscle layer; double-labelling for NOS and VIP showed that VIP coexists in a major part of the intrinsic neurones. NADPH diaphorase-histochemistry showed the same pattern of nitrergic neurones and nerves as NOS-immunocytochemistry. 8. It is concluded that a cyclic GMP- and a cyclic AMP-dependent pathway for relaxation is present in both the circular and longitudinal muscle layer of the pig gastric fundus. NO appears to contribute to short-lasting as well as sustained NANC relaxations. A peptide, possibly VIP, may be involved during sustained stimulation at lower frequencies of stimulation.     

Involvement of peptide histidine isoleucine in non-adrenergic non-cholinergic relaxation of the rat gastric fundus induced by high-frequency neuronal firing

The role of peptide histidine isoleucine (PHI) as a neurotransmitter of the inhibitory motor neurones, the physiological role of PHI and vasoactive intestinal polypeptide (VIP) in the non-adrenergic non-cholinergic (NANC) relaxation and the relative amounts of VIP- and PHI-like immunoreactivity (LI) co-released by neuronal activation were investigated in the rat gastric fundus.Longitudinal muscle strips from the rat gastric fundus precontracted by the thromboxane receptor agonist U46619 (0.1 micro M) were studied in organ baths under conditions of muscarinic receptor blockade by atropine (1 micro M) and adrenergic neurone blockade by guanethidine (5 micro M) ("NANC conditions"). Concentration-response curves were plotted for both amplitude and area under the curve (AUC) of the relaxant responses induced by VIP (0.3 nM-0.3 micro M), PHI (0.3 nM-1 micro M) and peptide histidine valine [PHV(1-42); 0.3 nM-1 micro M]. All three peptides were more potent in the curve based on amplitude than in that based on the AUC of relaxation. In addition, VIP was 5.3 and 7 times more potent than PHI and PHV(1-42), respectively, in producing relaxation expressed as amplitude, and 2.7 and 2.8 times, respectively, in producing relaxation expressed as AUC. PHI and PHV(1-42) behaved as partial agonists with respect to VIP in producing relaxation expressed as AUC. Electrical field stimulation (EFS; 120 mA, 1 ms, 4-32 Hz, pulse trains of 2 min) evoked frequency-dependent relaxant responses. Alpha-chymotrypsin (1 u/ml or 3 u/ml), an anti-VIP serum (1:100 or 1:50) and an anti-PHI serum (1:25), slightly reduced the amplitude, but greatly inhibited the AUC of the NANC relaxation induced by EFS (13 Hz) [approximately 72%, 47% and 28% less than that seen in time controls or with normal rabbit serum (1:100 or 1:25), respectively]. EFS (8-32 Hz) caused significant, frequency-dependent increases in the outflow of VIP- and PHI-LI from the strips. The EFS-induced release of VIP-LI was approximately 20% of the PHI-LI release.These findings indicate that PHI is involved in EFS-induced NANC relaxation of the rat gastric fundus, the major physiological role of VIP and PHI is the maintenance of smooth muscle relaxation and VIP is co-released in equimolar amounts mainly with PHI.     

Effect of LY 83583 on relaxation induced by non-adrenergic non-cholinergic nerve stimulation and exogenous nitric oxide in the rat gastric fundus.

In the rat gastric fundus, the reported cGMP-lowering agent LY 83583 (10(-5) M) inhibited the relaxation induced by nitric oxide (NO), without altering the response to isoprenaline, vasoactive intestinal polypeptide, sodium nitroprusside or electrical field stimulation of inhibitory non-adrenergic non-cholinergic neurones, which are thought to release NO. Incubation with superoxide dismutase partially prevented the effect of LY 83583. When added during a relaxation maintained by continuous NO infusion, LY 83583 reversed the relaxation. It is concluded that LY 83583 inactivates exogenous NO through the generation of superoxide anions.     

