The role of cyclic AMP in non-adrenergic non-cholinergic contraction in guinea-pig bronchi.

1. We investigated the role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in non-adrenergic non-cholinergic (NANC) contraction in guinea-pig bronchial strips. 2. Forskolin (3 nM to 1 microM) reduced NANC contraction induced by electrical field stimulation (EFS) in a concentration-dependent fashion (-log EC50 was 7.22 +/- 0.12 M and maximum inhibition was 100 +/- 0.01%). However, forskolin (less than 1 microM) did not alter the contraction induced by substance P (SP, 1 microM). 3. Dibutyryl cyclic AMP (1 mM) also reduced NANC contractions induced by EFS (100 +/- 0.01%) without significant effect on SP (1 microM)-induced contractions. In contrast, dibutyryl cyclic GMP (1 mM) was without effect against either NANC or SP-induced contractions. 4. Both the beta 2-adrenoceptor agonist, procaterol (0.1 nM to 3 nM) and theophylline (100 nM to 1 mM) concentration-dependently reduced EFS-induced NANC contractions without significant effect on SP (1 microM)-induced contractions. 5. In contrast to forskolin, procaterol and theophylline, both sodium nitroprusside and cromakalim inhibited the EFS-induced contractions only at those concentrations that similarly reduced the contractions induced by SP (1 microM). 6. These results suggest that cyclic AMP may mediate pre-junctional inhibition of NANC contractions in guinea-pig bronchi.     

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.

     

Evidence that an atypical beta-adrenoceptor mediates the prejunctional inhibition of non-adrenergic non-cholinergic contraction in guinea-pig bronchi.

We investigated the effect of the putative beta 3 agonist BRL 35135 on non-adrenergic non-cholinergic (NANC) contractions in guinea-pig bronchial strips. BRL 35135 (10(-9) to 10(-6) M) did not alter the baseline tension but reduced NANC contractions induced by electrical field stimulation (EFS) in a concentration-dependent fashion without having a significant effect on the contraction induced by substance P (10(-6) M). BRL 35135 (10(-6) M) also reduced the contraction induced by capsaicin (10(-7) M). Likewise, BRL 37344 (10(-9) to 10(-6) M) reduced NANC contractions induced by EFS in a concentration-dependent fashion. While BRL 37344 up to concentrations of 10(-8) M did not alter the contraction induced by SP (10(-6) M), BRL 37344 (10(-8) M) significantly inhibited NANC contractions induced by EFS and capsaicin (10(-7) M), (P less than 0.01). The inhibitory effect of BRL 35135 (10(-6) M) on NANC contractions induced by EFS was not significantly altered by the non-selective beta-adrenoceptor antagonists, propranolol and pindolol (P greater than 0.10), by the beta 1-selective antagonists, atenolol and metoprolol (P greater than 0.20) (10(-8) to 10(-6) M), or by the alpha-adrenoceptor antagonist, phentolamine (10(-7) to 10(-5) M) (P greater than 0.50). These results suggest that beta 3 agonists exert a prejunctional inhibitory action on NANC contractions.     

Regulation by phosphodiesterase isoenzymes of non-adrenergic non-cholinergic contraction in guinea-pig isolated main bronchus.

