The role of nitric oxide in cholinergic neurotransmission in rat trachea

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1 Br. J. Pharmacol. (I 993), 11, Br. J. Pharmacol. (1993), 11, '." Macmillan Press Ltd, 1993 The role of nitric oxide in cholinergic neurotransmission in rat trachea Kiyohisa Sekizawa, Takeyasu Fukushima, *Yasushi Ikarashi, *Yuji Maruyama & 'Hidetada Sasaki Department of Geriatric Medicine, Tohoku University School of Medicine, Sendai 98 and *Department of Neuropsychopharmacology (Tsumura), Gunma University School of Medicine, Maebashi 371, Japan 1 We have investigated the role of nitric oxide (NO) in cholinergic contraction in rat trachea. 2 Methylene blue (1O nm to 3 flm) potentiated cholinergic contraction induced by electrical field stimulation (FS) at 5 Hz in a concentration-dependent fashion. At a concentration of 3 i1m, methylene blue decreased responses to log FS frequency, producing 5% of maximum contraction from a control value of.74 ±.9 Hz to.3 ±.5 Hz without a significant effect on concentration-response curves to acetylcholine (ACh). 3 NG-monomethyl-L-arginine (L-NMMA; 1 1AM) also potentiated cholinergic contraction induced by FS at 5 Hz (131.5 ± 4.6% of control) without having any effect against ACh (3 jim)-induced contractions. Likewise, L-NMMA (111M) significantly increased FS (5 Hz)-evoked release of ACh from tracheal segments into the bath solution (51.4 ± 4. pmol ml-' in the presence of L-NMMA and 35. ± 1.8 pmol ml-' in the absence of L-NMMA, respectively). 4 Administration of NO (present in acidified soluton of NaNO2) (1 nm to 1 tm) and sodium nitroprusside (1 nm to 1 ytm) concentration-dependently reduced FS (5 Hz)-induced cholinergic contractions without having a significant effect on ACh (3 tlm)-induced contractions. These results were unaffected by prior exposure of the tissues to L-NMMA (1 ftm). 5 Dibutyryl cyclic GMP (3 mm) also reduced cholinergic contractions induced by FS at 5 Hz (7.1 ± 3.6% of control) without any significant effect on ACh (3 f1m)-induced contractions. 6 Pretreatment of tissues with capsaicin (3 1LM) or a-chymotrypsin (1 u ml-') failed to inhibit methylene blue (3 JLM)-induced potentiation of responses to FS at 5 Hz. 7 These results suggest that an endogenous NO-like factor may mediate prejunctional inhibition of cholinergic contraction through a cyclic GMP-dependent mechanism in rat trachea. Keywords: Nitric oxide; vasoactive intestinal peptide; non-adrenergic inhibitory nerve; acetylcholine release Introduction There is evidence that nitric oxide (NO) is a mediator of non-adrenergic non-cholinergic (NANC) stimulation-induced relaxations of the anococcygeus muscle of the rat (Li & Rand, 1989; Gillespie et al., 1989) and mouse (Gibson et al., 199) and of rat gastric fundus strips (Li & Rand, 199). In the airway, NO plays a role in NANC relaxation of tracheal smooth muscle in guinea-pigs (Tucker et al., 199; Li & Rand, 1991). Vasoactive intestinal polypeptide (VIP), another candidate for NANC inhibitory neurotransmitters, has been shown to inhibit cholinergic neurotransmission in ferret (Sekizawa et al., 1988), cat (Aikawa et al., 199; Hakoda & Ito, 199; Xie et al., 1991) and dog (Hakoda & Ito, 199; Xie et al., 1991) trachea as well as relaxing airway smooth muscle (Altiere & Diamond, 1984; Palmer et al., 1986). However, the modulatory effect of NO on cholinergic neurotransmission is not known. We have therefore investigated the effects of Nc-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis and methylene blue, an inhibitior of NO-activated guanylate cyclase (Ignarro, 1989), on cholinergic contractions evoked by electrical field stimulation (FS), to determine whether endogenous NO has a modulatory action on cholinergic neurones. We have also investigated the effects of the agonist NO and sodium nitroprusside (SNP) which is known to release NO (Moncada, 1992) on FS-induced cholinergic contraction. ' Author for correspondence. Methods Male Sprague-Dawley rats (Funabashi Farm, Shizuoka, Japan), weighing 25 to 3 g, were anaesthetized with sodium pentobarbitone (5 mg, i.p.). The trachea was removed rapidly and transverse rings (3 mm long) were cut from it. These rings were mounted in organ baths containing 5 ml Krebs- Henseleit solution maintained at 37 C. Isometric tension was continuously measured with a force transducer (TB 612-T, Nihon Koden, Japan) connected to a pen recorder (HORIZ- 8K, San-ei Co., Japan). Tracheal rings were placed between two rectangular platinum electrodes (6 x 4 mm) for FS. These tissues were stretched initially to a tension of 1 g for 3s and thereafter maintained for 6 min under a resting tension of.5 g, which was found to be optimal for measuring changes in tension. FS (supramaximal voltage of 5 V and.5 ms duration) was applied for 3 s. All experiments were carried out in the presence of indomethaciin (1 SAM), propranolol (1 tlm) and phentolamine (1 pm). The contractile responses induced under these conditions were completely abolished by tetrodotoxin (1 jam) or by atropine (1 jam), confirming the neural and cholinergic nature of the response. xperimental protocol To determine the baseline responses, tissues were stimulated electrically five times at 5 Hz at 5 min intervals. We then added increasing concentrations of methylene blue (from 1 nm to 3 jam) and repeated the stimulation 1 min after the administration of each concentration of methylene blue, when the effect of methylene blue on the response to FS reached its maximum. Methylene blue was administered after the preceding contraction had returned to the baseline. In

2 NO AND CHOLINRGIC CONTRACTION 817 preliminary studies, we found that methylene blue caused a concentration-dependent increase in contractions induced by FS at 5 Hz. However, because methylene blue itself changed the baseline tension at concentrations higher than 1 JAM, we studied the effect of 3 JM methylene blue on stimulus frequency-response curves to FS. To determine the effects of methylene blue on stimulus frequency-response curves we stimulated tissues by varying the stimulus frequencies from 1 Hz to 5 Hz. After the first frequency-response curve was completed, we performed the second one 1 min after the administration of methylene blue at a concentration of 3 gm. To determine whether the effects of methylene blue are mediated by postjunctional cholinergic mechanisms, we studied the effects of methylene blue (3 JM) on the cumulative concentration-response curves to acetylcholine (ACh) (from 1 nm to 1 mm). Likewise, we tested the effects of L-NMMA on the contractile response to FS at 5 Hz and ACh (3 JAM), which produced a contractile response equivalent to that produced by FS at 5 Hz. L-NMMA (1 I1M) was added 2 min before the response to FS or ACh was elicited. ffects of the agonists NO (from 1 nm to 1 mm), SNP (from 1 nm to.1 mm) and dibutyryl guanosine 3':5'-cyclic monophosphate (db cyclic GMP, 3 mm) on the contractile responses to FS at 5 Hz and ACh (3 JAM) were also studied. Because ACh (3 JM)-induced contractions required 2 min to reach maximum, NO was added immediately before ACh, but 2 min before the more rapidly developing response to FS. Other agonists were added 1 min before stimulation with either FS or ACh. To evaluate further whether endogenous NO released during FS modified the agonist-induced effects on the contractile response to FS, we tested NO (1 JM), sodium nitroprusside (SNP) (1 JAM) and db cyclic GMP (3 mm) on contractions induced by FS at 5 Hz in the presence of L-NMMA (1 JM). To examine the effect of endogenously released VIP and sensory neuropeptides such as substance P and neurokinin A on FS-induced contractions, we tested the effects of a- chymotrypsin (1 u ml-') and capsaicin (3 jam) on methylene blue (3 gm)-induced potentiating actions on contractions induced by FS at 5 Hz. Tissues were pretreated with a- chymotrypsin for 15 min before the study. In the case of capsaicin, tissues were pretreated for 2 min and were washed out before the study. Measurement of ACh release To examine whether endogenous NO inhibits ACh release from rat trachea, we performed parallel studies on paired rings of the same length (3 mm) from the same animal. One randomly chosen tracheal ring was pretreated with L-NMMA (1 gm) and the other was pretreated with the