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Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus
Regulatory Peptides 98 (2001) 27–32 www.elsevier.com / locate / regpep
Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus Maria Caterina Baccari*, Franco Calamai Department of Physiology, University of Florence, Viale G.B. Morgagni 63, 50134 Florence, Italy Received 5 May 2000; received in revised form 4 October 2000; accepted 16 October 2000
Abstract The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N G -nitro arginine ( L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by a-chymotrypsin. a-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by a-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe 6 ,Leu 17 ]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO. 2001 Elsevier Science B.V. All rights reserved. Keywords: Neuromodulation; VIP; Nitric oxide
1. Introduction Different non-adrenergic, non-cholinergic (NANC) inhibitory systems are involved in the relaxant responses of the gastrointestinal tract. Purines, nitric oxide (NO) and polypeptides all seem to contribute to the inhibitory motility patterns elicited either ‘in vivo’ or ‘in vitro’ in the different region of the gut [1–4]. Vasoactive intestinal polypeptide (VIP) and its structurally homologous pituitary adenylate cyclase-activating polypeptide (PACAP) are considered, among polypeptides, as the main inhibitory neurotransmitters released from NANC fibres [5–7]. PACAP exists in two amidated forms, a 38 amino acid *Corresponding author. Tel.: 1 39-55-423-7311; fax: 1 39-55-4379506. E-mail address: [email protected] (M.C. Baccari).
peptide (PACAP-38) and a 27 amino acid peptide (PACAP-27) [8], both identified in gut extracts, although PACAP-38 appears to predominate [9]. PACAP immunoreactivity has been found in neurons of the myenteric plexus [5,10] and PACAP release by nerve stimulation has been reported in the guinea pig stomach [11]. PACAP receptors have also been identified on smooth muscle [12,13] and relaxant responses to exogenous PACAP have been reported in several regions of the gastrointestinal tract [7,14–16]. However, a neuromodulatory role for PACAP in the enteric nervous system has also been described: contractile effects have been reported in the ileum through the release of acetylcholine and substance P [17,18] and a stimulant action by PACAP on excitatory enteric motor pathways involved in peristalsis has been suggested in the small intestine [19]. In the present experiments we tested the motor effects of
0167-0115 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 00 )00225-1
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VIP and PACAP-38 in the longitudinal muscle strips of the mouse stomach. The effects of PACAP 6-38, as a PACAP38 antagonist, and VIP 10-28 and [D-p-Cl-Phe 6 ,Leu 17 ]-VIP, as VIP antagonists, on the electrical field stimulation (EFS)-induced NANC inhibitory responses were also investigated.
subsequent applications of CCh was no less than 15 min, during which repeated and prolonged washes with Krebs– Henseleit solution were done. Drug concentrations are given as final bath concentrations (molar).
2.2. Calculations and statistical analysis 2. Materials and methods Experiments were carried out on male (C57BL / 10SnJ) 6–10-week-old mice (Jackson Laboratory, Maine, USA) killed by cervical dislocation. The stomach was rapidly dissected from the abdomen and two strips were cut in the direction of the longitudinal muscle layer from each gastric fundus. One end of each strip was tied to a platinum rod while the other was connected to a force displacement transducer (Grass model FT03) by a silk thread for continuous recording of isometric tension. The transducer was coupled to a polygraph (Sanborn model 7700). Muscle strips were mounted in 5-ml double-jacketed organ baths containing Krebs–Henseleit solution, gassed with 95% O 2 –5% CO 2 mixture, of the following composition (mM): NaCl 118, KCl 4.7, MgSO 4 1.2, KH 2 PO 4 1.2, NaHCO 3 25, CaCl 2 2.5 and glucose 10 (pH 7.4). Prewarmed water (378C) was circulated through the outer jacket of the tissue bath via a constant-temperature circulator pump. The temperature of the Krebs–Henseleit solution in the organ bath was maintained within 60.58C. Electrical field stimulation (EFS) was applied via two platinum wire rings (2 mm diameter, 5 mm apart) through which the preparation was threaded. Electrical impulses (rectangular waves, 80 V, 4–16 Hz, 0.5 ms, for 15 s) were provided by a Grass model S8 stimulator. Strips were allowed to equilibrate for 1 h under an initial load of 0.8 g. During this period, repeated and prolonged washes of the preparations with Krebs–Henseleit solution were done to avoid accumulation of metabolites in the organ baths.