1. We have investigated the role of phosphodiesterase isoenzymes in modulating electric field stimulation (EFS), substance P and capsaicin-induced contraction of the guinea-pig isolated main bronchus. 2. Non-adrenergic non-cholinergic contractile responses were elicited by EFS (3 Hz, 20 s) in the guinea-pig isolated main bronchus in the presence of the non-selective muscarinic antagonist, atropine (0.1 microM), the non-selective beta-adrenoceptor antagonist, propranolol (1 microM), the neutral endopeptidase inhibitor, thiorphan (10 microM) and the cyclo-oxygenase inhibitor, indomethacin (5 microM). The type III, type III/IV, type IV and type V phosphodiesterase isoenzyme inhibitor, SKF 94836, benzafentrine, Ro-20-1724 and zaprinast respectively, significantly attenuated the contractile response to EFS. The IC50 (95% confidence limits) value for SKF 94836, benzafentrine, Ro-20-1724 and zaprinast was 8.3 microM (0.89-78); 0.7 microM (0.1-4.5); 0.5 microM (0.2-1.2) and 13 microM (2-87) respectively. 3. The phosphodiesterase isoenzyme inhibitors, SKF 94836, Ro-20-1724 and zaprinast, partially attenuated the contractile response to substance P (10 nM). Benzafentrine significantly inhibited the contractile response to substance P, yielding an IC50 value of 1.9 microM (0.9-3.8). 4. The phosphodiesterase isoenzyme inhibitor, Ro-20-1724 (0.1-100 microM) failed to reduce significantly the contractile potency of capsaicin (P > 0.05). In contrast, SKF 94836 (1 microM), benzafentrine (10 microM) and zaprinast (100 microM) significantly reduced the contractile potency of capsaicin (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin gene-related peptide (CGRP)-enhanced non-adrenergic non-cholinergic contraction of guinea-pig proximal colon.

1. We have investigated the effect of calcitonin gene-related peptide (CGRP) on non-adrenergic, non-cholinergic (NANC) excitatory transmission to the longitudinal muscle of the guinea-pig proximal colon. 2. In the presence of atropine (0.3 microM), guanethidine (5 microM), hexamethonium (100 microM) and indomethacin (3 microM), electrical field stimulation (EFS, 1 Hz, 0.3 ms for 10 s) produced tetrodotoxin-(300 nM)-sensitive contractions which were reduced by the combined administration of FK 888 (10 microM) and MEN 10,376 (0.3 microM), to block tachykinin NK1 and NK2 receptors, respectively. Thus, the EFS-induced NANC contractions are a tachykinin-mediated response. 3. CGRP, at concentrations higher than 0.1 nM, caused an increase in the electrically-evoked, NANC contractions in a concentration-dependent manner and at 10 nM produced a maximal effect (pEC50 = 9.20 +/- 0.17, n = 6). 4. 5-Hydroxytryptamine (5-HT, 1-100 nM) also caused an increase in the EFS-induced NANC contractions in a concentration-dependent manner and at 30 nM produced a maximal effect (pEC50 = 8.06 +/- 0.09, n = 4), but calcitonin (10-100 nM) failed to enhance the EFS-induced NANC responses. Moreover, a 5-HT4 receptor antagonist, DAU 6285 (3 microM) abolished the enhancing action of 5-HT (30 nM). 5. The combined administration of FK 888 (10 microM) plus MEN 10,376 (0.3 microM) abolished the enhancement of EFS-induced NANC contractions by CGRP (10 nM), but DAU 6285 (3 microM) had no effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of NG-nitro-L-arginine on non-adrenergic non-cholinergic relaxation in the guinea-pig gastric fundus.