vehicle of L-NMMA. The tracheal rings were mounted in the 2 ml organ bath and maintained for 6 min in the same condition as in the case of tension measurement. Then, the contents of the bath were emptied, the bath was refilled with buffer (same contents), and either L-NMMA (1 JAM) or the vehicle of L-NMMA was added into the bath. Twenty minutes after the addition of L-NMMA, FS at 5 Hz (5 V and.5 ms duration) was applied for 3 min. All experiments were carried out in the presence of indomethacin (1 JM), propranolol (1 jam), phentolamine (1 JAM) and neostigmine (1 JAM) (Baker et al., 1992). Neostigmine was present to prevent hydrolysis of ACh by endogenous acetylcholinesterase activity. After the end of FS, the incubation solution was stored at - 8 C until analysis. We also examined the release of ACh from tracheal rings (3 mm long) during 3 min incubation without FS. In the analysis, lol of a 2 pm solution of ethylhomocholine (HC), as an internal standard for ACh, was added to 1 ml of the incubation solution and mixed. The mixture was filtered with a.45 JAm-millipore filter and 1 JAl aliquots were subjected to analysis by liquid chromatography according to the procedure previously described (Ikarashi et al., 1984; 1992). The system consisted of a pump, an injector with a 1 JLI sample loop and a work station for the data processing used in conjunction with an amperometric detector with a platinum electrode (IRICA Instrument Inc., Kyoto, Japan). The analytical column was a Bioanalytical Systems Acetylcholine Separation Column (6 mm x 4 mm i.d., 3 JAm, polystyrene-based packing materials, Bioanalytical Systems, BAS, West Lafayette, IN, U.S.A.). An immobilized column (5 mm x 4 mm i.d., BAS) containing acetylcholinesterase (ACh) and choline oxidase (ChO) was used for post-column reaction. A glassy carbon pre-column (IRICA) was used for removing interfering peaks except for targeted compounds. The column temperatures were maintained at 35 C by means of a temperature controller (IRICA). The mobile phase comprised a.5 M phosphate buffer, ph 8.4 containing 1 mm DTA-2Na and.4 mm sodium octanesulphonate. The flow rate was.7 ml min-'. The electrode potential was set at +.5 V relative to a Ag/AgCl reference electrode. The principle of the technique is based upon the separation of HC and ACh in the separation column, followed by their enzymatic conversion through post-column reaction with ACh and ChO to hydrogen peroxide, which is detectable electrochemically by platinum electrode. Under the chromatographic conditions, the retention times of HC and ACh were 5.75 and 1.64 min, respectively. A calibration curve for the determination of ACh is linear between and 8 pmol ml-'. The minimum detection limit in the injected sample was at least.25 pmol for ACh. In preliminary experiments, the addition of indomethacin (1 JAM), propranolol (1 JM), phentolamine (1 JAM), neostigmine (1 JM) or L-NMMA (1 JM) to the Krebs-Henseleit buffer did not create retention peaks. The following drugs were used: tetrodotoxin, atropine sulphate, indomethacin, methylene blue, capsaicin, a-chymotrypsin, vasoactive intestinal peptide, N2, 2'-O-dibutyryl guanosine 3': 5'-cyclic monophosphate, ACh iodide (Sigma), ( ± )-propranolol hydrochloride, sodium nitroprusside dehydrate (Wako Pure Chemicals), phentolamine (Ciba Geigy). NG-monomethyl-L-arginine was kindly given by Dr K. Aisaka (Suntory Limited, Osaka, Japan). HC iodide as IS was synthesized from iodoethane and 3-dimethylamino-1-propanol. Values are expressed as mean ± s.e.mean. Statistical analysis was performed by one way analysis of variance. Significance was accepted at P <.5. Results Methylene blue up to concentrations of 3 JAM did not change the baseline tension. However, methylene blue (from 1 nm to 3 JM) caused a concentration-dependent increase in the contractile response to FS at 5 z (Figure 1). FS frequencyresponse curves were evaluated by mean ( ± s.e.mean) log frequency of the stimulation producing 5% of maximal contraction to FS (S5). FS frequency-response curves were reproducible and S5 did not differ significantly between the first and second frequency-response curves in the absence of methylene blue (.74 ±.7 Hz vs..75 ±.1 Hz; P <.5, n = 5). However, methylene blue (3 JAM) shifted the mean frequency-response curve to the left and significantly reduced S5 (.74 ±.9 Hz vs..3 ±.5 Hz; P<.1, n = 7) (Figure 2a). In contrast to FS-induced contractions, ACh-induced contractions were not affected by methylene blue (3 JM) (- log C5 = 5.45 ±.49 M vs ±.38 M; P<.5, n = 7) (Figure 2b). L-NNMA (1 JAM) significantly increased the contractile response to FS at 5 Hz (131.5 ± 4.6% of control, P<.1, n = 7) but it had no significant effect on the contractile response to exogenously administered ACh (3 JAM) ( % of control, P>.3, n = 7). The amount of acetylcholine in the bath solution was below the detection limit without FS. However, FS at 5 Hz caused a significant

3 818 K. SKIZAWA et al. 2 o 15 CU ** Methylene blue (-log M) Figure 1 Concentration-response curves to methylene blue in the contractile response to electrical field stimulation (FS) at 5 Hz. Data are shown as mean ± s.e.mean of values from 7 rats. Significant differences from control values are indicated by **P<.1 and ***P<.1.,o x C. a 1-I 5 - release of ACh from tracheal segments into the bath solution and this release of ACh was significantly enhanced by L- NMMA (1 flm) (51.4 ± 4. pmol ml-' in the presence of L-NMMA vs. 35. ± 1.8 pmol ml-' in the absence of L- NMMA; P<.1, n=7) (Figure 3). Administration of NO (present in acidified solution of NaNO2) and SNP decreased the amplitude of the contractile responses to both FS at 5 Hz and ACh (3,UM) (Figure 4a,b). The inhibition of each was concentration-dependent. NO and SNP at 1lOM inhibited FS-induced contractions (67. ± 4.8% of control and 72. ± 3.6% of control respectively; P<.1) but did not significantly alter the contractile response of exogenously administered ACh (3 pm) (95.1 ± 4.7% of control and 96.1 ± 3.2% of control, respectively; P>.2). Likewise, inhibitory effects of NO (1O"M) and SNP (1 tim) on FS-induced contractions did not differ significantly in the presence or absence of L-NMMA (1 gm) (71. ± 5.2% of control in the presence of L-NMMA vs. 67.±4.8% of control in the absence of L-NMMA and % of control in the presence of L-NMMA vs. 72. ± 3.6% of control in the absence of L-NMMA, respectively; P>.2). NO at 1 mm and SNP at.1 mm inhibited FS-induced cholinergic contractions (14.6 ± 3.% of control and 51.2 ± 3.1% of control, respectively; P<.1) and ACh-induced contractions (56.8 ± 5.% of control and 79. ± 4.% of control, respectively; P <.1). Db cyclic GMP (3 mm) significantly inhibited the contractile response to FS at 5 Hz both in the presence and absence of L-NMMA (1 tam) (67.2 ± 4.3% of control and 7.1 ± 3.6% of control, respectively; P<.1, n = 5) without affecting ACh (3 lim)-induced contractions (11.8 ± 4.9% of control; P>.5, n = 5). Pretreatment of tissues with either capsaicin (3 JAM) or a-chymotrypsin (1 u ml-) failed to inhibit methylene blue (3 tilm)-induced potentiating responses to FS at 5 Hz (168.2 ± 5.% of control in the absence of treatment, ± 4.9% of control in the presence of capsaicin and ± 7.2% of control in the presence of a-chymotrypsin, respectively, P>.2, n = 5). - I I I I I I I Impulse frequency (Hz) 6- T 1- x 5- C 1- U 'a CZ 3 C.) -I Acetylcholine (-log M) Figure 2 (a) ffects of methylene blue on the frequency-response curve to electrical field stimulation (FS). Data are shown as mean ± s.e.mean of values from 7 rats. FS at 5 Hz caused contractions of 1.2 ±.4 g in control conditions. Methylene blue (3 p.m; ) shifted the control frequency-response curve () to the left. (b) Concentration-response curves to acetylcholine (ACh) in the presence (@) and absence () of methylene blue (3 JAM). Data are shown as mean ± s.e.mean of values from 7 rats. Maximal contraction induced by ACh was 1.1 ±.3 g in control conditions. 3 -A (-).±1 I' L-NMMA (1 FM) Figure 3 lectrical field stimulation (5 Hz)-evoked release of acetylcholine in the presence of N-monomethyl-L-arginine (L-NMMA, 1 JM; solid column) and the vehicle of L-NMMA (open column). Data are shown as mean ± s.e.mean of values from 7 rats. Significant difference between columns is indicated by **P<.1.

4 ol a 1T ~~~~~~~~~~~~ o 5 ) wa~~~~~~~** I\ b NO- (-log M) C\ 75- < Sodium nitroprusside (-log M) Figure 4 (a) Concentration-response curves to nitric oxide (NO, present in acidified solution of NaNO2) in the contractile responses to electrical field stimulation (FS) at 5 Hz () and acetylcholine (3 tim; ). Data are shown as mean ± s.e.mean of values from 7 rats. Significant differences from control values are indicated by *P<.5, **P<.1 and ***P<.1. (b) Concentration-response curves to sodium nitroprusside (SNP) in the contractile responses to electrical field stimulation (FS) at 5 Hz () and acetylcholine (3ytM; ). Data are shown as mean ± s.e.mean of values from 7 rats. Significant differences from control values are indicated by *P<.5, **P<.1 and ***P<.1. Discussion In the presence of adrenoceptor antagonists and indomethacin, FS caused contractions which were abolished by atropine and tetrodotoxin, suggesting that these responses resulted from cholinergic nerve stimulation. Since these contractions were significantly potentiated by L-NMMA, a specific inhibitor of NO production (Palmer et al., 1988; Moncada et al., 1989: Johns et al., 199), inhibition of these contractions was mediated by an NO-like factor. In addition, exogenous administration of NO and SNP, an agent releasing NO (Moncada, 1992) reduced the cholinergic contraction induced by FS. Furthermore, methylene blue, an agent believed to inhibit the activation of NO-activated guanylate cyclase (Martin et al., 1985; Ignarro et al., 1989) potentiated the cholinergic contraction induced by FS, an opposite effect observed to that with the stable cyclic GMP analogue, NO AND CHOLINRGIC CONTRACTION 819 db cyclic GMP. These results indicate the endogenous NOlike factor modulates cholinergic contraction through a cyclic GMP-dependent mechanism. In order to establish whether NO elicits its inhibitory effects prejunctionally on neuronal terminals or postjunctionally on the acetylcholine receptors of the airway smooth muscle, we compared the effects of L-NMMA and methylene blue on the contractile response to FS with the effects that they had on the contractile response to ACh. Both L-NMMA and methylene blue potentiated contractions induced by FS without a significant effect on contractions induced by ACh. Likewise, exogenous NO and SNP in concentrations that did not alter the contractile response to ACh inhibited similar contractions induced by FS. Furthermore, L-NMMA enhanced release of ACh from tracheal segments induced by FS. These results indicate that an endogenous NO-like factor inhibits the release of ACh from cholinergic nerves. Brave et al. (1991) showed that L-NG-nitro-arginine potentiates contractions induced by FS but has no effect on ACh release in the guinea-pig trachea. They suggested that NO released along with ACh during FS acts directly on smooth muscle to produce effects opposite to those of ACh. In the present study, we used the rat trachea and therefore a species difference may explain the difference between the present study and that of Brave et al. (1991). However, the present study cannot rule out the contribution of a postsynaptic relaxant effect of endogenously released NO on FS-induced contractions. Although the precise nature of the NANC inhibitory neurotransmitter in the airway is still debated, VIP is believed to be the most likely candidate. VIP has been shown to inhibit prejunctionally cholinergic contractions in the airway (Sekizawa et al., 1988: Aikawa et al., 199; Hakoda & Ito, 199; Xie et al., 1991). However, a-chymotrypsin which inhibits the relaxant action of VIP in the airway (Li & Rand, 1991) did not alter methylene blue-induced potentiating responses to FS, suggesting that prejunctional inhibition of cholinergic contractions is not mediated via VIP in rat trachea. Likewise, sensory neuropeptides such as substance P and neurokinin A are unlikely to be the mechanism for prejunctional inhibition of cholinergic contractions because pretreatment of tissues with capsaicin failed to alter methylene blue-induced potentiating effects on cholinergic contractions. The source of NO released during FS is unknown in the present study. However, it is possible that NO could be released from nerves, endothelial cells lining blood vessels or other cell types that express NO synthase (Moncada et al., 1991). NO causes relaxation of airway smooth muscle (Tucker et al., 199; Li & Rand, 1991) and pulmonary artery (Liu et al., 1991). The receptor for NO in several tissues and cells including nerves appears to be soluble guanylate cyclase (Rapoport & Murad, 1983; Moncada, 1992). Cyclic GMP is reported to inhibit noradrenaline release from sympathetic nerve endings in canine vascular smooth muscle (Greenberg et al., 199). Our data suggest that the effects of NO on cholinergic nerve endings are also mediated via a cyclic' GMP-dependent mechanism. In conclusion, our study suggests that an endogenous NOlike factor released during FS may mediate prejunctional inhibition of cholinergic contraction through a cyclic GMPdependent mechanism. The authors thank Dr K. Aisaka for gifts of L-NMMA and Mr G. Crittenden for correcting the manuscript. References AIKAWA, T., SKIZAWA, K., ITABASHI, S., SASAKI, H. & TAKI- SHIMA, T. (199). Nonadrenergic inhibitory nerves attenuate neurally mediated contraction in cat bronchi. J. Appl. Physiol., 69, ALTIR, R.J. & DIAMOND, L. (1984). 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