Multiple inhibitory mechanisms mediate non-adrenergic non-cholinergic relaxation in the circular muscle of the guinea-pig colon

Summary The mechanisms responsible for nerve-mediated, non-adrenergic, non-cholinergic (NANC) relaxation in mucosa-free circular muscle strips from the proximal colon of the guinea-pig were investigated. Electrical field stimulation (EFS, 1–20 Hz, trains of 5 s duration, 100 V, 0.25 ms pulse width) in the presence of atropine (1 mol/l) and guanethidine (3 mol/l) evoked a triphasic motor response consisting of. (a) a primary relaxation, (b) a rebound contraction and (c) a secondary relaxation. These three responses were abolished by tetrodotoxin (1 mol/l). Both apamin (0.01–0.3 mol/l), a known blocker of low conductance, calcium-activated potassium channels in smooth muscles, and L-nitroarginine (L-NOARG) (1–100 mol/l), a known blocker of nitric oxide (NO) synthase, increased the tone of the strips. Maximum effects on tone were observed with 0.1 mol/l apamin (21 ± 3% of KCl-induced contraction) and 30 mol/l L-NOARG (26 ± 4% of KCl response). The combined administration of 0.1 mol/l apamin and 30 mol/l L-NOARG produced an increase in tone (47 ± 5% of KCl response) that was larger than that produced by either compound alone. Neither apamin (0.1 mol/l) nor L-NOARG (30 mol/l) affected the isoprenaline-induced relaxation.Apamin (0.1 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies without affecting the secondary relaxation. Apamin also enhanced the rebound contraction at a frequency of 1 Hz. L-NOARG (30 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies, had no effect on the rebound contraction and inhibited the secondary relaxation evoked at frequencies of 1–5 Hz, but not 10–20 Hz. L-arginine (300 mol/l) reversed the effect of L-NOARG on tone and the inhibitory effect on the EFS-evoked relaxation. In the presence of apamin and L-NOARG, the primary relaxation was suppressed at all frequencies; the secondary relaxation was inhibited at 1–5 Hz and unchanged at 10–20 Hz, as observed with L-NOARG alone. We conclude that three distinct mechanisms mediate the NANC relaxation of the circular muscle of the proximal colon of the guinea-pig in response to EFS. One mechanism can be operationally defined as apamin-sensitive and a second as L-NOARG-sensitive, the latter implying a possible role of NO as an inhibitory transmitter. A third NANC inhibitory mechanism, which is apamin- and L-NOARG-resistant, is also suggested.Correspondence to: C. A. Maggi at the above address     

Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway

1. Electrical field stimulation (EFS) of guinea-pig isolated main bronchi induced a non-adrenergic non-cholinergic (NANC) contractile response. Nociceptin (0.01–1 μM) significantly inhibited the contractile response to EFS (P<0.01), but not to capsaicin (P>0.05).
2. The μ-, δ- and κ-opioid receptor antagonists, naloxone (0.3 μM), naltrindole (3 μM) and nor-binaltorphimine (1 μM), respectively, did not significantly affect the inhibitory effect of nociceptin (0.03 μM; P>0.05).
3. The novel nociceptin antagonist, [Phe¹ψ(CH₂-NH)Gly²]nociceptin(1–13)NH₂ (0.03–1 μM); the σ ligands, carbetapentane (30 μM), 3-phenylpiperidine (30–100 μM) and (+)-cyclazocine (10–100 μM) significantly reversed the inhibitory effect of nociceptin (0.03 μM, P<0.05). In contrast, rimcazole, did not significantly reverse the inhibitory effect of nociceptin (0.03 μM) at any concentration tested (P>0.05).
4. EFS of guinea-pig bronchial preparations significantly increased SP-LI release above basal SP-LI (P<0.05). In the presence of nociceptin (1 μM), EFS induced a significant increase in SP-LI release above basal SP-LI release (P<0.05). Nociceptin caused a 59±11% (n=5) inhibition of EFS-induced release of SP-LI.
5. Nociceptin reduces the release of sensory neuropeptides induced by EFS, but not capsaicin, from guinea-pig airways. These experiments provide further evidence for a role for nociceptin in regulating the release of sensory neuropeptides in response to EFS.

     

Involvement of nitric oxide in non-adrenergic non-cholinergic relaxation and action of vasoactive intestinal polypeptide in circular muscle strips of the rat gastric fundus.

