Nonadrenergic Noncholinergic Responses Research Articles

Neuropeptide Y modulates non-adrenergic, non-cholinergic neural bronchoconstriction in vivo and in vitro

Non-adrenergic, non-cholinergic (NANC) neural mechanisms regulate airway tone in guinea-pigs, and it is possible that they may be regulated by other nerves. We have investigated effects of neuropeptide Y (NPY), a co-transmitter of adrenergic nerves, on the excitatory (bronchoconstrictor) NANC (e-NANC) response elicited both in vivo (via bilateral vagal nerve stimulation) and in vitro (via electrical field stimulation [EFS]), and on inhibitory (bronchodilator) NANC (i-NANC) responses in upper trachea elicited by EFS. NPY inhibited the e-NANC to vagal stimulation in vivo in a dose-dependent manner (90.0 ± 3.4% inhibition at 500 ug/kg), but failed to alter the bronchoconstrictor response to exogenous substance P (SP). NPY also inhibited the e-NANC response to EFS in main and hilar bronchi (57.2 ± 8.6% in main and 46.34 ± 7.5% in hilar bronchi at 10 −6M), whilst having no effect on the contractile response to SP. In contrast NPY had no effect on the i-NANC response in vitro. Thus, NPY exerts a powerful inhibitory action on tachykinin release from peripheral endings of capsaicin-sensitive airway sensory nerves, but has no effect on the i-NANC neural mechanisms. This suggests that adrenergic nerves may modulate e-NANC responses in the airways.

Epithelial influence on the tracheal non‐adrenergic inhibitory response

1. The role of epithelium in the non-adrenergic non-cholinergic (NANC) inhibitory response to electrical nerve stimulation as well as to the putative neurotransmitter, vasoactive intestinal polypeptide (VIP), was evaluated in guinea-pigs anaesthetized with chloralose-urethane. 2. The tracheal pouch, an in vivo method to demonstrate the NANC inhibitory response, was used in all experiments. The relaxation response was measured as a pressure drop (cm of H2O) in the pouch. The animals were given atropine (5 mg kg-1) and propranolol (1 mg kg-1) intraperitoneally to block adrenergic and cholinergic responses in the pouch. Cervical vagi were isolated and cut craneally. The distal ends were positioned on bipolar electrodes for subsequent stimulation with 5V pulses of 2 ms duration at 15 Hz for a total of 90 s. 3. The reproducibility of NANC responses to two consecutive nerve stimulations at 20 min apart was determined in group 1. 4. In group 2 pouch relaxations to electrical nerve stimulations were determined before and after complete epithelial denudation (determined by histological and pharmacological methods) of the pouch. 5. To determine the influence of relaxant prostaglandins synthesized by the epithelial cells, the effect of indomethacin (given either intravenously or into the pouch) on the pouch relaxation due to NANC stimulation was studied in groups 3 and 4 respectively. 6. In group 5, in order to distinguish between the effects of epithelium removal and prostaglandins, the animals were pretreated with indomethacin (i.v.) 30 min before the experiment. The pouch relaxation to nerve stimulation was then determined before and after the removal of epithelium. 7. The reproducibility of the pouch relaxations to consecutive single doses of VIP 10(-9) M at 20 min apart was determined in group 6. 8. In group 7, the pouch relaxation to a single dose of VIP was determined before and after intravenous indomethacin administration, but with the pouch epithelium left intact. 9. The effect of epithelium removal on VIP-induced pouch relaxation was determined in group 8. VIP-induced response was determined before and after the epithelium removal. 10. The study showed that both epithelium removal and indomethacin administration independently had significant effects on the decrease of the pouch relaxation (NANC inhibitory response). However, epithelium removal significantly diminished pouch relaxation despite indomethacin pretreatment suggesting a second, non-arachidonic dependent, mechanism (possibly via a relaxing factor) whereby epithelium maintains airway homeostasis.(ABSTRACT TRUNCATED AT 400 WORDS)