2.1. Drugs The following drugs were used: guanethidine sulphate, carbachol (CCh), N G -nitro-L-arginine ( L-NNA), 1H[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ), tetrodotoxin (TTX), apamin, a-chymotrypsin, vasoactive intestinal polypeptide (VIP), VIP 10-28, [D-p-ClPhe 6 ,Leu 17 ]-VIP, pituitary adenylate cyclase-activating peptide (PACAP-38), PACAP 6-38. All drugs were obtained from Sigma Chemical (St. Louis, MO), except for PACAP 6-38 which was obtained from Bachem (Bubendorf, Switzerland). The peptides were dissolved in a medium containing 0.1% BSA to avoid their absorption to glass and plastic apparatus. When contraction elicited by CCh reached a plateau, EFS or drugs were applied. The interval between two
Relaxant responses are expressed as percentage decrease relative to the muscular tension induced by 1 3 10 26 M CCh. Amplitude values of fast relaxations refer to the maximal peak obtained during the stimulation period. Amplitude values of sustained relaxations refer to the maximal peak, obtained following the stimulation period, with respect to prestimulus level. Wilcoxon test was applied for statistical analysis of data. Differences were considered significant at P , 0.05. Results are given as 6S.E. The number of muscle strip preparations is designated by n in the results.
3. Results As recently observed [20], EFS (4–16 Hz), applied during the plateau phase of the sustained increase in tension induced by 1 3 10 26 M CCh, in the presence of 1 3 10 26 M guanethidine, caused (n 5 26) relaxant responses whose amplitude increased by increasing stimulation frequency. These inhibitory responses consisted of a fast decrease in strip tension that reached the maximal peak (mean amplitude 30.863% at 4 Hz; 4764% at 8 Hz; 67.463% at 16 Hz) during the stimulation period (Figs. 1 and 2). At the lowest stimulation frequency employed (4 Hz) the fast relaxation was followed, at the end of the stimulation period, by a rapid return to the baseline. By increasing the stimulation frequency (8–16 Hz) strip tension no longer suddenly returned to the initial level at the end of the stimulation period but a further, sustained inhibitory component appeared (mean amplitude and duration: 12.163% for 3569.2 s at 8 Hz; 47.764% for 105618.8 s at 16 Hz) (Figs. 1 and 2). All relaxant responses to EFS were abolished by 1 3 10 26 M TTX. The NO synthesis inhibitor L-NNA (2 3 10 24 M) abolished (n 5 6), after 5–10 min of contact time, the EFS-induced fast relaxation without affecting the amplitude of the sustained relaxant responses. The same results were obtained in the presence of the guanylate cyclase inhibitor ODQ (1 3 10 26 M) (n 5 3). Addition of a-chymotrypsin (10 U / ml) to the bath medium (n 5 8) caused a slight increase (0.560.02%) of strip tension and after 20 min of contact time increased the amplitude of the EFS-induced fast relaxation at 8–16 Hz stimulation frequency (Fig. 3) and reduced by 97.162% the sustained one (Fig. 1). Apamin (1 3 10 26 M) did not statistically significantly
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Fig. 1. Effects of PACAP 6-38 on the EFS- and VIP-induced relaxant responses (upper records) and of VIP 10-28 on the relaxation to EFS and PACAP-38 (lower records) on CCh precontracted strips. PACAP 6-38 completely abolished the sustained response that follows the fast one at the highest stimulation frequency and enhances the amplitude of the fast inhibitory response. PACAP 6-38 also abolished the relaxant response to VIP. VIP 10-28 did not influence the amplitude of the fast or the slow component of the response to EFS nor influenced the relaxant response to PACAP-38.
