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Contribution of Nitric Oxide and Substance P to Nonadrenergic, Noncholinergic Transmission in the Guinea Pig Ileum
ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00394-7 All rights reserved
Gen. Pharmac. Vol. 31, No. 1, pp. 101–105, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Contribution of Nitric Oxide and Substance P to Nonadrenergic, Noncholinergic Transmission in the Guinea Pig Ileum Chr. Ivancheva,* D. Itzev, I. Lolova and R. Radomirov Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str., bl. 23, 1113 Sofia, Bulgaria [Tel: 1359-2-71-91-08; Fax: 1359-2-71-91-09; E-mail: [email protected]] ABSTRACT. 1. The possible contribution of the nonadrenergic noncholinergic (NANC) transmitters nitric oxide (NO) and substance P (SP) to the contractility of guinea pig isolated ileum was studied by the responses of the longitudinal muscle to electrical field stimulation (0.8 msec, 40 V, 1–20 Hz, 20 sec) of the intrinsic nerves and by the presence and distribution of NADPH-diaphorase- and SPpositive nerve structures in the myenteric plexus. 2. The electrically elicited, tetrodotoxin (0.3 mM)-sensitive responses, in the presence of phentolamine (5 mM), propranol (5 mM), and atropine (3 mM) consisted of relaxation, followed by twitch and tonic contraction on which phasic contractions were superimposed. 3. NG-nitro-L-arginine (L-NNA; 0.1 mM or 0.5 mM), an inhibitor of NO synthesis abolished the relaxation. L-arginine (0.1 mM), a substrate for NO synthesis, but not D-arginine, restored it. L-NNA concentration dependently increased the twitch and tonic contractions. Sodium nitroprusside (1 mM or 10 (M), an exogenous donor of NO, was without effect on the electrically evoked responses. 4. AP 13.2 ACOH (AP; 0.1 mM or 10 mM), a blocker of SP receptors, frequency dependently inhibited or even prevented the twitch and tonic contractions. AP concentration-dependently increased the relaxation or reversed the responses to electrical stimulation into a deep relaxation. 5. The concentration-response curve for SP (1 nM–0.1 mM) was shifted to the right by AP, the EC50 values being 5.260.4 nM and 88.063.0 nM, respectively. The effects of SP were not altered by L-NNA (0.1 mM). 6. These findings, supported by morphological data about distribution of NADPH-diaphorase-positive nerve cell bodies and processes and SP-positive varicose fibers, suggest the contribution of NO and SP to NANC transmission. It appears that NO inhibits prejunctionally the SP transmission whereas SP counteracts the NO effect at the postjunctional level. gen pharmac 31;1:101–105, 1998. 1998 Elsevier Science Inc. KEY WORDS. Nitric oxide, substance P, NANC transmission, ileum contractility
INTRODUCTION The transmitters resistant to adrenergic and cholinergic blockers have been called nonadrenergic, noncholinergic (NANC) transmitters and different NANC nerve-mediated mechanisms have been suggested. Recently evidence has been presented that adenosine-5-triphosphate (ATP) (Burnstock, 1972, 1986; Hole, 1992; Hoyle and Burnstock, 1989), vasoactive intestinal peptide (VIP) (Goyal and Rattan, 1980; Lundberg, 1996), and nitric oxide (NO) (Bult et al., 1990; Moncada et al., 1989; Rand, 1992; Rand and Li, 1995; Shuttleworth et al., 1991) contribute to the NANC inhibitory transmission in the gastrointestinal system (He and Goyal, 1993; Soediono and Burnstock, 1994; Zagorodnyuk and Maggi, 1994). Because the contractile and relaxant events represent a complex result of nerve influences on the smooth muscle (Hoyle and Burnstock, 1989; Wood, 1987), we tested the contribution to enteric NANC transmission (Grider, 1989; Pernow, 1983) of NO, an inhibitory transmitter and substance P (SP), an excitatory transmitter. *To whom correspondence should be addressed. Received 8 April 1997; accepted 22 August 1997.
The tissue used was the guinea pig ileum, which has inhibitory and excitatory NANC innervation (Bauer and Kuriyama, 1982). Recent studies of ours have shown that the relaxant and contractile components of the electrically induced NANC nerve-mediated responses of the longitudinal muscle develop consequently (Ivancheva et al., in press). The initial component of these responses, the relaxation, is most probably NO dependent, which suggests that NO could be a modulator of NANC neurotransmitter effects (Ivancheva and Radomirov, 1996). To verify this suggestion with respect to NOinduced modulation of SP effects, we evaluated the role of NO and SP in the electrically evoked NANC responses by drugs influencing the NO and SP effects. MATERIALS AND METHODS
Animals and isolated preparations Male guinea pigs (250–280 g) given food and water ad lib but starved for 12–14 hr before the experiments were stunned by a blow on the neck and exsanguinated by severing the carotid arteries. Part of the ileum, 18–20 cm proximal to the ileocaecal sphincter, was removed and cleaned with modified Krebs solution in room temperature. To retain the myenteric plexus intact, 20-mm-long segments were cut out.
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Contractile activity The segments were placed along the longitudinal axis in 10-ml organ baths containing modified Krebs solution (in mM): NaCl 120, KCl 5.9, NaHCO3 15.4, NaH2 PO4 1.2, MgCl2 1.2, CaCl2 2.5, and glucose 11.5, aerated by 95% O and 5% CO2 (pH 7.2) at 378C. The changes in the isometric tension of the longitudinal muscle induced by drugs or electrical field stimulation were measured by a strain gauge after 1-hr equilibration under a load of 1 g. Preparations in which the repeated administration of 10 nM acetylcholine induced equal-in-amplitude contractile responses were used in the experiments.
Electrical stimulation Electrical field stimulation (EFS) (Paton and Zar, 1968) of the intrinsic nerves was achieved with rectangular pulses (0.8 msec, 40 V, 1–20 Hz) from Stimulator ST 02 (Experimetria Ltd, Hungary). The stimuli were applied for 20 sec by a pair of platinum electrodes (0.45 mm thick) diametrically opposed on the organ bath walls. The response to EFS is a complex of changes in the contractility of the longitudinal muscle and consists of more than one component. The components occurring during the EFS were examined. Adreno- and cholinoceptor blockers, which are thought to act preferentially at postjunctional receptor sites, were used to obtain NANC responses to the transmural stimulation.
Drugs Acetylcholine hydrochloride (Germed), phentolamine and propranolol hydrochloride (Ciba-Geigy), atropine sulfate (Merck), tetrodotoxin (TTX, Sankyo), NG-nitro-L-arginine (L-NNA), L-arginine, D-arginine, sodium nitroprusside (SNP) (all from Sigma), substance P (Wellcome), and AP 13.2 ACOH supplied by Ferring GMBH (Kiel, Germany) as substances blocking the SP receptors were used. The concentrations stated are the final bath values.
Evaluation of results and statistics The amplitude of the components of the responses elicited by the EFS or drug treatment was measured in linear units (mm) and recalculated as force in milinewtons, and comparisons by percentage versus the controls were made. Concentration–response curves for SP were obtained. The curves in the region between 16% and 84% of the maximum effects were subjected to regression analyses, and the EC50 values (concentration required to produce 50% of the maximum contractile effect) were calculated. The data were assessed for significance of the differences using the Student’s paired t-test at P,0.05. To understand whether a correlation exists between the effects of NO-ergic and SP-ergic drugs and the presence of endogenous NO and SP, we studied the presence and distribution of NADPH-diaphorase(NADPH-d) and SP-containing structures in untreated fixed segments, using histo- and immunohistochemical techniques (Scherer-Singler et al., 1983; Vacca et al., 1980). RESULTS
NANC responses to EFS In the presence of phentolamine (5 mM), propranolol (5 mM) and atropine (3 mM), the NANC responses of the longitudinal muscle during EFS (0.8 msec, 40 V, 1–20 Hz) applied for 20 sec consisted
FIGURE 1. Longitudinal muscle of guinea pig ileum. Responses to EFS (0.8 msec, 40 V, 1–20 Hz for 20 sec) (—) in the absence (a,b) control and in the presence of drugs: NG-nitro-L-arginine; (c) L-NNA; L-arginine; (d); L-Arg; and AP 13.2 ACOH (e,f,g) AP.
of a relaxation followed by a fast twitch and a slow tonic contraction on which phasic contractions were superimposed. The twitch and tonic contractions frequency dependently increased, whereas the relaxation reached a maximum at 10 Hz EFS (Fig. 1A, B; Fig. 2A). Neither the amplitude of the different components of the responses to EFS nor the tone of the preparations was considerable changed during 75- to 90-min period of stimulations. Significant differences between the responses serving as controls before the drug treatment in the different experiments were not observed. Tetrodotoxin (0.1 mM, 15 min) prevented the responses to EFS (Fig. 2B, C and D).
Effects of drugs on the NANC responses L-NNA, an inhibitor of NO synthesis, L-arginine, a substrate of NO synthesis, D-arginine, and SNP, an exogenous donor of NO, were used to investigate the role of NO in these NANC responses to EFS. L-NNA (0.1 mM or 0.5 mM) did not alter the spontaneous contractility but abolished the relaxation of all responses, except for that evoked by 10-Hz EFS (Fig. 1C; Fig. 2B). L-NNA increased the twitch and tonic contraction as the effect was more pronounced on the twitch of the responses to EFS at frequency <5 Hz (Fig. 2C and D). There was no difference in the effectiveness of the two concentrations of L-NNA (Fig. 2B, C, and D). L-arginine (0.5 mM, 15 min), but not D-arginine (0.5 mM, 15 min), gradually restored relaxation, twitch, and tonic contraction (Fig. 1D; Fig. 2B, C, and D). SNP (1 mM or 10 mM, 15 min) decreased the smooth muscle tone but was without effect on the responses to EFS. The role of SP in the NANC responses to EFS was studied using AP, an SP receptor blocker and evaluating the effects of SP on the spontaneous NANC contractility in the absence and in the presence of AP or L-NNA. AP greatly affected the responses to EFS. AP at a concentration of 0.1 mM (15 min) increased the relaxation (Fig. 1E; Fig. 2B) and prevented the twitch and tonic contractions of the responses to EFS at frequency of <5 Hz (Fig. 1E; Fig. 2C and D). The pattern of the responses to 10-Hz or 20-Hz EFS was unchanged, but the twitch and tonic contractions were strongly decreased (Fig. 1F; Fig. 2C and D). In the presence of AP, 10 mM, the response to EFS consisted in a deep relaxation, and contractions were no longer observed (Fig. 1G; Fig. 2B, C, and D).
NO and Substance P in NANC Transmission
103 plexus, and positive fibers innervated the circular and longitudinal muscle and rarely formed pericellular endings in the ganglia (Fig. 3A). SP-positive varicose fiber formed rich meshwork around SPnegative nerve cell bodies in the ganglia. Many varicose SP-positive fibers were observed in the nerve bundles between the myenteric ganglia and the muscle layers (Fig. 3B).
DISCUSSION
FIGURE 2. Longitudinal muscle of guinea pig ileum. Frequencyresponse curves to EFS (0.8 msec, 40 V, 1–20 Hz for 20 sec) in the (a) absence and in the presence of drugs (b,c,d): NG-nitro-Larginine (L-NNA), L-arginine (L-Arg), AP 13.2 ACOH (AP), and tetrodotoxin (TTX). Values refer to mean6SEM. At least eight different preparations were used to obtain each of these values. Points indicate significant difference from the respective control response (Student’s t-test for paired data, P,0.05). Designations: (Relax.)5relaxation; (Contr.)5contraction.
Effects of SP on the spontaneous NANC contractility The contractile response to SP (1 nM–0.1 mM) was similar to the contraction in response to EFS. Concentration–response curves for SP (obtained in a noncumulative manner) in the absence and in the presence of AP (0.1 mM) showed parallelism. AP shifted to the right the concentration–response curves to SP (EC50 5.260.4 nM and 88.063.0 nM, respectively, mean6SEM, n55), suggesting that AP acts as competitive antagonist of SP. The concentration–response curve to SP was not altered after L-NNA (0.1 mM) (EC50 4.760.3 nM), thus suggesting that L-NNA does not influence the receptors mediating the contractile effect of SP.
NADPH-d- and SP-positive nerve structures The morphological study revealed that many NADPH-d positive nerve cell bodies and their processes were located in the myenteric
This study shows that the ileum NANC responses to EFS consisting of relaxation followed by twitch and tonic contraction are mediated by inhibitory and excitatory pathways. The finding that the relaxation of the NANC response was depressed by L-NNA, an inhibitor of NO synthesis (Mulsch and Busse, 1990) and restored by L-arginine indicates its nitrergic nature. This is in accordance with the data about NO-mediated ileum relaxation (Osthaus and Galligan, 1992; Williams and Parsons; 1995) and supports the evidence for the role of NO as an inhibitory transmitter in the intestine (Bult et al., 1990; Kilbinger and Wolf, 1994; Rand, 1992; Rand and Li, 1995; Shuttleworth et al., 1991). During L-NNA treatment, the twitch and tonic contractions were increased demonstrating an NO-exerted modulation of the effects of excitatory transmitter(s). A concept about nitrergic modulation of excitatory pathways, cholinergic (Bartho and Lefebvre, 1995; Tanobe et al., 1994) or SP-ergic (Gustafsson et al., 1990; Wiklund et al., 1993) has recently been advanced. Obviously, the elimination of NO affects the noncholinergic excitatory transmission(s) underlying the twitch and tonic contractions in response to stimulation of the intrinsic nerves (Radomirov and Venkova, 1988). The arguments favoring the view that the twitch and tonic contractions are, at least in part, SP-mediated and that NO could modulate the SP effects are the following: (1) the blocker of SP receptors, AP, concentration dependently suppresses or even abolishes the twitch and tonic contractions that are most probably mediated via tachykinin NK receptors identified in the guinea pig intestines (Maggi et al., 1994); (2) AP competitively antagonizes the effect of SP; (3) the inhibition of NO synthesis by L-NNA does not change the effect of applied SP; and (4) L-NNA increases the EFS-evoked, AP-sensitive contractions, twitch and tonic, suggesting the elimination of the inhibitory action of NO on the SPmediated excitatory effects at the prejunctional level. NO causes relaxation of smooth muscle through stimulation of soluble guanylate cyclase (Moncada et al., 1989). However, the highly diffusible nature of NO suggests the possibility of prejunctional sites of action, too (Shuttleworth et al., 1991). Inhibitory prejunctional effects of NO on cholinergic (Kilbinger and Wolf, 1994) and sympathetic (Kasakov et al., 1995) transmissions have been reported recently. Blockade of SP receptors by AP induced a concentration-dependent increase in the NO-mediated relaxation of the NANC responses to EFS, demonstrating that the elimination of the excitatory effects of SP facilitated the postjunctional inhibitory effect of NO. The morphological observations about neuronal and neuromuscular connections are in accordance with the data concerning the distribution of NADPH-d and SP-positive nerve structures in the guinea pig intestine (Schultzberg et al., 1980; Young et al., 1992) and suggest interactions between them. In conclusion, the pharmacological characterization of the NANC responses to transmural stimulation and the morphological findings speak for the contribution of NO and SP to the NANC nerve-mediated activity in the longitudinal muscle of the guinea pig
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FIGURE 3. Myenteric plexus of guinea pig ileum. NADPH-diaphorase staining (A). The arrows showed the neurites of the positive neurons running into the primary nerve bindles of the plexus. SP immunoreactivity (B). Positive varicose fibers encircle the myenteric neurons and run in secondary and tertiary bundles of the plexus. Scale bar550 mm.
ileum. It appears that NO exerts a prejunctional inhibitory action on SP transmission and that the excitatory effect of SP counteracts the inhibitory effect of NO at the postjunctional level. This study was supported by National Science Fund, Grants L-408 and L-550.
References Bartho L. and Lefebvre R. A. (1995) Nitric oxide-mediated contraction in enteric smooth muscle. Arch. Int. Pharmacodyn. 329, 53–66. Bauer V. and Kuriyama H. (1982) Evidence for non-adrenergic non-cholinergic innervation in the guinea pig ileum. J. Physiol. 330, 375–391. Bult H., Boeckxtaens G. E., Pelckmans P. A., Jordaens F. H., Van Maercke Y. M. and Herman A. G. (1990) Nitric oxide as inhibitory non-adrenergic non-cholinergic neurotransmitter. Nature 345, 346–347. Burnstock G. (1972) Purinergic nerves. Pharmacol. Rev. 24, 509–581. Burnstock G. (1986) The changing face of autonomic neurotransmission. Acta Physiol. Scand. 126, 67–91. Goyal R. K. and Rattan S. (1980) VIP as possible neurotransmitter of nonadrenergic, non-cholinergic inhibitory neurons. Nature 288, 378–380. Grider J. R. (1989) Tachykinins as transmitters of ascending contractile component of the peristaltic reflex. Am. J. Physiol. 257, G709–G714. Gustafsson L. E., Wiklund C. U., Wiklund N. P., Persson M. G. and Moncada S. (1990) Modulation of autonomic neuroeffector transmission by nitric oxide in guinea pig ileum. Biochem. Biophys. Res. Commun. 173, 106–110. He X. D. and Goyal R. K. (1993) Nitric oxide is involved in the peptide VIP associated inhibitory junction potential in the guinea pig ileum. J. Physiol. 461, 485–499. Hoyle C. H. V. and Burnstock G. (1989) Neuromuscular transmission in the gastrointestinal tract. In Handbook of Physiology, Sect.6: The Gastrointestinal System, Vol.1 (Edited by Macklouf G. M.), pp. 435–464. Am. Physiol. Soc., Washington, DC. Hoyle C. H. V. (1992) Transmission: Purines. In The Autonomic Nervous System. Vol.1. Autonomic Neuroeffector Mechanisms (Edited by Burnstock G. and Hoyle C. H. V.), pp. 367–407. Harwood Academic Publishers, Chur, Switzerland. Ivancheva Chr. and Radomirov R. (1996) Met-enkephalin dependent nitrergically mediated relaxation in the guinea pig ileum. Methods Find. Exp. Clin. Pharmacol., 18, 521–525. Ivancheva Chr., Pencheva N. and Radomirov R. (in press) Pattern of nonadrenergic, noncholinergic responses during short- or long-lasting electrical stimulation in guinea pig ileum. Gen. Pharmacol. Kasakov L., Cellek S. and Moncada S. (1995) Characterization of nitrergic
neurotransmission during short- and long-term electrical stimulation of rabbit anococcygeus muscle. Br. J. Pharmac. 115, 1149–1154. Kilbinger H. and Wolf D. (1994) Increase by NO synthase inhibitors of acetylcholine release from guinea pig myenteric plexus. Naunyn-Schmiedeberg’s Arch. Pharmacol. 349, 543–545. Lundberg J. M. (1996) Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol. Rev. 48, 113– 178. Maggi C. A., Patacchini R., Meini S., Quartara L., Sisto A., Potier E., Giuliani A. and Giachetti A. (1994) Comparison of tachykinin NK1 and NK2 receptors of the guinea pig ileum and proximal colon. Br. J. Pharmacol. 112, 150–160. Moncada S., Palmer R. M. J. and Higgs E. A. (1989) Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication. Biochem. Pharmacol. 38, 1709–1715. Mulsch A. and Busse R. (1990) N-nitro-L-arginine (N-[imuno (nitroamino) methyl[-L-ornithidine] impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. Naunyn Schmiedeberg’s Arch. Pharmacol. 341, 143–147. Osthaus L. E. and Galligan J. J. (1992) Antagonists of nitric oxide synthesis inhibit nerve-mediated relaxations of longitudinal muscle in guinea pig ileum. J. Pharmacol. Exp. Ther. 260, 140–145. Paton W. D. M. and Zar A. (1968) The origin of acetylcholine released from guinea pig intestine and longitudinal muscle strip. J Physiol. 194, 13–33. Pernow B. (1983) Substance P. Pharmacol. Rev. 35, 85–141. Radomirov R. and Venkova K. (1988) Pharmacological characteristics of the postsynaptically mediated contractile responses of guinea pig ileum to long-lasting electrical field stimulation. Neuropharmacology 27, 729– 735. Rand M. J. (1992) Nitrergic transmission: nitric oxide as a mediator of NANC neuroeffector transmission. Clin. Exp. Pharmacol. Physiol. 19, 147–169. Rand M. J. and Li C. G. (1995) Nitric oxide as a neurotransmitter in peripheral nerves: nature of transmitter and mechanism of transmission. Annu. Rev. Physiol. 57, 659–682. Scherer-Singler U., Vincent S. R., Kimura H. and McGeer E. G. (1983) Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J. Neurosci. Methods 9, 229–234. Schultzberg M., Hokfeld T., Nilsson G., Terenius L., Rehfeld, J. F., Brown M., Elde R., Goldstein M. and Said S. (1980) Distribution of peptide and catheholamine-containing neurons in the gastro-intestinal tract of rat and guinea pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/
NO and Substance P in NANC Transmission cholecystokinin, neurotensin and dopamine b-hydro-xylase. Neuroscience 5, 689–744. Shuttleworth C. W. R, Murphy R. and Furness J. B. (1991) Evidence that nitric oxide participates in non-adrenergic non-cholinergic inhibitory transmission to intestinal muscle in the guinea pig. Neurosci. Lett. 130, 77–80. Soediono P. and Burnstock G. (1994) Contribution of ATP and nitric oxide to NANC inhibitory transmission in rat pyloric sphincter. Br. J. Pharmacol. 113, 681–686. Tanobe Y., Fujimura, M. and Toda, N. (1994) Nitric oxide as a putative non-adrenergic non-cholinergic neurotransmitter in dog duodenal muscle. Biomed. Res. 15, (suppl.2), 183–185. Vacca L. L., Abrahams S. J. and Naftch N. E. (1980) A modified peroxidaseantiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord. J. Histochem. Cytochem. 28, 297–307.
105 Wiklund C. U., Olgart, C., Wiklund N. P. and Gustafsson L. E. (1993) Modulation of cholinergic and substance P-like neurotransmission by nitric oxide in the guinea pig ileum. Br. J. Pharmacol. 110, 833–839. Williams S. J. and Parsons M. E. (1995) Nitric oxide, an enteric nonadrenergic relaxant transmitter: evidence using phosphodiesterase V and nitric oxide inhibition. Br. J. Pharmacol. 116, 1789–1796. Wood J. D. (1987) Physiology of enteric nervous system. In Physiology of Gastrointestinal Tract (Edited by Johnson L. R.), pp. 67–109. Raven, New York. Young H. M., Furness J. B., Shuttleworth C. W. R., Brendt D. S. and Snyder S. H. (1992) Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea pig intestine. Histochemistry 97, 345–378. Zagorodnyuk V. and Maggi C. A. (1994) Electrophysiological evidence for different release mechanisms of ATP and NO as inhibitory NANC transmitters in guinea pig colon. Br. J. Pharmacol. 112, 1077–1082.
Gen. Pharmac. Vol. 31, No. 1, pp. 101–105, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Contribution of Nitric Oxide and Substance P to Nonadrenergic, Noncholinergic Transmission in the Guinea Pig Ileum Chr. Ivancheva,* D. Itzev, I. Lolova and R. Radomirov Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str., bl. 23, 1113 Sofia, Bulgaria [Tel: 1359-2-71-91-08; Fax: 1359-2-71-91-09; E-mail: [email protected]] ABSTRACT. 1. The possible contribution of the nonadrenergic noncholinergic (NANC) transmitters nitric oxide (NO) and substance P (SP) to the contractility of guinea pig isolated ileum was studied by the responses of the longitudinal muscle to electrical field stimulation (0.8 msec, 40 V, 1–20 Hz, 20 sec) of the intrinsic nerves and by the presence and distribution of NADPH-diaphorase- and SPpositive nerve structures in the myenteric plexus. 2. The electrically elicited, tetrodotoxin (0.3 mM)-sensitive responses, in the presence of phentolamine (5 mM), propranol (5 mM), and atropine (3 mM) consisted of relaxation, followed by twitch and tonic contraction on which phasic contractions were superimposed. 3. NG-nitro-L-arginine (L-NNA; 0.1 mM or 0.5 mM), an inhibitor of NO synthesis abolished the relaxation. L-arginine (0.1 mM), a substrate for NO synthesis, but not D-arginine, restored it. L-NNA concentration dependently increased the twitch and tonic contractions. Sodium nitroprusside (1 mM or 10 (M), an exogenous donor of NO, was without effect on the electrically evoked responses. 4. AP 13.2 ACOH (AP; 0.1 mM or 10 mM), a blocker of SP receptors, frequency dependently inhibited or even prevented the twitch and tonic contractions. AP concentration-dependently increased the relaxation or reversed the responses to electrical stimulation into a deep relaxation. 5. The concentration-response curve for SP (1 nM–0.1 mM) was shifted to the right by AP, the EC50 values being 5.260.4 nM and 88.063.0 nM, respectively. The effects of SP were not altered by L-NNA (0.1 mM). 6. These findings, supported by morphological data about distribution of NADPH-diaphorase-positive nerve cell bodies and processes and SP-positive varicose fibers, suggest the contribution of NO and SP to NANC transmission. It appears that NO inhibits prejunctionally the SP transmission whereas SP counteracts the NO effect at the postjunctional level. gen pharmac 31;1:101–105, 1998. 1998 Elsevier Science Inc. KEY WORDS. Nitric oxide, substance P, NANC transmission, ileum contractility
INTRODUCTION The transmitters resistant to adrenergic and cholinergic blockers have been called nonadrenergic, noncholinergic (NANC) transmitters and different NANC nerve-mediated mechanisms have been suggested. Recently evidence has been presented that adenosine-5-triphosphate (ATP) (Burnstock, 1972, 1986; Hole, 1992; Hoyle and Burnstock, 1989), vasoactive intestinal peptide (VIP) (Goyal and Rattan, 1980; Lundberg, 1996), and nitric oxide (NO) (Bult et al., 1990; Moncada et al., 1989; Rand, 1992; Rand and Li, 1995; Shuttleworth et al., 1991) contribute to the NANC inhibitory transmission in the gastrointestinal system (He and Goyal, 1993; Soediono and Burnstock, 1994; Zagorodnyuk and Maggi, 1994). Because the contractile and relaxant events represent a complex result of nerve influences on the smooth muscle (Hoyle and Burnstock, 1989; Wood, 1987), we tested the contribution to enteric NANC transmission (Grider, 1989; Pernow, 1983) of NO, an inhibitory transmitter and substance P (SP), an excitatory transmitter. *To whom correspondence should be addressed. Received 8 April 1997; accepted 22 August 1997.
The tissue used was the guinea pig ileum, which has inhibitory and excitatory NANC innervation (Bauer and Kuriyama, 1982). Recent studies of ours have shown that the relaxant and contractile components of the electrically induced NANC nerve-mediated responses of the longitudinal muscle develop consequently (Ivancheva et al., in press). The initial component of these responses, the relaxation, is most probably NO dependent, which suggests that NO could be a modulator of NANC neurotransmitter effects (Ivancheva and Radomirov, 1996). To verify this suggestion with respect to NOinduced modulation of SP effects, we evaluated the role of NO and SP in the electrically evoked NANC responses by drugs influencing the NO and SP effects. MATERIALS AND METHODS
Animals and isolated preparations Male guinea pigs (250–280 g) given food and water ad lib but starved for 12–14 hr before the experiments were stunned by a blow on the neck and exsanguinated by severing the carotid arteries. Part of the ileum, 18–20 cm proximal to the ileocaecal sphincter, was removed and cleaned with modified Krebs solution in room temperature. To retain the myenteric plexus intact, 20-mm-long segments were cut out.
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Contractile activity The segments were placed along the longitudinal axis in 10-ml organ baths containing modified Krebs solution (in mM): NaCl 120, KCl 5.9, NaHCO3 15.4, NaH2 PO4 1.2, MgCl2 1.2, CaCl2 2.5, and glucose 11.5, aerated by 95% O and 5% CO2 (pH 7.2) at 378C. The changes in the isometric tension of the longitudinal muscle induced by drugs or electrical field stimulation were measured by a strain gauge after 1-hr equilibration under a load of 1 g. Preparations in which the repeated administration of 10 nM acetylcholine induced equal-in-amplitude contractile responses were used in the experiments.
Electrical stimulation Electrical field stimulation (EFS) (Paton and Zar, 1968) of the intrinsic nerves was achieved with rectangular pulses (0.8 msec, 40 V, 1–20 Hz) from Stimulator ST 02 (Experimetria Ltd, Hungary). The stimuli were applied for 20 sec by a pair of platinum electrodes (0.45 mm thick) diametrically opposed on the organ bath walls. The response to EFS is a complex of changes in the contractility of the longitudinal muscle and consists of more than one component. The components occurring during the EFS were examined. Adreno- and cholinoceptor blockers, which are thought to act preferentially at postjunctional receptor sites, were used to obtain NANC responses to the transmural stimulation.
Drugs Acetylcholine hydrochloride (Germed), phentolamine and propranolol hydrochloride (Ciba-Geigy), atropine sulfate (Merck), tetrodotoxin (TTX, Sankyo), NG-nitro-L-arginine (L-NNA), L-arginine, D-arginine, sodium nitroprusside (SNP) (all from Sigma), substance P (Wellcome), and AP 13.2 ACOH supplied by Ferring GMBH (Kiel, Germany) as substances blocking the SP receptors were used. The concentrations stated are the final bath values.
Evaluation of results and statistics The amplitude of the components of the responses elicited by the EFS or drug treatment was measured in linear units (mm) and recalculated as force in milinewtons, and comparisons by percentage versus the controls were made. Concentration–response curves for SP were obtained. The curves in the region between 16% and 84% of the maximum effects were subjected to regression analyses, and the EC50 values (concentration required to produce 50% of the maximum contractile effect) were calculated. The data were assessed for significance of the differences using the Student’s paired t-test at P,0.05. To understand whether a correlation exists between the effects of NO-ergic and SP-ergic drugs and the presence of endogenous NO and SP, we studied the presence and distribution of NADPH-diaphorase(NADPH-d) and SP-containing structures in untreated fixed segments, using histo- and immunohistochemical techniques (Scherer-Singler et al., 1983; Vacca et al., 1980). RESULTS
NANC responses to EFS In the presence of phentolamine (5 mM), propranolol (5 mM) and atropine (3 mM), the NANC responses of the longitudinal muscle during EFS (0.8 msec, 40 V, 1–20 Hz) applied for 20 sec consisted
FIGURE 1. Longitudinal muscle of guinea pig ileum. Responses to EFS (0.8 msec, 40 V, 1–20 Hz for 20 sec) (—) in the absence (a,b) control and in the presence of drugs: NG-nitro-L-arginine; (c) L-NNA; L-arginine; (d); L-Arg; and AP 13.2 ACOH (e,f,g) AP.
of a relaxation followed by a fast twitch and a slow tonic contraction on which phasic contractions were superimposed. The twitch and tonic contractions frequency dependently increased, whereas the relaxation reached a maximum at 10 Hz EFS (Fig. 1A, B; Fig. 2A). Neither the amplitude of the different components of the responses to EFS nor the tone of the preparations was considerable changed during 75- to 90-min period of stimulations. Significant differences between the responses serving as controls before the drug treatment in the different experiments were not observed. Tetrodotoxin (0.1 mM, 15 min) prevented the responses to EFS (Fig. 2B, C and D).
Effects of drugs on the NANC responses L-NNA, an inhibitor of NO synthesis, L-arginine, a substrate of NO synthesis, D-arginine, and SNP, an exogenous donor of NO, were used to investigate the role of NO in these NANC responses to EFS. L-NNA (0.1 mM or 0.5 mM) did not alter the spontaneous contractility but abolished the relaxation of all responses, except for that evoked by 10-Hz EFS (Fig. 1C; Fig. 2B). L-NNA increased the twitch and tonic contraction as the effect was more pronounced on the twitch of the responses to EFS at frequency <5 Hz (Fig. 2C and D). There was no difference in the effectiveness of the two concentrations of L-NNA (Fig. 2B, C, and D). L-arginine (0.5 mM, 15 min), but not D-arginine (0.5 mM, 15 min), gradually restored relaxation, twitch, and tonic contraction (Fig. 1D; Fig. 2B, C, and D). SNP (1 mM or 10 mM, 15 min) decreased the smooth muscle tone but was without effect on the responses to EFS. The role of SP in the NANC responses to EFS was studied using AP, an SP receptor blocker and evaluating the effects of SP on the spontaneous NANC contractility in the absence and in the presence of AP or L-NNA. AP greatly affected the responses to EFS. AP at a concentration of 0.1 mM (15 min) increased the relaxation (Fig. 1E; Fig. 2B) and prevented the twitch and tonic contractions of the responses to EFS at frequency of <5 Hz (Fig. 1E; Fig. 2C and D). The pattern of the responses to 10-Hz or 20-Hz EFS was unchanged, but the twitch and tonic contractions were strongly decreased (Fig. 1F; Fig. 2C and D). In the presence of AP, 10 mM, the response to EFS consisted in a deep relaxation, and contractions were no longer observed (Fig. 1G; Fig. 2B, C, and D).
NO and Substance P in NANC Transmission
103 plexus, and positive fibers innervated the circular and longitudinal muscle and rarely formed pericellular endings in the ganglia (Fig. 3A). SP-positive varicose fiber formed rich meshwork around SPnegative nerve cell bodies in the ganglia. Many varicose SP-positive fibers were observed in the nerve bundles between the myenteric ganglia and the muscle layers (Fig. 3B).
DISCUSSION
FIGURE 2. Longitudinal muscle of guinea pig ileum. Frequencyresponse curves to EFS (0.8 msec, 40 V, 1–20 Hz for 20 sec) in the (a) absence and in the presence of drugs (b,c,d): NG-nitro-Larginine (L-NNA), L-arginine (L-Arg), AP 13.2 ACOH (AP), and tetrodotoxin (TTX). Values refer to mean6SEM. At least eight different preparations were used to obtain each of these values. Points indicate significant difference from the respective control response (Student’s t-test for paired data, P,0.05). Designations: (Relax.)5relaxation; (Contr.)5contraction.
Effects of SP on the spontaneous NANC contractility The contractile response to SP (1 nM–0.1 mM) was similar to the contraction in response to EFS. Concentration–response curves for SP (obtained in a noncumulative manner) in the absence and in the presence of AP (0.1 mM) showed parallelism. AP shifted to the right the concentration–response curves to SP (EC50 5.260.4 nM and 88.063.0 nM, respectively, mean6SEM, n55), suggesting that AP acts as competitive antagonist of SP. The concentration–response curve to SP was not altered after L-NNA (0.1 mM) (EC50 4.760.3 nM), thus suggesting that L-NNA does not influence the receptors mediating the contractile effect of SP.
NADPH-d- and SP-positive nerve structures The morphological study revealed that many NADPH-d positive nerve cell bodies and their processes were located in the myenteric
This study shows that the ileum NANC responses to EFS consisting of relaxation followed by twitch and tonic contraction are mediated by inhibitory and excitatory pathways. The finding that the relaxation of the NANC response was depressed by L-NNA, an inhibitor of NO synthesis (Mulsch and Busse, 1990) and restored by L-arginine indicates its nitrergic nature. This is in accordance with the data about NO-mediated ileum relaxation (Osthaus and Galligan, 1992; Williams and Parsons; 1995) and supports the evidence for the role of NO as an inhibitory transmitter in the intestine (Bult et al., 1990; Kilbinger and Wolf, 1994; Rand, 1992; Rand and Li, 1995; Shuttleworth et al., 1991). During L-NNA treatment, the twitch and tonic contractions were increased demonstrating an NO-exerted modulation of the effects of excitatory transmitter(s). A concept about nitrergic modulation of excitatory pathways, cholinergic (Bartho and Lefebvre, 1995; Tanobe et al., 1994) or SP-ergic (Gustafsson et al., 1990; Wiklund et al., 1993) has recently been advanced. Obviously, the elimination of NO affects the noncholinergic excitatory transmission(s) underlying the twitch and tonic contractions in response to stimulation of the intrinsic nerves (Radomirov and Venkova, 1988). The arguments favoring the view that the twitch and tonic contractions are, at least in part, SP-mediated and that NO could modulate the SP effects are the following: (1) the blocker of SP receptors, AP, concentration dependently suppresses or even abolishes the twitch and tonic contractions that are most probably mediated via tachykinin NK receptors identified in the guinea pig intestines (Maggi et al., 1994); (2) AP competitively antagonizes the effect of SP; (3) the inhibition of NO synthesis by L-NNA does not change the effect of applied SP; and (4) L-NNA increases the EFS-evoked, AP-sensitive contractions, twitch and tonic, suggesting the elimination of the inhibitory action of NO on the SPmediated excitatory effects at the prejunctional level. NO causes relaxation of smooth muscle through stimulation of soluble guanylate cyclase (Moncada et al., 1989). However, the highly diffusible nature of NO suggests the possibility of prejunctional sites of action, too (Shuttleworth et al., 1991). Inhibitory prejunctional effects of NO on cholinergic (Kilbinger and Wolf, 1994) and sympathetic (Kasakov et al., 1995) transmissions have been reported recently. Blockade of SP receptors by AP induced a concentration-dependent increase in the NO-mediated relaxation of the NANC responses to EFS, demonstrating that the elimination of the excitatory effects of SP facilitated the postjunctional inhibitory effect of NO. The morphological observations about neuronal and neuromuscular connections are in accordance with the data concerning the distribution of NADPH-d and SP-positive nerve structures in the guinea pig intestine (Schultzberg et al., 1980; Young et al., 1992) and suggest interactions between them. In conclusion, the pharmacological characterization of the NANC responses to transmural stimulation and the morphological findings speak for the contribution of NO and SP to the NANC nerve-mediated activity in the longitudinal muscle of the guinea pig
104
Chr. Ivancheva et al.
FIGURE 3. Myenteric plexus of guinea pig ileum. NADPH-diaphorase staining (A). The arrows showed the neurites of the positive neurons running into the primary nerve bindles of the plexus. SP immunoreactivity (B). Positive varicose fibers encircle the myenteric neurons and run in secondary and tertiary bundles of the plexus. Scale bar550 mm.
ileum. It appears that NO exerts a prejunctional inhibitory action on SP transmission and that the excitatory effect of SP counteracts the inhibitory effect of NO at the postjunctional level. This study was supported by National Science Fund, Grants L-408 and L-550.
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