L‐NG‐nitro‐arginine and its methyl ester are potent inhibitors of non‐adrenergic, non‐cholinergic transmission in the rat anococcygeus

1. The effects of L-NG-nitro-arginine (L-NOARG) and some other arginine analogues on non-adrenergic, non-cholinergic (NANC) relaxations of the rat anococcygeus muscle were investigated. 2. L-NOARG (5-200 microM) produced concentration-related inhibition of the NANC response; 100 microM L-NOARG produced 90% inhibition. 3. L-Arginine (5-200 microM) produced a concentration-related reversal of the inhibitory effect of 20 microM L-NOARG; a five fold excess of L-arginine (100 microM) was required to obtain the maximum reversal of 90%. D-Arginine (100 microM) produced no such reversal, but significant reversal was produced by L-citrulline, L-arginine-L-aspartate, L-homoarginine and L-arginine-methyl-ester (all at 100 microM). 4. L-NG-nitro-arginine-methyl-ester (L-NAME; 5-200 microM) also reduced NANC relaxations, with a potency similar to that of L-NOARG; both L-NOARG and L-NAME were some ten times more potent than L-NG-monomethyl-arginine (L-NMMA). Like L-NOARG, the effects of L-NAME (20 microM) were reversed by 100 microM L- but not D-arginine. 5. Neither L-NOARG nor L-NAME (both 20 microM) affected submaximal relaxations induced by 10 microM sodium nitroprusside or 20 microM hydroxylamine. 6. D-NOARG, L-NG-tosyl-arginine and L-N alpha-(t-butyl-oxycarbonyl)-NG-nitro-arginine (all at 100 microM) had no effect on NANC relaxations. 7. Thus, in the rat anococcygeus, L-NOARG and L-NAME are more potent than L-NMMA as prejunctional inhibitors of NANC transmission. The reversibility of the effect of L-NOARG by arginine analogues suggests that the NANC system of the anococcygeus shows similarities to the endogenous nitrate system recently described in the brain.

Modulation of cholinergic neurotransmission in guinea‐pig airways by opioids

1. Opioid receptors have been localised on sensory fibres in the vagus nerve and opioids have previously been shown to inhibit non-adrenergic, non-cholinergic (NANC) neurotransmission in guinea-pig bronchi in vitro and in vivo. We have now investigated whether an inhibitory effect could be demonstrated on cholinergic neurotransmission. 2. Electrical field stimulation (EFS) (8 Hz, 0.5 ms, 40 V for 20 s) produced only a rapid, cholinergic response in the upper trachea but in the lower trachea and main bronchi a cholinergic response which was atropine-sensitive and a longer lasting NANC contraction that was atropine-insensitive was demonstrated. This slow contraction could be blocked by tetrodotoxin and capsaicin pretreatment. 3. [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO), a selective mu-opioid receptor agonist, inhibited the cholinergic response to EFS at 8 Hz in a dose-dependent manner in main bronchi (IC50 = 113 nM with a maximal inhibition of 35.7 +/- 5.6% 10 microM, n = 5). In the lower trachea, DAMGO inhibited the cholinergic response to a similar extent (inhibition of 35.8 +/- 3.5% at 10 microM, n = 5). However, DAMGO had no effect on the contractile response to exogenously applied acetylcholine in the main bronchi. By contrast, opioids had no inhibitory effect on cholinergic neurotransmission in the upper trachea. DAMGO (1 microM) inhibited the cholinergic response to EFS in a frequency-dependent manner in the main bronchi with greater inhibition at lower frequencies of stimulation. 4. The delta-opioid receptor agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) significantly inhibited the cholinergic component of the constrictor response to EFS at 8 Hz in the bronchi but at the highest dose used (10 microM). U-50,488H, a Kappa-receptor agonist, had no inhibitory effect on the cholinergic constrictor component in the main bronchi (10microM). 5. DAMGO also inhibited the NANC responses to EFS in the main bronchi in a dose-dependent manner (with an IC50 = 36 nm and a maximal inhibition of 63.4 + 8.3%, at 1 microM, n = 5). DAMGO had no effect on contractile responses to exogenously applied substance P (SP). DPDPE (10 microM) was less effective in inhibition of the NANC bronchoconstriction with a maximal inhibition of 29.2 + 4.2% (n = 7), and U-50,488H (1O microM) had no inhibitory effect. 6. After capsaicin pretreatment, which depleted sensory nerves of neuropeptides, the inhibitory effect of DAMGO (1 microM) on cholinergic constriction in main bronchi at 8 Hz was only 13.4 + 1.9% (n = 13) compared with 32.9 + 4.0% (n = 9) inhibition in vehicle-treated controls (P < 0.001). 7. Opioids may reduce the cholinergic neural responses in airways partly via an inhibitory action on excitatory NANC nerves and partly by a direct effect on cholinergic neurotransmission. The opioid receptor involved is of the mu-opioid receptor subtype.