We examined the characteristics of the non-adrenergic non-cholinergic (NANC) nerve induced relaxation and the possible interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) on the basal tone of the circular muscle of the rat gastric fundus. Electrically induced NANC relaxations were partly inhibited by N(omega)-nitro-L-arginine (100 microM), whereas sodium nitroprusside (SNP; 10 microM) and VIP (5 nM) induced relaxations were not affected. 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; 5 microM) also inhibited the responses to electrical stimuli to a similar extent as N(omega)-nitro-L-arginine but not VIP. However, AMT plus N(omega)-nitro-L-arginine did not give an additional inhibition above that of each drug alone on NANC relaxations, and dexamethasone (10 microM) had no effect on NANC nerve induced relaxations. 1H-[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM), a selective inhibitor of guanylate cyclase, abolished the responses to NANC nerve stimulation and SNP, while VIP responses were not influenced. N-ethylmaleimide (100 microM), an adenylate cyclase inhibitor, attenuated relaxations to NANC nerve stimulation, VIP and isoproterenol (1 nM), while having no effect on those to SNP, but in combination with N(omega)-nitro-L-arginine, there was no additional inhibition on the responses to nerve stimulation. Alpha-chymotrypsin (10 u ml(-1)) severely diminished VIP induced relaxations, but did not reduce electrical responses. In conclusion, these results suggest that NO is involved in the relaxations induced by short-term electrical stimulation. However, another possible unidentified transmitter that can trigger the accumulation of cyclic GMP is not entirely ruled out and there is no interaction between NO and VIP in the circular muscle strip of the rat gastric fundus, even in the basal state of the tissue.     

Neither morphine nor clonidine influence the non-adrenergic, non-cholinergic inhibitory response in the rat gastric fundus

Morphine and clonidine are among the few agents reported to modulate non-adrenergic, non-cholinergic (NANC) responses in different tissues. They were therefore tested for their effect on the NANC inhibitory response in the rat gastric fundus. Neither morphine (10(-5) M), nor clonidine (10(-5) M in the presence of prazosin 10(-6) M to avoid its own relaxatory effect) modified the NANC response in the rat gastric fundus.     

Neurotransmitters of the non-adrenergic non-cholinergic relaxation of proximal stomach

The proximal third of the stomach (fundus plus oral corpus) relaxes during swallowing so that it can hold large amounts of food with limited increases in intraluminal pressure. This mechanism has been called "receptive relaxation" and is mediated by a vago-vagal reflex. When the food bolus reaches the stomach, gastric relaxation is maintained by another reflex starting from mechanoreceptors in the gastric wall. This second mechanism has been named "adaptive relaxation" or "gastric accommodation" and involves both intramural and vagal reflex pathways, whose inhibitory neurons are always intramural. There was initially a great deal of controversy about the identity of the neurotransmitter/s released by inhibitory neurons, but at present nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are considered to be the most likely candidates. Several lines of evidence indicate that adenosine triphosphate (ATP) might be implicated too. It seems that these neurotransmitters are co-released from the inhibitory motor neurons and are responsible for the different features of the NANC relaxation induced by low- or high-frequency neuronal firing. NO (and perhaps ATP) would be responsible for the rapid beginning and the initial rapid development of the relaxation evoked by neuronal firing at low- or high-frequency and VIP for the long duration of the relaxation evoked by high-frequency neuronal activation. This review will deal mainly with the physiological characteristics and pharmacological features of the NANC relaxation of the proximal stomach and the evidences favoring or excluding a role as inhibitory neurotransmitters of ATP, NO and VIP in different species.     

Effect of diabetes on relaxations to non-adrenergic, non-cholinergic nerve stimulation in longitudinal muscle of the rat gastric fundus.

1. The effect of 8-week streptozotocin-induced diabetes has been examined on relaxations to non-adrenergic, non-cholinergic (NANC) nerve stimulation in longitudinal strips of rat gastric fundus. 2. In the presence of noradrenergic and cholinergic blockade and raised tissue tone, electrical field stimulation (0.5-4 Hz, 30 s trains) induced frequency-dependent relaxations that were significantly smaller in gastric fundus strips from diabetic rats than in strips from control rats. 3. NG-nitro-L-arginine methyl ester (NAME, 100 microM) significantly reduced NANC relaxations in muscle strips from both control and diabetic rats, but the reduction was greater in muscle strips from diabetic rats than in those from control rats at frequencies of 2 and 4 Hz. alpha-Chymotrypsin (1 u ml-1) slightly reduced relaxations to nerve stimulation in muscle strips from both control and diabetic rats. 4. The duration of NANC nerve relaxations (1-4 Hz, 30 s trains) was smaller in muscle strips from diabetic rats than in those from control rats. The duration of NANC relaxations was reduced by alpha-chymotrypsin (1 u ml-1) in muscle strips from control rats but not in muscle strips from diabetic rats. 5. Relaxations to both nitric oxide (NO; 1-30 microM) and vasoactive intestinal polypeptide (VIP; 0.1-30 microM) were concentration-dependent and did not differ between muscle strips from control and diabetic rats. 6. The results suggest that streptozotocin-induced diabetes impairs relaxations to NANC nerve stimulation in the rat gastric fundus, which are largely mediated by NO and to a lesser extent by VIP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that adenosine 5'-triphosphate is the third inhibitory non-adrenergic non-cholinergic neurotransmitter in the rat gastric fundus

In the rat gastric fundus, non-adrenergic, non-cholinergic (NANC) relaxations are mediated by nitric oxide (NO), vasoactive intestinal polypeptide (VIP), and a third, as yet unidentified, neurotransmitter. The possible involvement of adenosine 5'-triphosphate (ATP) in the NANC relaxations was examined using pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (PPADS), apamin and desensitization to alpha,beta-methylene ATP. NANC responses were studied in the absence and presence of N(G)-nitro-L-arginine methyl ester (NAME; 100 microM) and alpha-chymotrypsin (1 u ml(-1)), to inhibit responses to NO and VIP, respectively. PPADS (100 microM), apamin (1 microM) and desensitization to alpha,beta-methylene ATP (10 microM, three additions) all significantly (P<0.05) reduced NANC relaxations to electrical field stimulation (0.5 - 4 Hz, 30 s trains) in longitudinal strips of rat gastric fundus and almost abolished the residual relaxation remaining in the presence of NAME and alpha-chymotrypsin. PPADS had no effect on responses to the NO-donor, sodium nitroprusside (SNP), or VIP. Apamin slightly reduced relaxations to SNP, but did not affect those to VIP, whereas desensitization to alpha,beta-methylene ATP markedly reduced responses to both SNP and VIP. The effects of PPADS and apamin in this study provide strong evidence that the third inhibitory NANC neurotransmitter in the rat gastric fundus is ATP.     

Comparison of the effect of vasoactive intestinal polypeptide and non-adrenergic non-cholinergic neurone stimulation in the cat gastric fundus

The possible involvement of vasoactive intestinal polypeptide (VIP) in the non-adrenergic non-cholinergic (NANC) relaxation of the cat gastric fundus was studied in circular and longitudinal muscle strips. Cumulative transmural stimulation induced a frequency-dependent relaxation, while cumulative administration of VIP induced a concentration-dependent relaxation. Tetrodotoxin almost completely antagonized the relaxation induced by transmural stimulation (1 Hz), but did not influence the relaxation induced by VIP (10(-7) M); the latter was not influenced by hexamethonium or propranolol plus phentolamine. Trypsin (30 min incubation) and VIP antiserum (1 h incubation) prevented the relaxation induced by VIP and reduced that induced by transmural stimulation, but did not influence the relaxation induced by isopropylnoradrenaline. Two putative VIP receptor antagonists, [AcTyr1]hGRF-(1-40)OH and [4Cl-D-Phe6,Leu17]VIP, did not influence the relaxation induced by VIP or transmural stimulation. These results are compatible with the hypothesis that VIP is involved in the NANC relaxation of the cat gastric fundus, although participation of a non-VIP component cannot be excluded.     

Adenosine modulation of non-adrenergic non-cholinergic neurotransmission in isolated guinea-pig atria

Summary Transmural stimulation of non-adrenergic, non-cholinergic sensory nerves in guinea-pig atria, isolated from reserpine-pretreated animals, in the presence of atropine and the beta-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. Adenosine (0.1–10 M) concentration-dependently reduced the cardic response to transmural nerve stimulation, without modifying the response to exogenous calcitonin gene-related peptide; the inhibitory effect of adenosine was antagonized by 1 M 8-phenyltheophylline. Moreover, the cardiac response to field stimulation was enhanced by 8-phenyltheophylline (0.1, 1 M) and by adenosine deaminase (1 g/ml), but was reduced by dipyridamole (1 M). These findings indicate the presence of inhibitory adenosine receptors on cardiac sensory nerves and suggest a modulatory effect of endogenous adenosine on cardiac non-adrenergic, non-cholinergic neurotransmission.Send offprint requests to A. Rubino at the above address