1. The influence of NG-nitro-L-Arginine (L-NNA) on non-adrenergic non-cholinergic (NANC) relaxations induced by electrical field stimulation was investigated in circular muscle strips of the guinea-pig gastric fundus. 2. In the presence of 10(-6) M atropine and 4 x 10(-6) M guanethidine, electrical field stimulation (40 V, 1 ms, 0.125-16 Hz) with 10 s trains at 5 min intervals induced short-lasting, frequency-dependent relaxations. Continuous stimulation, with cumulative increase of the stimulation frequency, induced sustained frequency-dependent relaxations. Both types of response were abolished by 3 x 10(-6) M tetrodotoxin. 3. L-NNA (10(-5) M and 10(-4) M) concentration-dependently reduced both types of NANC response. Pre-incubation with 2 x 10(-3) M L-arginine prevented the inhibitory action of 10(-5) M L-NNA and partially antagonized that of 10(-4) M L-NNA. D-arginine (2 x 10(-3) M) did not protect against the inhibitory effect of L-NNA. 4. L-NNA did not consistently influence the basal tone of the tissues. L-Arginine and D-arginine likewise did not influence basal tone; they also had no influence on the electrically-induced NANC relaxations. 5. NO (10(-6)-10(-4) M) induced short-lasting concentration-dependent relaxations, while vasoactive intestinal polypeptide (VIP, (10(-9)-10(-7) M) induced more sustained relaxations, that developed at a slower rate. The NO- and VIP-induced relaxations were not influenced by 10(-4) M L-NNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Evidence that tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitation and contraction in the circular muscle of guinea-pig duodenum.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), apamin (0.1 microM) and L-nitroarginine (L-NOARG, 30 microM), electrical field simulation (EFS) produced a nonadrenergic, noncholinergic (NANC) excitatory junctional potential (e.j.p.), action potentials and contraction of the circular muscle of the guinea-pig proximal duodenum, recorded by the single sucrose gap technique. 2. The selective tachykinin (TK) NK1 receptor antagonist, GR 82,334 (30 nM-3 microM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. Similarly, the selective NK2 receptor antagonists, MEN 10,627 (30 nM-3 microM) and GR 94,800 (100 nM-10 microM), both produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. GR 82,334 inhibited the electrical and mechanical NANC responses to EFS in an almost parallel manner, while MEN 10,627 and GR 94,800 were more effective in inhibiting the mechanical than the electrical response to EFS. 3. Activation of the NK1 or NK2 receptor by the selective agonists, [Sar9]substance P (SP) sulphone and [beta Ala8]neurokinin A (NKA) (4-10), respectively (0.3 microM each), produced depolarization, action potentials and contractions. GR 82,334 selectively inhibited the responses to [Sar9]SP sulphone, without affecting the responses to [beta Ala8]NKA (4-10). MEN 10,627 and GR 94,800 inhibited or abolished the responses to [beta Ala8]NKA (4-10), without affecting the responses to [Sar9]SP sulphone. 4. Nifedipine (1 microM) abolished the action potentials and contraction produced either by EFS or by the TK receptor agonists [Sar9]SP sulphone or [beta Ala8]NKA (4-10). 5. In the presence of nifedipine, the NANC e.j.p. produced by EFS was biphasic: in the majority of strips tested (21 out of 29) an early fast phase of depolarization was followed by a second slow component. The combined administration of GR 82,334 and GR 94,800 (3 microM each) reduced both components, the slow phase being inhibited to a greater extent than the fast phase. 6. The P2 purinoreceptor antagonist, suramin (100 microM) reduced the fast phase of the e.j.p. produced by EFS in the presence of nifedipine, without affecting the slow phase. The combined administration of suramin, GR 82,334 and GR 94,800 produced a nearly complete blockade of the e.j.p. produced by EFS in the presence of nifedipine. 7. When tested in the absence of apamin and L-NOARG, EFS induced a NANC inhibitory junction potential (i.j.p.) followed by an e.j.p., and the selective P2Y receptor agonist, adenosine-5'-O-(2-thiodiphosphate) (ADP beta S, 10 microM), produced membrane hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of non-adrenergic, non-cholinergic neural bronchoconstriction in guinea-pig airways via GABAB-receptors.

1. Evidence suggests that gamma-aminobutyric acid (GABA) and its receptors are present in the peripheral nervous system. We have now investigated the effect of GABA and related substances on non-adrenergic, non-cholinergic (NANC) neurally-evoked bronchoconstriction in the anaesthetised guinea-pig. 2. Bilateral vagal stimulation (5 V, 5 ms, 3 or 5 Hz) for 30 s, after propranolol (1 mg kg-1 i.v.) and atropine (1 mg kg-1 i.v.) evoked a NANC bronchoconstrictor response manifest as a mean tracheal pressure rise of 21.9 +/- 1.04 cmH2O (n = 70). The bronchoconstrictor response was reproducible for any given animal. 3. GABA (10 micrograms-10 mg kg-1 i.v.) did not alter basal tracheal pressure but reduced the NANC bronchoconstrictor response to vagal stimulation in a dose-dependent manner (ED50 = 186 micrograms kg-1 with a maximal inhibition of 74 +/- 3.4% at 10 mg kg-1). Neither the opioid antagonist naloxone (1 mg kg-1 i.v.) nor the alpha-adrenoceptor antagonist phentolamine (2.5 mg kg-1 i.v.) had any significant effect on the inhibitory response produced by GABA (500 micrograms kg-1). 4. GABA-induced inhibition was not antagonised by the GABAA-antagonist bicuculline (2 mg kg-1 i.v.). 5. The GABAB-agonist baclofen (10 micrograms-3 mg kg-1 i.v.) caused a dose-dependent inhibition of the NANC response (ED50 = 100 micrograms kg-1 with a maximal inhibition of 35.5 +/- 2.8% at 3 mg kg-1). The GABAA-agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-C] pyridin-3-ol (THIP), also inhibited the NANC bronchoconstrictor response.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-NG-nitro arginine inhibits non-adrenergic, non-cholinergic relaxations of guinea-pig isolated tracheal smooth muscle.