L‐NG‐monomethyl arginine and l‐NG‐nitro arginine inhibit non‐adrenergic, non‐cholinergic relaxation of the mouse anococcygeus muscle

1. The effects of L-NG-monomethyl arginine (L-NMMA) and L-NG-nitro arginine (L-NOARG) on non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus were investigated. 2. L-NMMA (10-200 microM) produced a concentration-related inhibition of the NANC response; the inhibitory effect of 50 microM L-NMMA was completely reversed by L-arginine but not D-arginine (both 100 microM). 3. L-NOARG (1-50 microM) also produced a concentration-related inhibition of the NANC response and was some 30-50 times more potent than L-NMMA; again, the effects of 10 microM L-NOARG were reversed by 100 microM L-, but not D-, arginine. By itself 100 microM L-arginine did not relax the tissue, but did cause a slight potentiation of the NANC response. 4. Sodium nitroprusside (0.01-10 microM), hydroxylamine (0.1-100 microM), sodium azide (1-100 microM) and nitric oxide (3-120 microM) all relaxed carbachol-induced tone; relaxations to submaximal concentrations of these nitrovasodilators were unaffected by either 50 microM L-NMMA or 10 microM L-NOARG. 5. L-NOARG 10 microM did not inhibit, but rather potentiated, contractions of the mouse anococcygeus due to stimulation of its sympathetic nerves. 6. The inhibitory effects of 10 microM L-NOARG on NANC relaxations were reversed by L-arginine (by 131%), L-citrulline (by 75%), L-arginine methyl ester (by 46%) and L-homoarginine (by 22%), but were unaffected by a variety of other amino acids and their derivatives (all at 100 microM). 7. The results provide strong evidence that NANC relaxations of the mouse anococcygeus are mediated by an endogenous nitrate material, probably derived from L-arginine, and confirm that L-NOARG provides a very useful and potent drug for the investigation of endogenous nitrate function.

The effects of pyrogallol and hydroquinone on the response to NANC nerve stimulation in the rat anococcygeus and the bovine retractor penis muscles

1. The effects of pyrogallol and hydroquinone on the bovine retractor penis (BRP) and rat anococcygeus muscles to non-adrenergic, non-cholinergic (NANC) nerve stimulation have been examined. Both drugs at a concentration of 10(-4) M significantly reduced the response in the rat anococcygeus muscle but had no effect in the BRP muscle. 2. The inhibition of the NANC response in the rat anococcygeus muscle by pyrogallol was completely reversed by superoxide dismutase suggesting it was due to the generation of superoxide anions. 3. Pyrogallol inhibited the response to nitric oxide (NO) in the rat anococcygeus muscle but not that to 3-isobutyl-1-methyl-xanthine (IBMX) which confirmed a selective action. 4. These results suggest that the NANC neurotransmitter in the rat anococcygeus muscle is susceptible to superoxide anions and may be NO or a substance that can liberate NO.