influence (n 5 7) either the fast or the sustained phase of the EFS-induced relaxations. Addition of 1 3 10 27 M VIP (n 5 8) or 1 3 10 27 M PACAP-38 (n 5 8) on the plateau phase elicited by CCh, caused TTX-insensitive sustained relaxant responses (mean amplitude 69.364.9% and 6565.2% for VIP and PACAP38, respectively) that were both abolished by a-chymotrypsin (10 U / ml) (Fig. 1). These direct smooth muscle responses were not influenced by either L-NNA (2 3 10 24 M), suggesting that nitric oxide is not involved in these relaxations, or by apamin (1 3 10 26 M). Addition to the bath medium of PACAP 6-38 (2 3 10 25 M) caused (n 5 8) a slight increase (0.460.03%) in strip tension. PACAP 6-38 enhanced, at the highest stimulation frequency employed (8–16 Hz), the amplitude of the EFS-induced fast relaxation (Figs. 2 and 3) and reduced by 96.563% the amplitude of the sustained one (Fig. 2). PACAP 6-38 (2 3 10 25 M) also abolished the relaxation caused by 1 3 10 27 M VIP (n 5 5) (Fig. 2) and by 1 3 10 27 M PACAP-38 (n 5 4). Addition to the bath medium of VIP 10-28 (2 3 10 25 M) (n 5 10) or [D-p-Cl-Phe 6 ,Leu 17 ]-VIP (2 3 10 25 M) (n 5 10), did not cause any effects on smooth muscle tension. Furthermore they did not influence the amplitude of either the fast (Fig. 3) or sustained inhibitory response
to EFS nor relaxation to 1 3 10 27 M PACAP-38 (Fig. 2) and 1 3 10 27 M VIP.
4. Discussion As previously observed [20] in CCh precontracted longitudinal muscle strips of the mouse gastric fundus and in the presence of guanethidine, EFS evokes fast relaxations during the stimulation period and sustained relaxant responses after the end of the stimulation period itself. These latter are fully manifested at the highest stimulation frequency employed. Therefore, the EFS-induced NANC inhibitory motility obtained in the longitudinal muscle strips from the mouse gastric fundus does not differ to that observed in other animal species either ‘in vitro’ or ‘in vivo’ [1,2,21–23] suggesting that more than one neurotransmitter appears to be involved in these responses. The abolition by the NO synthesis inhibitor, L-NNA, or by the guanylate cyclase inhibitor, ODQ, of the EFS-induced fast response indicates that NO is responsible for this phase of relaxation. In the present experiments VIP and PACAP both cause TTX-insensitive prolonged relaxations indicating a direct muscular relaxant effect. The lack of influence of the NO
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Fig. 2. Effects of a-chymotrypsin on inhibitory responses elicited by EFS (upper records), VIP (middle records) and PACAP-38 (bottom records) on CCh precontracted strips. a-Chymotrypsin (10 U / ml), after 20 min of contact time, completely abolished the sustained relaxation elicited by EFS at the highest stimulation frequency employed. Note also the increase in amplitude of the fast relaxant response elicited during EFS. a-Chymotrypsin (10 U / ml), after 20 min of contact time, also completely abolished the direct inhibitory responses caused by VIP and PACAP-38.
synthesis inhibitor, L-NNA, on direct relaxation elicited either by VIP or PACAP-38 suggests that, in the longitudinal muscle strips from the gastric fundus of the mouse, the action of these peptides is not mediated by the release of NO at the effector level. Contrasting results on the influence of NO synthesis inhibitors on the relaxation induced by both VIP and PACAP in the gastrointestinal smooth muscle preparations have been reported [24–27] and they might be referred to the experimental method employed [28]. In the longitudinal muscle strips from the mouse gastric fundus, the abolition of the EFS-induced delayed relaxant response by a-chymotrypsin suggests that peptides are responsible for this kind of relaxant response and that their release occurs at the highest stimulation frequency employed. An inhibitory neurotransmitter role and a fre-
quency-dependent release have been reported for both VIP and PACAP-38 [11,29]. In the present experiments the abolition of the EFS-induced sustained relaxations by PACAP 6-38, compared with the lack of effects of VIP 10-28 and [D-p-Cl-Phe 6 -Leu 17 ]-VIP, might suggest that PACAP-38 is responsible in the longitudinal muscle strips from the mouse gastric fundus for the EFS-induced sustained relaxations. However, the lack of effects of apamin, an appropriate tool to distinguish PACAP-specific actions compared to VIP [30], on either the EFS or PACAP-38 induced sustained relaxation does not support a role for PACAP-38 itself in the EFS-induced sustained relaxation. The observation that PACAP 6-38 blocks both relaxation to VIP and PACAP-38 may indicate that, in the mouse gastric fundus, PACAP-38 interfere with VIP by acting on VIP receptors responsible for sustained relaxant
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Fig. 3. Effects of a-chymotrypsin (10 U / ml), PACAP 6-38 (2 3 10 25 M), VIP 10-28 (2 3 10 25 M) and [D-p-Cl-Phe 6 ,Leu 17 ]-VIP (2 3 10 25 M) on EFS-induced fast relaxations. Amplitude values represent percentage decreases relative to the tension induced by 1 3 10 26 M CCh. Values are means6S.E.; n 5 6–8 muscle strip preparations. *P , 0.05, significant compared with controls.