The effects of L-NG-nitro arginine (L-NOARG) on alpha-chymotrypsin-resistant, non-adrenergic, non-cholinergic (NANC) relaxations of guinea-pig tracheal smooth muscle have been examined. L-NOARG (1-100 microM), but not D-NOARG (100 microM), inhibited the NANC relaxations in a concentration-related manner. The effects of L-NOARG were partially reversed by L-arginine but not D-arginine. L-NOARG was without effect on acetylcholine-induced contractile responses of the trachea or on relaxations produced by vasoactive intestinal peptide, sodium nitroprusside or isoprenaline. These results suggest that an endogenous nitrate may contribute to NANC relaxations of tracheal smooth muscle.     

Characterization of histamine-H3 receptors controlling non-adrenergic non-cholinergic contractions of the guinea-pig isolated ileum.

1. In the presence of atropine, mepyramine and ranitidine, electric field stimulation of the guinea-pig isolated ileum longitudinal muscle-myenteric plexus preparation resulted in a two component non-adrenergic non-cholinergic contraction. The initial contraction had a duration of approximately 1 s whereas the second contraction lasted approximately 10 s. The second contraction was completely inhibited by tetrodotoxin (0.2 x 10(-6) M) with minimal effect on the initial contraction. Phentolamine (3 x 10(-6) M), propranolol (3 x 10(-6) M) and hexamethonium (10(-4) M), did not significantly reduce either component of the contractile response. 2. The neurokinin NK1 receptor antagonists, GR82334 and GR71251, produced concentration-related (EC50 = 564 and 173 nM respectively) inhibitions of the second contraction with no effect on the initial contraction. The neurokinin NK2 receptor antagonists MEN 10207 and Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2 (R 396), 1 x 10(-9)-10(-5) M, were without effect on either component of the contractile response. 3. Concentration-related inhibitions of the second contraction, with no effect on the initial contraction, were observed after inclusion of the histamine H3 receptor agonists (R)-alpha-methylhistamine (pD2 = 7.6), N alpha-methylhistamine (pD2 = 7.7) and N alpha,N alpha-dimethylhistamine (pD2 = 6.3). Histamine also inhibited the second contraction (pD2 = 6.2) in a concentration-related manner but produced a lower maximum inhibitory effect than the other agonists tested. 4. Inclusion of the H3 receptor antagonists, thioperamide, burimamide, impromidine and phenylbutanoylhistamine, caused parallel concentration-related rightward shifts in the concentration-response curve to (R)-alpha-methylhistamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Non-adrenergic, non-cholinergic neural activation in guinea-pig bronchi: powerful and frequency-dependent stabilizing effect on tone.

1. We examined non-adrenergic, non-cholinergic (NANC) stimulation for its stabilizing effect on bronchial smooth-muscle tone with respect to its regulatory power and the effect of variations in neural impulse frequency. 2. The guinea-pig isolated main bronchus (n = 4-12) was pretreated with indomethacin (10 microM) and incubated with atropine (1 microM) and guanethidine (10 microM). Electrical field stimulation (EFS: 1200 mA, 0.5 ms, 240 s) was applied at various levels of tone prior to EFS: first without tone, then at a moderate tone induced by histamine (0.3 microM) and, finally, at a high tone induced by histamine (6 microM). Three different stimulation frequencies (1, 3 or 10 Hz) were used in order to produce moderate to near-maximum contractile and relaxant NANC neural responses. Both the contractile and the relaxant NANC responses were tetrodotoxin-sensitive in the guinea-pig isolated main bronchus (3 Hz). 3. Without tone prior to EFS, NANC activation (1, 3 or 10 Hz) induced a pronounced contractile response. At a moderate level of tone prior to EFS, NANC activation induced a less pronounced contractile response. At the highest level of tone prior to EFS, NANC activation induced a relaxant response. All these NANC responses adjusted the tone towards a similar level and this 'stabilization level' was 56(6)% at 1 Hz, 65(3)% at 3 Hz and 56(5)% at 10 Hz, expressed as a percentage of the maximum histamine-induced (0.1 mM) tone in each airway preparation. 4. There was a difference of approximately 90% of maximum between the highest and the lowest tone level prior to NANC activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Conditional involvement of muscarinic M1 receptors in vagally mediated contraction of guinea-pig bronchi

The involvement of ganglionic muscarinic M₁ receptors in vagally induced bronchoconstriction in guinea-pig airways is controversial. Therefore, we studied the effects of the M₁-selective muscarinic receptor antagonist pirenzepine on vagus nerve (VNS, preganglionic) and electrical field stimulation (EFS, postganglionic)-induced contractions of the guinea-pig main bronchus under various experimental conditions.Using identical stimulation parameters for VNS and EFS (8V, 30 Hz, 0.5 ms, 5s every min), the amplitude of the VNS-induced twitch contractions was 30.4% of the EFS-induced responses, and pirenzepine showed 2.3-fold selectivity (pIC₅₀-values 6.45 and 6.09, respectively) to inhibit vagally induced contractions. With the stimulation frequency for EFS lowered to match contraction levels obtained using VNS, pirenzepine was equipotent to inhibit both types of response at M₃ receptor-selective concentrations, suggesting that M₁ receptors are not involved. By contrast, when the stimulation episode was prolonged until plateau contraction (10–20 s), in the presence of the nicotinic antagonist hexamethomum (5 M), the M₂ receptor antagonist AQ-RA 741 (0.1 M) and the -adrenoceptor antagonist timolol (1 M), and again using matched VNS- and EFS-induced contraction levels, pirenzepine inhibited nerve stimulation-evoked responses in a biphasic manner, yielding (pIC₅₀-values of 8.12 indicative of M₁ receptor blockade) and 6.43 (indicative of M₃ receptor blockade) for the first and second phase, respectively, while postganglionic stimulation showed a purely monophasic inhibition (pIC₅₀ = 6.32).These results show that facilitatory muscarinic M₁ receptors are involved in vagally mediated contraction of guinea-pig bronchi, under conditions of elevated neurotransmission and partial nicotinic receptor blockade.     

The non-adrenergic, non-cholinergic response counteracts changes in guinea-pig airway tone with and without sympathetic activation.

1. We examined whether the non-adrenergic, non-cholinergic (NANC) neural response can counteract changes in smooth-muscle tone with and without simultaneous sympathetic activation in guinea-pig airways. 2. Isolated airway preparations were pretreated with indomethacin (10 microM) and incubated with either atropine (1 microM) and guanethidine (10 microM) or atropine (1 microM) alone. The response to electrical field stimulation (EFS: 1200 mA, 0.5 ms, 3 Hz for 240 s) was studied at various levels of tone prior to EFS: first without induced tone, then at a moderate tone induced by histamine (0.3 microM) and finally at a high tone induced by histamine (6 microM). 3. The response to EFS was a contraction when the tone prior to EFS was low and a relaxation when the tone prior to EFS was high. These responses converged towards a similar level of tone, in the distal trachea and in the main bronchus, with and without guanethidine. 4. The mean (s.e. mean) level of tone towards which the responses to EFS converged was lower after incubation with atropine alone compared with incubation with atropine and guanethidine, both in the distal trachea [8 (1)% compared with 30 (6)% of maximum tone] and in the main bronchus [28 (4)% compared with 57 (2)% of maximum tone]. In separate experiments, the guanethidine-induced effect on the responses to EFS was imitated by propranolol (1 microM) but not by prazosin (0.3 microM) and yohimbine (1 microM). 5. These findings indicate that the NANC neural response can counteract changes in airway smooth-muscle tone via a contraction or via a relaxation, depending on the tone prior to activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine receptors involved in the inhibitory control of non-adrenergic non-cholinergic neurotransmission in guinea-pig atria belong to the A1 subtype

Summary We have previously shown that endogenous adenosine inhibits non-adrenergic, non-cholinergic (NANC) neurotransmission in isolated guinea-pig atria. In the present study the effect of adenosine analogues, such as N⁶cyclopentyladenosine (CPA), 5 N-ethylcarboxamide adenosine (NECA), 2 chloroadenosine (2-CADO), R- and S-phenylisopropyladenosine (R- and S-PIA) on the cardiac response to transmural nerve stimulation has been tested in order to characterize the subtype of adenosine receptor involved in the inhibitory control of NANC neurotransmission. The effect of the adenosine antagonist 8-phenyltheophylline (8-PT) was then tested against CPA and NECA.The prototypical A-1 selective agonist CPA was the most active agonist, reducing the response to the stimulation of NANC nerves with an IC₅₀ value of 2.8 nM; RPIA, NECA and 2-CADO showed IC₅₀ values of 9.5, 13.7 and 35 nM respectively. S-PIA was the least active agonist, showing an IC₅₀ value (306 nM) about 30-fold greater than that of R-PIA (9.5 nM). None of the agonists tested was able to modify cardiac response to exogenous CGRR Furthermore, 8-PT competitively antagonized the effect of CPA and NECA with very close pA₂ values (6.77±0.01 and 6.63±0.08 respectively). From these findings we concluded that prejunctional inhibitory adenosine receptors on capsaicin sensitive sensory nerves of cardiac tissue belong to the A-1 subtype. Send offprint requests to A. Rubino at the above address     

Control of non-adrenergic non-cholinergic reflex motor responses in circular muscle of guinea-pig small intestine by Met-enkephalin

1 A triple organ bath method allowing the synchronous recording of the motor activity of the circular muscle layer belonging to the oral and anal segments of guinea-pig small intestine adjacent to an electrically stimulated middle segment was developed to study the ascending and descending reflex motor responses. 2 Electrical field stimulation (0.8 ms, 40 V, 5 Hz, 10 s) applied to the middle part of the segments elicited tetrodotoxin (1 microm)-sensitive ascending and descending contractile responses of the nonstimulated parts, oral and anal, respectively. The ascending contraction was more pronounced as compared with the descending contraction. 3 In the presence of phentolamine (5 microm), propranolol (5 microm) and atropine (3 microm) a significant decrease in the amplitude of the ascending contraction was seen and a descending relaxation, instead of a contraction was observed. 4 Met-enkephalin applied at a single concentration (0.1 microm) or cumulatively (0.001-1 microm) inhibited both non-adrenergic non-cholinergic (NANC) descending relaxation and ascending contraction with similar efficacy but different potency, IC50 being 5.9 +/- 0.3 and 39.0 +/- 4 nm, respectively. Naloxone (0.5 microm) prevented the effects of Met-enkephalin. 5 L-NNA (0.5 mm), an inhibitor of nitric oxide synthesis, increased the ascending contraction and strongly reduced but not abolished the descending relaxation. l-Arginine (0.5 mm) restored the motor responses to the initial level in l-NNA-pretreated preparations, d-Arginine (0.5 nm) had no effects. 6 Met-enkephalin (0.1 microm) depressed the l-NNA-dependent increase of the ascending contraction and failed to change the l-NNA-resistant part of the descending relaxation. 7 Met-enkephalin did not alter spontaneous NANC mechanical activity. SNP (1 or 10 microm), an exogenous donor of nitric oxide, caused a concentration-dependent relaxation. The effects of SNP persisted in Met-enkephalin (0.1 microm)-pretreated preparations. 8 NANC reflex ascending contraction and descending relaxation were synchronously induced by a local nerve stimulation indicating a functional coactivation of NANC orally projected excitatory and anally directed inhibitory pathways. Acting prejunctionally, Met-enkephalin provided a negative controlling mechanism inhibiting both ascending and descending, mainly nitric oxide mediated, reflex responses. A higher sensitivity of the descending relaxation to Met-enkephalin was observed suggesting an essential role of opioid(s) in reducing the efficacy of descending motor activity.     

Evidence that sensory neurons participate in the non-cholinergic, non-adrenergic contractile response of the guinea-pig ileum

The possible role of non-cholinergic, non-adrenergic (NCNA) nerves in responses of the guinea-pig terminal ileum to transmural nerve stimulation (TNS) and that of sensory nerves in NCNA responses were investigated. The action of acetylcholine was almost abolished in the presence of histamine, whereas the contractions elicited by TNS were changed to frequency-dependent contraction followed by a secondary relaxation. Guanethidine did not alter the contractions or secondary relaxations. Atropine abolished the action of acetylcholine and transiently suppressed the responses to low (up to 2 Hz) and attenuated (by about 50%) those to high (4 to 20 Hz) frequency stimulation. The remaining complex NCNA response was the sum of the excitatory and inhibitory responses. During desensitization to capsaicin, and in its presence, the NCNA contractions were reduced, whereas the relaxations were not significantly enlarged. The present results suggest that besides the cholinergic innervation, the excitatory and inhibitory NCNA innervation also participates in the responses of the guinea-pig ileum to TNS even without suppression of cholinergic and adrenergic transmission, and that the sensory nerves are, at least to some extent, involved in the NCNA excitatory response.     

Inhibition of excitatory non-adrenergic non-cholinergic bronchoconstriction in guinea-pig airways in vitro by activation of an atypical 5-HT receptor.

1. The effect of 5-hydroxytryptamine (5-HT) was studied on excitatory neurally mediated non-adrenergic non-cholinergic (NANC) contractions evoked by electrical field stimulation (EFS) in guinea-pig isolated bronchi. 2. 5-HT (0.1-100 microM) produced a concentration-dependent inhibition of the excitatory NANC response with 50.9 +/- 5.0% (n = 5, P < 0.01) inhibition at 100 microM. This inhibition was not significantly affected by the 5-HT2 antagonist, ketanserin (1 microM) when inhibitions (+/- ketanserin) at each concentration of 5-HT were compared by unpaired t tests; however, this concentration appeared to produce a leftward shift (approximately 10 fold) of the 5-HT concentration-inhibition curve. Ketanserin (1 microM) was effective in blocking bronchoconstriction evoked by activation of 5-HT2A receptors on airway smooth muscle. In the presence of ketanserin (1 microM) 5-HT (100 microM) evoked an inhibition of 57.4 +/- 5.9% (n = 5, P < 0.01) with an EC50 of 0.57 microM. 3. Inhibition evoked by 5-HT (0.1-100 microM) was unaffected by the alpha-adrenoceptor antagonist phentolamine (1 microM), the beta 2-adrenoceptor antagonist, ICI 118551 (0.1 microM), the 5-HT1A/B antagonist, cyanopindolol (1 microM) or the 5-HT3/4 antagonist, ICS 205-930 (1 microM). 4. Methiothepin (0.1 microM) produced an insurmountable inhibition of the effect of 5-HT (0.1-100 microM), reducing the maximum inhibition produced by 5-HT (100 microM) to 30.2 +/- 5.0% (n = 5, P < 0.001) and suggesting a non-competitive antagonism. Methiothepin inhibited the effect of 5-HT (10 microM) in a concentration-dependent manner with an IC50 of 81 nM.(ABSTRACT TRUNCATED AT 250 WORDS)