Histamine H 3-receptors modulate nonadrenergic noncholinergic neural bronchoconstriction in guinea-pig in vivo

We have investigated whether the histamine H 3-receptors influence nonadrenergic noncholinergic (NANC) bronchoconstriction in guinea-pig in vivo. Atropine, propranolol, mepyramine and cimetidine were administered to block the effects of β-adrenoceptor-, acetylcholine, H 1- and H 2-receptor-mediated responses, respectively. Vagal stimulation evoked a NANC bronchoconstrictor response. The selective H 3-agonist, α-methylhistamine (α-MeHA, 1–10 mg/kg i.v.) did not alter basal respiratory insufflation pressure, but reduced the NANC bronchoconstrictor response to vagal stimulation in dose-dependent manner (with a maximal inhibition of 46.0 ± 10.3%; mean ± S.E. at 10 mg/kg) (P < 0.02). Histamine itself also had a significant inhibitory effect on NANC responses with H 1- and H 2-blockade. The α-adrenoceptor antagonist phentolamine had no effect on the inhibitory response produced by α-MeHA, but the H 3-receptor antagonist thioperamide blocked the inhibitory effect of α-MeHA. α-MeHA had no inhibitory effect on bronchoconstriction induced by exogenous substance P (5–25 μg/kg i.v.). We conclude that H 3-receptors inhibit the release of transmitter from NANC nerves and that H 3-receptors might play a role in regulation of neurogenic inflammatory responses in the airways.

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. However, the dose of THIP required for this effect was high (3 mg kg- ') and the effect ( <10% inhibition) was small. 6. Substance P (SP; 5upgkg-1 or 25pgkg-1), produced a bronchoconstrictor response equivalent to that produced by NANC vagal stimulation. This response was significantly increased by injection of GABA. Baclofen had no significant effect on responses evoked by exogenous SP. 7. We conclude that GABA inhibits the release of transmitter from NANC nerves via an action at GABAB receptors and that GABA might play a role in the regulation of neurogenic responses in the airways.

Action of histamine on nerve mediated responses of the guinea-pig ileum.

The effect of histamine on the responses of the guinea-pig ileum to stimulation of intramural nerves and to some potential nonadrenergic, noncholinergic (NANC) neurotransmitters was analysed. During sustained tonic histamine contraction, electrical stimulation of all intramural nerves elicited a biphasic response (contraction followed by after-relaxation) and application of ATP and bradykinin caused relaxation of the ileum in contrast to their contractile effect on basal tension. Histamine reduced contractile and augmented relaxatory NANC responses, and prevented capsaicin from producing any contractile effect and from significantly influencing the NANC contractions. The present results suggest that, besides its direct effect, histamine activates intramural nerve fibres, mainly the sensory ones, and unmasks NANC relaxation thus modifying the mechanical activity.

Effects of Capsaicin on Nonadrenergic Noncholinergic Nerves in the Guinea Pig Atria

Transmural nerve stimulation (TNS) in vitro produced positive chronotropic and inotropic responses in the spontaneously beating right atria and electrically driven left atria of guinea pigs, respectively. Both the positive responses of the atria were mediated not only by adrenergic but also by nonadrenergic noncholinergic (NANC) nerves. Numerous calcitonin gene-related peptide (CGRP)-like immunoreactive nerves were found in the sinus node and the muscle layers of both atria of guinea pigs. Exogenously applied CGRP produced positive chronotropic and inotropic responses in a dose-dependent manner. In both the right and left atria, capsaicin (10(-5) M) induced positive chronotropic and inotropic responses. The second application of capsaicin did not cause any responses in the atria, indicating a rapid development of tachyphylaxis. When the tissues developed tachyphylaxis to capsaicin, the intensity of CGRP-like immunoreactivity greatly decreased in the sinus node and in both atria. In the atria that developed tachyphylaxis to capsaicin, the positive chronotropic and inotropic responses to NANC nerve stimulation diminished markedly, but the responses to exogenous CGRP and those attributed to adrenergic nerves were not affected. These results suggest that CGRP is the neurotransmitter of intracardiac NANC nerves and that capsaicin produces positive chronotropic and inotropic responses through the release of CGRP from NANC nerves in the guinea pig atria. It is also suggested that capsaicin depletes endogenous CGRP, thereby accounting for the rapid development of tachyphylaxis to capsaicin and the marked and selective diminution of TNS-induced NANC responses following application of capsaicin.