responses. The inefficacy of VIP 10-28 and [D-p-Cl-Phe 6 Leu 17 ]-VIP on relaxation to VIP confirms that they have not significant antagonistic properties in response to VIP, itself. The lack of effects of the NO synthesis inhibitor L-NNA, or the guanylate cyclase inhibitor ODQ, on the EFS-induced sustained responses suggests that NO is not involved in this phase of relaxation. A modulatory role for both VIP and PACAP has been reported [17–19,29]: in the present experiments, carried out on CCh precontracted strips, the increase in amplitude of the EFS-induced fast relaxation by a-chymotrypsin and PACAP 6-38 might indicate the presence of prejunctional peptidergic receptors modulating the release of the neurotransmitter responsible for the fast inhibitory response, that appears to be NO. The presence of PACAP receptors on nitrergic neurons has been recently reported in the rat colon [27]. However, since in our experiments PACAP 6-38 seems to interfere, at the muscular level, with VIP receptors we cannot exclude that the modulatory action is exerted by VIP. The inhibitory action of peptides on the release of the neurotransmitter responsible for the fast response may also account for the reduced amplitude of the EFS-induced fast relaxation observed in the dystrophic mice [20] in which sustained responses are well developed and a derangement of neurotransmitter release in favour of a peptidergic one has been suggested. In the guinea-pig gastric circular muscle strips PACAP 6-38 decreases rather than increases the amplitude of the relaxation to EFS [11]. The discrepancy observed may be related to different muscular layers and animal species employed. In fact, in the guinea-pig it appears that only one type of relaxant response is obtained that appears to be mediated by the concomitant release of more than one neurotransmitter since it is reduced but not abolished by L-NNA as well as by PACAP 6-38. In the rat distal colon
PACAP 6-38 and VIP 10-28 have been shown to reduce the amplitude of the EFS-induced fast response without affecting the sustained one [24]. In the mouse gastric fundus PACAP 6-38 not only abolished the slow, sustained relaxation but also increased the amplitude of the fast one at the highest stimulation frequency (i.e. when the peptide is released). Thus, as reported in the literature the variability of effects and the relative potencies of VIP and PACAP differ not only between species but also between various regions of the gut probably reflecting different sets of receptors [27] that differ in structure, ligand specificity and tissue distribution [31]: PAC1 receptors that exhibit high affinity for PACAP and low affinity for VIP and VPAC1 and VPAC2 that show high affinity for both VIP and PACAP. The present results suggest that in the mouse gastric fundus neuropeptides, other than being involved in the EFS-induced sustained relaxation, also modulate the release of the neurotransmitter responsible for the fast relaxation that appears to be NO. This may represent an important physiological mechanism in the control of gastric relaxation: when sustained relaxations are required, NANC fibres discharge at high frequency thus inducing peptide release and a slow, sustained relaxant response. Concomitantly, the release of the neurotransmitter, responsible for the fast short-lasting relaxation, characteristic of peristaltic movements, is inhibited by the peptides themselves.
Acknowledgements The financial support of Telethon-Italy (Grant no. 906 to F.C.) is gratefully acknowledged. This study was also supported by grants from the Ministero dell’Universita` e
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della Ricerca Scientifica e Tecnologica of Italy. The authors thank Alessandro Aiazzi, Salvatore Cammarata and Mario Dolfi for their skilled technical assistance and Adrio Vannucchi for the preparation of Figs. 1 and 2.
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References [17] [1] Baccari MC, Calamai F, Staderini G. Prostaglandin E2 modulates neurally induced nonadrenergic, noncholinergic gastric relaxations in the rabbit in vivo. Gastroenterology 1996;110:129–38. [2] Baccari MC, Iacoviello C, Calamai F. Nitric oxide as modulator of cholinergic neurotransmission in gastric muscle of rabbits. Am J Physiol 1997;273:G456–63. [3] Mashimo H, He XD, Huang PL, Fishman MC, Goyal RK. Neuronal constitutive nitric oxide synthase is involved in murine enteric neurotransmission. J Clin Invest 1996;98:8–13. [4] Ishiguchi T, Nishioka S, Takahashi T. Inhibitory neural pathway regulating gastric emptying in rats. J Auton Nerv Syst 2000;79:45– 51. ˚ ¨ [5] Sundler F, Ekblad E, Absood A, Hakanson R, Koves K, Arimura A. Pituitary adenylate cyclase activating peptide: a novel vasoactive intestinal peptide-like neuropeptide in the gut. Neuroscience 1992;46:439–54. [6] Shuttleworth CWR, Keef KD. Roles of peptides in enteric neuromuscular transmission. Regul Pept 1995;56:101–20. ¨ [7] Lauffer JM, Modlin IM, Tang LH. Biological relevance of pituitary adenylate cyclase-activating polypeptide (PACAP) in the gastrointestinal tract. Regul Pept 1999;84:1–12. [8] Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 1992;37:287–303. [9] Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy DH, Kitada C. Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 1991;129:2787–9. [10] Portbury AL, McConalogue K, Furness JB, Young HM. Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon. Cell Tissue Res 1995;279:385– 92. [11] Katsoulis S, Schmidt WE, Schwarzhoff R, Folsch UR, Jin JG, Grider JR, Makhlouf GM. Inhibitory transmission in guinea pig stomach mediated by distinct receptors for pituitary adenylate cyclase-activating peptide. J Pharmacol Exp Ther 1996;278:199– 204. [12] Mao YK, Wang YF, Moogk C, Fox-Threlkeld JET, Xiao Q, McDonald TJ, Daniel EE. Locations and molecular forms of PACAP and sites and characteristics of PACAP receptors in canine ileum. Am J Physiol 1998;274:G217–25. [13] Teng B, Murthy KS, Kuemmerle JF, Grider JR, Makhlouf GM. Selective expression of vasoactive intestinal peptide (VIP)2 / pituitary adenylate cyclase-activating polypeptide (PACAP)3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells. Regul Pept 1998;77:127–34. [14] Schworer H, Katsoulis S, Creutzfeldt W, Schmidt WE. Pituitary adenylate cyclase activating peptide, a novel VIP-like gut-brain peptide, relaxes the guinea-pig taenia caeci via apamin-sensitive
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potassium channels. Naunyn Schmiedeberg’s Arch Pharmacol 1992;346:511–4. Schworer H, Katsoulis S, Creutzfeldt W, Schmidt WE. Pituitary adenylate cyclase-activating peptide is a potent modulator of human colonic motility. Scand J Gastroenterol 1993;28:625–32. Katsoulis S, Schmidt WE, Folsch UR, Jin JG, Grider JR. Inhibitory neurotransmission by PACAP in guinea pig stomach is mediated by VIP/ PACAP-II receptors and by distinct apamin-sensitive receptors. Gastroenterology 1994;106:A519. Katsoulis S, Clemens A, Schworer H, Creutzfeldt W, Schmidt WE. PACAP is a stimulator of neurogenic contraction in the guinea pig ileum. Am J Physiol 1993;265:G295–302. Fox-Threlkeld JET, McDonald TJ, Woskowska Z, Iesaki K, Daniel EE. Pituitary adenylate cyclase-activating peptide as a neurotransmitter in the canine ileal circular muscle. J Pharmacol Exp Ther 1999;290:66–75. Heinemann A, Holzer P. Stimulant action of pituitary adenylate cyclase-activating peptide on normal and drug-compromised peristalsis in the guinea-pig intestine. Br J Pharmacol 1999;127:763–71. Baccari MC, Romagnani P, Calamai F. Impaired nitrergic relaxations in the gastric fundus of dystrophic (mdx) mice. Neurosci Lett 2000;282:105–8. G Lefebvre RA, Baert E, Barbier AJ. Influence of L-N -nitro arginine on non-adrenergic non-cholinergic relaxation in the guinea-pig gastric fundus. Br J Pharmacol 1992;106:173–9. Lefebvre RA, Smits GJM, Timmermans JP. Study of NO and VIP as non-adrenergic, non-cholinergic neurotransmitters in the pig gastric fundus. Br J Pharmacol 1995;116:2017–26. Takahashi T, Owyang C. Vagal control of nitric oxide and vasoactive intestinal polypeptide release in the regulation of gastric relaxation in rat. J Physiol 1995;484:481–92. Kishi M, Takeuchi T, Suthamnatpong N, Ishii T, Nishio H, Hata F, Takewaki T. VIP- and PACAP-mediated nonadrenergic, noncholinergic inhibition in longitudinal muscle of rat distal colon: involvement of activation of charybdotoxin- and apamin-sensitive K 1 channels. Br J Pharmacol 1996;119:623–30. Bayguinov O, Keef KD, Hagen B, Sanders KM. Parallel pathways mediate inhibitory effects of vasoactive intestinal polypeptide and nitric oxide in canine fundus. Br J Pharmacol 1999;126:1543–52. Yamamoto H, Kuwahara A, Fujimura M, Maeda T, Fujimiya M. Motor activity of vascularly perfused rat duodenum. 2. Effects of VIP, PACAP27 and PACAP38. Neurogastroenterol Motil 1999;11:235–41. Ekblad E. Pharmacological evidence for both neuronal and smooth muscular PAC1 receptors and a VIP-specific receptor in rat colon. Regul Pept 1999;85:87–92. Dick JM, Van Geldre LA, Timmermans JP, Lefebvre RA. Investigation on the interaction between nitric oxide and vasoactive intestinal polypeptide in the guinea-pig gastric fundus. Br J Pharmacol 2000;129:751–63. Grider JR, Makhlouf GM. Prejunctional inhibition of vasoactive intestinal peptide release. Am J Physiol 1987;253:G7–G12. Jin JG, Katsoulis S, Schmidt WE, Grider JR. Inhibitory transmission in tenia coli mediated by distinct vasoactive intestinal peptide and apamin-sensitive pituitary adenylate cyclase activating peptide receptors. J Pharmacol Exp Ther 1994;270:433–9. Harmar T, Lutz E. Multiple receptors for PACAP and VIP. Trends Pharmacol Sci 1994;15:97–9.
Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus Maria Caterina Baccari*, Franco Calamai Department of Physiology, University of Florence, Viale G.B. Morgagni 63, 50134 Florence, Italy Received 5 May 2000; received in revised form 4 October 2000; accepted 16 October 2000
Abstract The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N G -nitro arginine ( L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by a-chymotrypsin. a-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by a-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe 6 ,Leu 17 ]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO. 2001 Elsevier Science B.V. All rights reserved. Keywords: Neuromodulation; VIP; Nitric oxide
1. Introduction Different non-adrenergic, non-cholinergic (NANC) inhibitory systems are involved in the relaxant responses of the gastrointestinal tract. Purines, nitric oxide (NO) and polypeptides all seem to contribute to the inhibitory motility patterns elicited either ‘in vivo’ or ‘in vitro’ in the different region of the gut [1–4]. Vasoactive intestinal polypeptide (VIP) and its structurally homologous pituitary adenylate cyclase-activating polypeptide (PACAP) are considered, among polypeptides, as the main inhibitory neurotransmitters released from NANC fibres [5–7]. PACAP exists in two amidated forms, a 38 amino acid *Corresponding author. Tel.: 1 39-55-423-7311; fax: 1 39-55-4379506. E-mail address: [email protected] (M.C. Baccari).
peptide (PACAP-38) and a 27 amino acid peptide (PACAP-27) [8], both identified in gut extracts, although PACAP-38 appears to predominate [9]. PACAP immunoreactivity has been found in neurons of the myenteric plexus [5,10] and PACAP release by nerve stimulation has been reported in the guinea pig stomach [11]. PACAP receptors have also been identified on smooth muscle [12,13] and relaxant responses to exogenous PACAP have been reported in several regions of the gastrointestinal tract [7,14–16]. However, a neuromodulatory role for PACAP in the enteric nervous system has also been described: contractile effects have been reported in the ileum through the release of acetylcholine and substance P [17,18] and a stimulant action by PACAP on excitatory enteric motor pathways involved in peristalsis has been suggested in the small intestine [19]. In the present experiments we tested the motor effects of
0167-0115 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 00 )00225-1
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VIP and PACAP-38 in the longitudinal muscle strips of the mouse stomach. The effects of PACAP 6-38, as a PACAP38 antagonist, and VIP 10-28 and [D-p-Cl-Phe 6 ,Leu 17 ]-VIP, as VIP antagonists, on the electrical field stimulation (EFS)-induced NANC inhibitory responses were also investigated.
subsequent applications of CCh was no less than 15 min, during which repeated and prolonged washes with Krebs– Henseleit solution were done. Drug concentrations are given as final bath concentrations (molar).
2.2. Calculations and statistical analysis 2. