Nonadrenergic Noncholinergic Inhibitory Responses Research Articles

24: Effect of extrinsic denervation on NANC innervation with substance P and VIP in longitudinal smooth muscle of the rat jejunum

Obligate extrinsic denervation may contribute to enteric motor dysfunction of the transplanted gut. We showed that chronic extrinsic denervation decreased response to exogenous Substance P (Sub P) and abdominal operations transiently increased sensitivity to exogenous Vasoactive intestinal polypeptide (VIP) (Neurogastroenterol Motil 18:779, 2006). Aim: To determine effects of extrinsic denervation mediated by endogenous release of the nonadrenergic, noncholinergic (NANC) neurotransmitters Sub P and VIP. Methods: Jejunal longitudinal muscle strips were obtained from 3 groups of Lewis rats (n ≥ 6/group): NC (naive controls); 8 wks after jejunal and ileal transaction/anastomosis to disrupt enteric myoneuronal continuity (T/A-8), and after syngeneic (no immune rejection) jejunoileal transplantation (SBT-8). Endogenously released Sub P and VIP were studied using electrical field stimulation (EFS; voltage 10V, pulse width 0.5msec, duration 10sec) at 50Hz (excitatory) and 6Hz (inhibitory) frequencies under NANC-conditions (atropine, propranolol, phentolamine) with and without Sub P antagonist ([D-Pro2,D-Trp7,9]-Sub P), VIP antagonist ([D-p-Cl-Phe6,Leu17]-VIP), and the NO-synthase inhibitor L-NNA (L-NG-nitro arginine). Data are Mean±SEM in % of baseline contractile activity or seconds of inhibition. Results: EFS at 50 Hz increased contractile activity in NC but not in T/A-8 and SBT-8 and the Sub P antagonist blocked contractile activity during EFS at 50Hz in NC (146±18 vs. 85±22%; (without vs. with Sub P antagonist); p=0.04), SBT-8 (92±24 vs. 33±14%; p=0.005), but not in T/A-8 (98±43vs. 95±40 %; p=0.92). VIP antagonist had no effect. Inhibition caused by EFS at 50Hz was of longer duration in SBT-8 vs. NC (5.1±0.7 vs. 2.9±0.2sec; p<0.02). Neither Sub P nor VIP antagonist altered duration of inhibition. EFS at 6Hz inhibited contractile activity markedly in all groups (NC: 46±12; T/A-8: 23±12; SBT-8: 29±11%; p<0.02); this inhibition was reversed by L-NNA in all groups (NC: 103±11; T/A-8: 77±22; SBT-8: 66±29%; p<0.02), but not by the VIP antagonist. In the presence of both L-NNA and VIP antagonist, contractile activity at 6Hz was greater in NC compared to T/A-8 and SBT-8 (154±24 vs. 66±18 and 52±20%; p<0.02), while the duration of inhibition was longer in SBT-8 vs. NC (7.2±.0.6 vs. 4.4±0.3sec; p<0.02). Conclusions: Our data suggest that the chronic effects of denervation as induced by T/A and SBT may blunt the release of excitatory NANC neurotransmitters; Sub P release after SBT is preserved. In contrast, changes in VIP signaling are acute and reversible. Changes in the NANC inhibitory response after extrinsic denervation appear to be mediated via NO rather than VIP. These effects to extrinsic denervation may alter enteric motor function after small bowel transplantation.

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea.

Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons. NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method. alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections. In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.

Phosphodiesterase IV and neutral endopeptidase in airways from developing and allergen sensitized rabbits

Phosphodiesterase IV (PDE IV) and neutral endopeptidase (NEP) may modulate the neurally mediated nonadrenergic noncholinergic inhibitory (NANCi) response. This response is not present in normal rabbits until 2 weeks of age. Allergen sensitization and challenge of fully grown 13-week old rabbits decreases the NANCi response. Our goal was to assess NANCi as a function of age and allergen sensitization. Tracheal smooth muscle (TSM) rings from normal 1-, 2-, and 13-week old rabbits plus ragweed immune as well as ragweed immune/challenged (I/C) 13-week old rabbits were assessed. Colorimetric assays of PDE IV and NEP activity were conducted on TSM from each group. NANCi responses were obtained in the presence and absence of Ro 20-1724 (PDE IV inhibitor) and/or phosphoramidon (Phos; NEP inhibitor) after contraction of TSM with neurokinin A. In normal TSM, there was no difference in PDE IV as a function of age. Conversely, NEP decreased significantly from 1 to 13 weeks of age. The immune and I/C groups had decreases in NEP and increases in PDE IV that were significant. Neither Ro 20-1724 nor Phos alone or together increased NANCi responses in TSM from 1- or 2-week old rabbits. However, both enhanced relaxation in TSM from normal, immune, and I/C 13-week old rabbits with an additive effect when drugs were combined. This work demonstrates (1) normal maturational changes in NEP but not PDE IV within TSM of this species; (2) modulation of the NANCi response by inhibitors of PDE IV and NEP in 13- but not 2-week old rabbits; (3) increased PDE IV and decreased NEP levels in the immune and I/C groups with reconstitution of NANCi responses by the combination of inhibitors. We conclude that mediation of the NANCi response is different in normal 2- and 13-week old rabbits. Both PDE IV and NEP modulated relaxation in fully grown rabbits, but had no effect at the younger age. Furthermore, both ragweed sensitization alone and ragweed challenge of immune rabbits altered NANCi via increases in PDE IV and decreases in NEP.