Calcitonin gene-related peptide as potential neurotransmitter in guinea pig right atrium.

The potential neurotransmitter role of calcitonin gene-related peptide (CGRP) in cardioacceleratory nonadrenergic noncholinergic (NANC) nerves was examined in the guinea pig right atrium in vitro. In the presence of atropine, a muscarinic antagonist, and atenolol, a beta-adrenoceptor antagonist, transmural nerve stimulation (TNS) of the isolated right atrium caused a positive chronotropic response, which is slow in both onset and decay. This TNS-induced slow response was assumed to be mediated by NANC nerves in the right atrium since tetrodotoxin inhibited the response. Dense distribution of CGRP-like immunoreactive (CGRP-I) nerves was demonstrated in the sinus node. Exogenously applied CGRP exerted a positive chronotropic effect on the isolated right atrium in a dose-dependent manner. Both CGRP-I nerves and NANC response induced by TNS were not affected by surgical sympathectomy and reserpine pretreatment but were abolished by the pretreatment of animals with capsaicin. The results suggest that CGRP is the neurotransmitter of cardioacceleratory NANC nerves in the right atrium of the guinea pig.

P-203 - Intramural nerve-mediated inotropic response of the rat and guinea-pig left atria : Demonstration of ∝-adrenergic and nonadrenergic noncholinergic responses

P-203 - Intramural nerve-mediated inotropic response of the rat and guinea-pig left atria : Demonstration of ∝-adrenergic and nonadrenergic noncholinergic responses

Mechanisms underlying electrical and mechanical responses of the bovine retractor penis to inhibitory nerve stimulation and to an inhibitory extract.

The response of the bovine retractor penis (BRP) to stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves and to an inhibitory extract prepared from this muscle have been studied using intracellular microelectrode, sucrose gap and conventional mechanical recording techniques. Both inhibitory nerve stimulation and inhibitory extract hyperpolarized the membrane potential and relaxed spontaneous or guanethidine (3 X 10(-5) M)-induced tone. These effects were accompanied by an increase in membrane resistance. Following membrane potential displacement from an average value of -53 +/- 7 mV (n = 184; Byrne & Muir, 1984) inhibitory potentials to nerve stimulation were abolished at approximately -30 mV; there was no evidence of reversal. Displacement by inward hyperpolarizing current over the range -45 to -60 mV increased the inhibitory response to nerve stimulation and to inhibitory extract; at more negative potential values (above approximately -60 mV) the inhibitory potential decreased and was abolished (approximately -103 mV). There was no evidence of reversal. Removal of [K+]o reversibly reduced hyperpolarization to nerve stimulation and inhibitory extract. No enhancement was observed. Increasing the [K+]o to 20 mM reduced the inhibitory potential to nerve stimulation but this was restored by passive membrane hyperpolarization. Inhibitory potentials were obtained at membrane potential values exceeding that of the estimated EK (-49 mV). [Cl-]o-free or [Cl-]o-deficient solutions reduced and abolished (after some 20-25 min) the hyperpolarization produced by inhibitory nerve stimulation or inhibitory extract. The inhibitory potential amplitude following nerve stimulation was not restored by passive displacement of the membrane potential from -26 to -104 mV approximately. Ouabain (1-5 X 10(-5) M) reduced then (45-60 min later) abolished the inhibitory potential to nerve stimulation. The effects of this drug on the extract were not investigated. It is concluded that the inhibitory response to nerve stimulation and extract in the BRP may involve several ionic species. However, unlike that in gastrointestinal muscles the NANC response in the BRP is accompanied by an increased membrane resistance and does not primarily involve K+. The underlying mechanisms for the inhibitory response to both NANC nerve stimulation and inhibitory extract appear to be similar, compatible with the view that the latter may contain the inhibitory transmitter released from these nerves in this tissue.