Involvement of purinergic nerves in the NANC inhibitory junction potentials in pigeon oesophageal smooth muscle

1. Electrical field stimulation (EFS) (0.5 ms in train of 2-32 Hz for 300 ms) in smooth muscle of pigeon oesophagus, in the presence of atropine (1 microm) and guanethidine (1 microm), elicited an inhibitory response consisting of a transient hyperpolarization (inhibitory junction potential, IJP) associated with muscle relaxation. 2. Sodium nitroprusside (SNP, 100 microm) induced hyperpolarization correlated to mechanical relaxation. 3. The nitric oxide (NO) synthase inhibitor N(omega)-nitro-l-arginine (from 0.1 to 100 microm) caused a concentration-dependent reduction of electromechanical response to EFS indicating a role for NO in this response. 4. Apamin (1 microm) reduced both IJP and relaxation to EFS but was without effect on the response to SNP indicating a role for purines, which are also blocked by apamin. 5. Adenosine, AMP, ADP and ATP (all from 1 microm to 1 mm) application caused transient hyperpolarization and muscular relaxation with the following order of potency: adenosine > AMP > ADP > ATP. 6. Inhibitory responses evoked by purines are TTX (1 microm) insensitive but they were inhibited by apamin. This indicates that a purine component for the non-adrenergic non-cholinergic (NANC) response exists but the purine receptor site is not located on the neurone. 7. Overall these results suggest that NANC inhibitory response elicited by EFS presents two different components apamin-sensitive, probably purines-mediated and apamin-insensitive probably NO-mediated as apamin only partially block the response to EFS.

Iron deficiency suppresses ileal nitric oxide synthase activity

Intestinal motility disorders are more common in women of childbearing age who are prone to iron deficiency anemia. The neurotransmitters nitric oxide (NO) and acetylcholine (ACh) play a key role in ileal smooth muscle relaxation and contraction, respectively. Iron-containing heme is known to be a cofactor for nitric oxide synthase (NOS), the enzyme responsible for NO production. Therefore we tested the hypothesis that iron deficiency would downregulate ileal NOS activity without affecting the ileum's response to ACh. Twelve adult female prairie dogs were fed either an iron-supplemented (Fe+) (200 ppm) (n = 6) or an iron-deficient (Fe−) (8 ppm) (n = 6) diet for 8 weeks. Ileal circular muscle strips were harvested to measure responses to ACh and electrical field stimulation. Under nonadrenergic noncholinergic (NANC) conditions, Nω-nitro- l-arginine ( l-NNA), an NOS inhibitor, and VIP 10–28, a vasoactive intestinal peptide (VIP) inhibitor, were added prior to electrical field stimulation. NANC inhibitory responses are expressed as a percentage of optimal relaxation from EDTA. The excitatory response to ACh was similar in both groups (1.1 ± 0.3 N/cm 2 vs. 1.5 ± 0.3 N/cm 2, P = 0.45). The inhibitory response to electrical field stimulation under NANC conditions was greater in the Fe+ group (34.7 ± 2.9%) compared to the Fe− group (23.9 ± 3.2%; P<0.01). l-NNA eliminated the inhibitory response in the Fe+ group (0.02 ± 0.02%) but not in the Fe− group (8.38 ± 2.15%; P <0.01). VIP 10–28 led to greater relaxation in the Fe+ animals (45.8 ± 6.6%) than in the Fe− animals (23.4 ± 5.8%; P <0.05). Both l-NNA and VIP 10−28 had no inhibitory response (0.02 ± 0.02%) in the Fe+ animals, whereas the Fe− animals had some residual inhibition (2.54 ± 1.04%; P <0.05). These data suggest that ileal NANC relaxation is due to NOS and that iron deficiency results in (1) decreased NANC relaxation, (2) a compensatory relaxation due to a non-NOS, non-VIP mechanism, and (3) a normal excitatory response. We conclude that iron deficiency suppresses ileal NOS activity.

Iron deficiency suppresses ileal nitric oxide synthase activity

Intestinal motility disorders are more common in women of childbearing age who are prone to iron deficiency anemia. The neurotransmitters nitric oxide (NO) and acetylcholine (ACh) play a key role in ileal smooth muscle relaxation and contraction, respectively. Iron-containing heme is known to be a cofactor for nitric oxide synthase (NOS), the enzyme responsible for NO production. Therefore we tested the hypothesis that iron deficiency would downregulate ileal NOS activity without affecting the ileum’s response to ACh. Twelve adult female prairie dogs were fed either an ironsupplemented (Fe+) (200 ppm) (n = 6) or an iron-deficient (Fe-) (8 ppm) (n = 6) diet for 8 weeks. Ileal circular muscle strips were harvested to measure responses to ACh and electrical field stimulation. Under nonadrenergic noncholinergic (NANC) conditions, Nω-nitro-L-arginine (L-NNA), an NOS inhibitor, and VIP10-28, a vasoactive intestinal peptide (VIP) inhibitor, were added prior to electrical field stimulation. NANC inhibitory responses are expressed as a percentage of optimal relaxation from EDTA. The excitatory response to ACh was similar in both groups (1.1 ± 0.3 N/cm2 vs. 1.5 ± 0.3 N/cm2, P = 0.45). The inhibitory response to electrical field stimulation under NANC conditions was greater in the Fe+ group (34.7 ±2.9%) compared to the Fe-group (23.9 ±3.2%; P <0.01). L-NNA eliminated the inhibitory response in the Fe+ group (0.02 ± 0.02%) but not in the Fe-group (8.38 2 2.15%; P <O.Ol). VIP10-28 led to greater relaxation in the Fe+ animals (45.8 ± 6.6%) than in the Fe-animals (23.4 ±5.8%; P <0.05). Both L-NNA and VIP10-28 had no inhibitory response (0.02 ± 0.02%) in the Fe+ animals, whereas the Fe-animals had some residual inhibition (2.54 ±1.04%; P <0.05). These data suggest that ileal NANC relaxation is due to NOS and that iron deficiency results in (1) decreased NANC relaxation, (2) a compensatory relaxation due to a non-NOS, non-VIP mechanism, and (3) a normal excitatory response. We conclude that iron deficiency suppresses ileal NOS activity.

Characterization of non‐adrenergic, non‐cholinergic inhibitory responses of the isolated guinea‐pig trachea: differences between pre‐ and post‐ganglionic nerve stimulation

1 Differences in the mechanism of non-adrenergic, non-cholinergic (NANC) inhibitory responses to preganglionic- and post-ganglionic nerve stimulation were investigated in the guinea-pig isolated trachea. 2 Stimulation of the vagus nerve at frequencies above 4 Hz elicited NANC relaxation of the trachealis muscle. Responses to low frequencies of stimulation (4-8 Hz) were abolished by the nitric oxide (NO) synthase inhibitor L-NOARG (10 microM), while a L-NOARG resistant component was observed at higher stimulus frequencies. The L-NOARG-resistant component of NANC inhibitory responses to higher frequencies of vagus nerve stimulation were significantly attenuated by the proteinase alpha-chymotrypsin (2 U/ml), suggesting that a neuropeptide such as VIP may contribute to NANC responses. 3 When postganglionic nerves were stimulated by electrical field stimulation (EFS), responses were readily elicited at frequencies below 4 Hz. Like responses to vagus nerve stimulation, responses to low frequency (<4 Hz) EFS were abolished by L-NOARG while a L-NOARG-resistant component was apparent at higher stimulus frequencies. 4 The L-NOARG-resistant component of NANC inhibitory responses to EFS was sensitive to alpha-chymotrypsin only if stimuli were delivered in either long trains at a low frequency (4 Hz for 10-30 s) or short trains of high frequency (16 Hz for 2.5-7.5 s). 5 Responses to preganglionic nerve stimulation were approximately 35% of the amplitude of responses to EFS in the same preparations. 6 In conclusion, responses to preganglionic and postganglionic NANC inhibitory nerve stimulation in the guinea-pig trachea differ in maximum amplitude, frequency-response characteristics and the contributions of cotransmitters. We suggest that these differences may be explained by filtering of preganglionic input to postganglionic NANC neurons. These results have implications in all studies where EFS is considered to be representative of physiological stimulation of post-ganglionic nerve stimulation.

Effects of suramin on electrical and mechanical activities in antrum smooth muscle of the guinea-pig stomach.

The effects of suramin on electrical and mechanical responses produced by adenosine triphosphate (ATP), acetylcholine and transmural nerve stimulation were observed in antrum smooth muscle isolated from the guinea-pig stomach. Suramin (>10(-6) M) inhibited the non-adrenergic non-cholinergic inhibitory junction potential, with no alteration of the resting membrane potential, slow wave and the ATP-induced responses (hyperpolarization and inhibition of slow waves). The amplitude, but not the frequency, of spontaneous rhythmic contraction was inhibited by suramin (>10(-5) M), with no alteration of electrical responses of the membrane. Transmural nerve stimulation elicited cholinergic excitatory and non-adrenergic non-cholinergic inhibitory responses on the spontaneous contraction, and suramin inhibited only the latter. Suramin did not alter the ATP-induced inhibition of spontaneous contraction. The contractions produced by low concentrations (<10(-7) M), but not high concentrations (10(-6) - 10(-5) M), of acetylcholine were inhibited by suramin. It is concluded that in smooth muscle of the guinea-pig antrum, suramin inhibits contractions produced spontaneously and by low concentrations of acetylcholine, with no relation to the electrical responses of the membrane. Parallel inhibition by suramin of the electrical and mechanical responses elicited by excitation of non-adrenergic non-cholinergic inhibitory nerves may not be causally related to the inhibition of ATP-receptors.

Involvement of nitric oxide in the mediation of NANC inhibitory neurotransmission of guinea-pig trachea.

1. The involvement of nitric oxide (NO) in the non-adrenergic non-cholinergic inhibitory (NANC-i) neurotransmission was evaluated in guinea-pigs anaesthetized with chloralose-urethane, using a tracheal pouch preparation. 2. The tracheal pouch, a surgically isolated segment of trachea with intact nerve and blood supply, is an in situ method to demonstrate NANC-i response after complete cholinergic and adrenergic blockade using atropine (5 mg kg(-1)) and propranolol (1 mg kg(-1)), respectively. Cervical vagi and sympathetic trunks were isolated and cut cranially. The distal ends of the vagi were positioned on bipolar electrodes for subsequent stimulation with 5 V pulses for 2 ms duration at 15 Hz for a total of 90 s. The relaxation response was measured as a pressure drop (cm of H2O) in the pouch. Each experimental group was composed of six animals. 3. NANC-i responses to two consecutive nerve stimulations at 25 min apart were reproducible. 4. Pouch relaxation responses to electrical nerve stimulations were determined before and after incubation of the pouch with N(omega)-nitro-L-arginine methyl ester (L-NAME; 10(-5) M), a NO synthase (NOS) inhibitor, for 30 min. L-NAME significantly, but not completely, inhibited the NANC-i response of the pouch, suggesting involvement of NO in the NANC-i neurotransmission. 5. The pouch relaxations to vagal stimulations were inhibited significantly after incubation with oxyHb indicating that NO was released. 6. The amount of methaemoglobin (metHb) formed from oxyhaemoglobin (oxyHb) during vagal stimulation was measured by spectrophotometry. Comparison of the values between the control and after nerve stimulation indicated a trend (P = 0.07) toward greater metHb formation in the pouch perfusate after nerve stimulation. 7. NANC-i responses were not significantly inhibited by incubation of the pouch with either of the guanylate cyclase inhibitors, methylene blue or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). However, a trend toward significance (P < or = 0.07) was observed. 8. This study demonstrated that NO is involved in NANC-i neurotransmission. However, the findings did not conclusively support the contention that NO is the sole neurotransmitter of NANC inhibition. It is possible that NO produced relaxation of guinea-pig trachea through a cGMP-independent mechanism.

Human respiratory syncytial virus produces prolonged alterations of neural control in airways of developing ferrets.

A dysfunction of pathways that normally cause contraction or relaxation of airways has been proposed to explain heightened levels of responsiveness produced by various insults to the airway. For example, we previously reported (4) that infection of cotton rats with the human respiratory syncytial virus (HRSV) leads to a significant decrease in an airway's nonadrenergic noncholinergic inhibitory (NANCi) response shortly after the infection. In the present study we addressed the more chronic effects of HRSV infection on airway function in young ferrets during a period of rapid somatic growth. Animals 1 wk old received HRSV or uninfected cell culture medium intranasally. In vitro studies of airway function were performed on tracheal smooth muscle (TSM) segments at 4, 8, and 24 wk of age. To evaluate neurally mediated contractile responses, frequency-response curves to electrical field stimulation (EFS) were performed with results expressed in terms of the frequency causing 50% of the maximal contractile response (ES50). In addition, contractile responses of TSM to methacholine (MCh) were also assessed with results expressed as the concentration needed to produce 50% of the maximal contractile response (EC50). To gauge NANCi responses, TSM was contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. Relaxant responses to EFS were assessed at frequencies from 5 to 30 Hz, with results expressed as mean percent relaxation. We found increased contractile responses to EFS in infected animals compared with that in the control group in both 4- and 8-wk old animals (p = 0.001 and p = 0.008, respectively). This difference had resolved by 24 wk of age. There was no difference in TSM responses to MCh between the groups at any age. Although there were no NANCi responses in 4-wk-old ferrets from either group, NANCi responses were significantly decreased in 8-wk-old ferrets previously infected with HRSV in the first week of life (p = 0.0001). A significant difference persisted (p = 0.008), albeit to a lesser degree, at 24 wk of age. These findings demonstrate that HRSV produces prolonged alterations of TSM function in ferret airways in vitro.

Effect of aspiration of milk on mechanisms of neural control in the airways of developing rabbits.

We studied the effects of recurrent aspiration of milk on neural control of airways in young developing rabbits. Beginning at 1 week of age, rabbits received 0.5 ml/kg of whole milk or sterile physiologic saline intranasally while under light methoxyflourane anesthesia 5 days a week for a period of 3 weeks. At 4 and 8 weeks of age, in vitro studies of contractile and relaxant responses of tracheal smooth muscle (TSM) segments were evaluated. To assess the neurally mediated contractile responses, frequency response curves to electrical field stimulation (EFS) were performed with results expressed in terms of frequency of EFS causing 50% of the maximal contractile response (ES50) values. In addition, the contractile responsiveness of TSM to methacholine (MCh) as reflected by the concentration causing 50% of the maximal contractile response (EC50) values was also determined to evaluate the underlying cholinergic reactivity of this segment of airway. To assess nonadrenergic noncholinergic inhibitory (NANCi) responses, experiments were performed on TSM contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. EFS was delivered to the contracted tissue at stimulation frequencies ranging from 5 to 30 Hz with results expressed as mean percent relaxation. Recurrent aspiration of milk but not saline increased EFS-induced contractile responses, as shown by significantly lower ES50 values compared with the control group: P = 0.02 and P = 0.001 at 4 and 8 weeks of age, respectively. TSM responsiveness to MCh was no different between the two groups, suggesting that alterations in prejunctional mechanisms of neural control were most likely responsible for the increased contractile response to EFS. The NANCi responses were significantly decreased by milk aspiration at both 4 and 8 weeks of age, with the abnormalities less pronounced at the later time point. These findings demonstrate that repeated aspiration of milk leads to abnormal mechanisms of neural control within airways of developing rabbits. While aspiration of milk altered both contractile and relaxant responses to EFS, the former abnormalities became more pronounced with time while the latter appeared to be resolving. These observations suggest that injury to an airway early in development does not necessarily resolve with time but may persist, with functional abnormalities becoming more pronounced even after the airway insult has ceased.

VIP- and PACAP-mediated nonadrenergic, noncholinergic inhibition in longitudinal muscle of rat distal colon: involvement of activation of charybdotoxin- and apamin-sensitive K+ channels.

1. The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied. 2. An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP6-38, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 microM. 3. PACAP6-38 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIP10-28 at 3 microM, which inhibited the relaxation by 44%. 4. An antagonist of large conductance Ca(2+)-activated K+ channels, charybdotoxin, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM. 5. An antagonist of small conductance Ca(2+)-activated K+ channels, apamin, concentration-dependently inhibited the relaxation (58% at 1 microM). 6. Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS-induced relaxation, which is comparable to the extent of inhibition induced by PACAP6-38 plus VIP10-28. 7. The inhibitory effect of VIP10-28 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6-38 and charybdotoxin, but not apamin. 8. Exogenously added VIP (100 nM 1 microM) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP-induced relaxation VIP10-28, but not PACAP6-38 selectively inhibited the VIP-induced relaxation. 9. Exogenously added PACAP (10-100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP-induced relaxation. PACAP6-38, but not VIP10-28 selectively inhibited the PACAP-induced relaxation. 10. Apamin at 100 nM inhibited inhibitory junction potentials (i.j.ps) induced by a single pulse of EFS Apamin also inhibited a rapid phase, but not a delayed phase of i.j.ps induced by two pulses at 10 Hz. VIP10-28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIP10-28 abolished the i.j.ps induced by two pulses. 11. Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP-, but not VIP-induced hyperpolarization. 12. From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin- and apamin-sensitive K+ channels, respectively.

Human respiratory syncytial virus affects nonadrenergic noncholinergic inhibition in cotton rat airways

A dysfunction of the nonadrenergic noncholinergic inhibitory (NANCi) system has been invoked as a possible mechanism underlying or contributing to altered airway function. In the present study we assessed whether human respiratory syncytial virus (HRSV) infection affects the airways' neurally mediated contractile and relaxant (NANCi) responses in vitro. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from young adult cotton rats, a well-established model for HRSV infection. To assess NANCi responses, TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit, atropine (1 x 10(-6) M) and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation (EFS) was applied at stimulation frequencies ranging from 5 to 30 Hz. The NANCi responses were measured and expressed as the mean (+/- SE) percent relaxation. To evaluate neurally mediated contractile responses, full frequency response curves (0.5-30 Hz) to EFS were also performed. We found significantly decreased NANCi responses in TSM segments obtained from infected cotton rats (n = 12) compared with control animals (n = 9) (P < 0.002). Furthermore, the contractile responses to EFS were increased in infected animals compared with the control group (P = 0.0001). These findings demonstrate that HRSV infection leads to an enhanced contractile response to EFS and a significant decrease in NANCi response in cotton rat airways in vitro. This disruption of the neural control of airways may lead to the development of altered airway function.

Maturation of nonadrenergic noncholinergic inhibitory system in normal and allergen-sensitized rabbits.

We investigated the functional existence of the nonadrenergic noncholinergic inhibitory (NANCi) system in developing rabbit airways in vitro. Furthermore, we evaluated the effect of parenteral exposure to a specific allergen (ragweed) on the maturation of this neural pathway. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from normal and ragweed-sensitized New Zealand White rabbits at 1, 2, 4, and 12 wk of age. The TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit solution, atropine (1 x 10(-5) M), and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation was applied at stimulation frequencies ranging from 5 to 30 Hz to determine the frequency that produced maximal relaxation. The NANCi response to EFS was measured and expressed as the mean (+/- SE) percent relaxation at 20 Hz, because this stimulation frequency gave the maximal NANCi response at each age studied. TSM segments obtained from control rabbits at 1 wk of age did not demonstrate a NANCi response at the frequencies of stimulation used. By contrast, a reproducible NANC relaxation was demonstrated in TSM from 2-, 4-, and 12-wk-old rabbits. The magnitude of this response was 27 +/- 4.7 (n = 10), 29 +/- 4.8 (n = 9), and 37 +/- 4% (n = 18), respectively. The same experiments performed on TSM segments obtained from ragweed-sensitized animals gave significantly decreased values of NANCi response. In 2-, 4-, and 12-wk-old rabbits, the NANCi responses were 11.5 +/- 3.4 (n = 9), 11 +/- 2 (n = 13), and 16 +/- 4.2% (n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of atropine, pirenzepine, clonidine, and morphine on biphasic response of rat gastric fundus to field stimulation

1. Electrically evoked contractions in isolated strips of rat gastric fundus were inhibited by atropine ( IC 50 = 2.5 × 10 −9 M), pirenzepine ( IC 50 = 2.3 × 10 −8 M), clonidine ( IC 50 = 3.9 × 10 −8 M) and morphine ( IC 50 = 3.2 × 10 −7 M) in a dose-dependent manner. 2. The inhibitory effect of morphine was antagonized by naloxone (10 −6 M). The inhibitory effect of clonidine not only was not reversed by yohimbine but also was enhanced. Yohimbine per se inhibited these contractions ( IC 50 = 6.4 × 10 −6 M). 3. In presence of atropine (2 × 10 −6 M) and guanethidine (5 × 10 −6 M), electrical stimulation of isolated strips of rat gastric fundus produced a non-adrenergic, non-cholinergic (NANC) inhibitory response. 4. The NANC inhibitory response was decreased by morphine (10 −8−3 × 10 −6 M). In addition, morphine decreased the tone of the muscle. These effects of morphine was antagonized by naloxone (3 × 10 −6 M). 5. Clonidine up to 10 −6 M had no influence on the NANC inhibitory response but yohimibine per se (10 −7−3 × 10 −5 M) blocked it ( IC 50 = 3 × 10 −6 M). 6. These findings indicate that electrically evoked contractions in the rat gastric fundus were mediated by muscarinic receptors. In addition, the NANC inhibitory response in the isolated 5-trips of rat gastric fundus was blocked by morphine and yohimbine.

Effect of ω-agatoxin-IVA on autonomic neurotransmission

omega-Agatoxin-IVA, a peptide from the venom of the funnel-web spider Agelenopsis aperta and a P type Ca2+ channel inhibitor, was examined for effects on responses to nerve stimulation in isolated autonomic neuroeffector preparations from the rabbit, guinea-pig and rat. Ca(2+)-dependent, tetrodotoxin sensitive, noradrenergic excitatory responses of rabbit pulmonary artery, rat vas deferens, and anococcygeus muscles, and cholinergic guinea-pig myenteric plexus preparations (all highly sensitive to the N type Ca2+ channel inhibitor omega-conotoxin-GVIA) were unaffected by omega-agatoxin-IVA (100 nM). Similarly, the neurogenic response of rat bladder, which has cholinergic, and non-adrenergic non-cholinergic (NANC) excitatory components, and the NANC inhibitory response of rat jejunum (atropine 0.5 microM- and guanethidine 5.0 microM-treated), which are partially sensitive and insensitive to omega-conotoxin-GVIA, respectively, were unaffected by omega-agatoxin-IVA (100 nM). Neurogenic NANC inhibitory responses of the guinea-pig taenia caecum, and rat anococcygeus muscles (atropine- and guanethidine-treated, and tone raised with prostaglandin F2 alpha), were also insensitive to omega-agatoxin-IVA. These results suggest that P type Ca2+ channels, if present, play an insignificant role in supplying the Ca2+ necessary for neurotransmitter release in the peripheral autonomic nervous system.

Decrease in the airways' nonadrenergic noncholinergic inhibitory system in allergen sensitized rabbits

A decrease in the airways' nonadrenergic noncholinergic inhibitory (NANC-i) system is one of the mechanisms that may contribute to allergen-induced changes in neural control within airways. We measured the airways' neurally mediated contractile and relaxant (NANC-i) responses in tracheal segments and left mainstem bronchus (LMB) from normal (control), immune (ragweed sensitized), and immune challenged rabbits. Immune rabbits were sensitized to mixed ragweed extract through parenteral injections from birth, while the immune challenged group had an additional airway exposure to aerosolized ragweed 48 hours prior to the in vitro studies. Neurally mediated contractile responses to electrical field stimulation (EFS) were increased in the immune challenged group, with the increase most significant in tracheal smooth muscle at a stimulation frequency of 20 Hz. To assess NANC-i responses, airway smooth muscle (ASM) segments from these groups were placed in tissue baths containing atropine (10(-6) M) and propranolol (5 x 10(-6) M). After contraction of the tissue with neurokinin A (NKA, 10(-5) M), the NANC-i response to EFS at 20 Hz was measured and reported as the mean (+/- SEM) percent relaxation. No significant differences were seen in the contractile responses of ASM segments to NKA among the three groups. The tracheal segments showed a significantly different NANC-i relaxation response among all groups: in the control group, 29.1 +/- 3.7; in the immune group 15.8 +/- 2.3%; and in the immune challenged group, 2.1 +/- 4.2%.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide as putative nonadrenergic noncholinergic inhibitory transmitter in the opossum sphincter of Oddi

The sphincter of Oddi has a typical nonadrenergic noncholinergic inhibitory innervation; however, the transmitter of this inhibition has not been identified. The aim of the present study was to evaluate whether metabolites of the L-arginine--nitric oxide synthase pathway mediate neural inhibition in the sphincter of Oddi of the opossum. Electrical field stimulation at various frequencies (3, 5, and 10 pulses/s), performed in the presence of guanethidine (10(-6) M) to exclude adrenergic responses, caused a slight, but significant excitatory response of the sphincter of Oddi. The responses were more pronounced at the duodenal side of the sphincter of Oddi than on the hepatic side. When the electrical field stimulation was repeated after blockading muscarinic receptors, using atropine (10(-6) M), a potent inhibitory response was obtained. The inhibitory response to each of the various stimulation parameters was similar. Addition of L-arginine methyl ester (L-NAME, 2 x 10(-4) M) abolished and reversed the inhibitory effect of electrical field stimulation, resulting in a potent stimulatory effect. Higher frequencies (5 and 10 pulses/s) were more potent in causing a stimulatory response than lower frequencies (3 pulses/s). The excitatory effect of electrical field stimulation was blocked or reversed to inhibition when the amino acid L-arginine (2 x 10(-3) M) was added to the bath. In a second series of experiments, the inhibitory effect of electrical field stimulation in the presence of atropine and guanethidine was not prevented after the addition of methylene blue (5 x 10(-5) M), a substance that, in vascular smooth muscle, has been demonstrated to block cyclic GMP dependent inhibitory responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for nitric oxide as an inhibitory neurotransmitter in rabbit isolated anococcygeus

The role of nitric oxide (NO) and ATP as putative inhibitory non-adrenergic, non-cholinergic (NANC) neurotransmitter was investigated in rabbit isolated anococcygeus after block of adrenergic and cholinergic responses, and raising tone with histamine. NANC nerve stimulation produced rapid relaxations which were completely abolished by tetrodotoxin. The magnitude of the NANC inhibitory responses was significantly reduced by the nitric oxide synthase inhibitors N G-nitro-L-arginine (NO-Arg) and N G-nitro-L-arginine methyl ester (L-NAME). This effect could be partially reversed by L-arginine but not by D-arginine. Oxyhaemoglobin inhibited NANC nerve responses and sodium nitroprusside mimicked the effects of NANC nerve stimulation. NO-Arg also reduced the magnitude of the inhibitory junction potentials recorded from the smooth muscle cells during NANC nerve stimulation. Exogenously applied ATP and adenosine each produced concentration dependent relaxations which were unaffected by the NO-synthase inhibitor NO-Arg. Relaxations to adenosine were virtually abolished by the P 1 purinoceptor antagonist 8-(p-sulphophenyl)theophylline. Relaxations to ATP were also significantly reduced, indicating that part of the response to exogenous ATP is due to its breakdown to adenosine and subsequent action on P 1 ourinoceptors. Relaxations of the tissue to ATP and adenosine were unaffected by the P 2 purinoceptor antagonist suramin. NANC nerve mediated responses were not significantly changed by either 8-(p-sulphophenyl)theophylline or suramin. These results suggest that NO is involved in inhibitory NANC neurotransmission in the rabbit isolated anococcygeus, but do not support a role for ATP as a NANC neurotransmitter in this tissue.

Mediators of nonadrenergic, noncholinergic inhibition in the proximal, middle and distal regions of rat colon

1. The mediators of non-adrenergic non-cholinergic (NANC) relaxation of the longitudinal muscle of rat proximal, middle and distal colon were examined in vitro. 2. Electrical transmural stimulation (TMS) of proximal, middle and distal segments of rat colon induced NANC relaxations which were inhibited by tetrodotoxin (1 microM), but not by atropine (1 microM) or guanethidine (4 microM). 3. In the proximal colon, L-nitro-arginine (N5-nitroamidino-L-2,5-diaminopentanoic acid) inhibited the TMS-induced NANC relaxation and L-arginine (1 mM) reversed this inhibition. Nitric oxide (0.3-10 microM) induced relaxation of the proximal segment. 4. NANC relaxation of the proximal segments was still evident after desensitization to vasoactive intestinal peptide (VIP). A VIP antagonist (VIP 10-28, 10 microM) had no effect on the TMS-induced NANC relaxation, which was also resistant to alpha-chymotrypsin (2 units ml-1) and a substance P antagonist ([D-Pro2, D-Trp7,9]substance P, 1 microM). 5. In the middle colon, L-nitro-arginine did not inhibit the TMS-induced NANC relaxation in 6 of 9 preparations tested and partially inhibited the relaxation in the other 3 preparations. L-Arginine did not reverse the partial inhibition. 6. Complete desensitization to VIP was not achieved in the middle colon. The VIP antagonist had no effect on the TMS-induced NANC relaxation. After alpha-chymotrypsin treatment of the segment, desensitization of the segments to substance P, or in the presence of the substance P antagonist, the TMS-induced NANC relaxation was augmented. 7. In the distal colon, L-nitro-arginine did not have any significant effect on the TMS-induced relaxation and nitric oxide did not induce relaxation. The VIP antagonist significantly inhibited TMS-induced NANC relaxation. Alpa-Chymotrypsin-treatment of the distal segments resulted in significant inhibition of NANC relaxation. No desensitization to substance P was achieved. Treatment with the substance P antagonist had no effect. 8. These results suggest that nitric oxide is the mediator of the NANC inhibitory response in the proximal region of rat colon; in the middle colon, substance P acts as an excitatory neurotransmitter, antagonizing the NANC relaxation caused by the mediator of the response, which is still uncertain. Our results suggest that that VIP is the most likely candidate as a NANC transmitter in the distal colon.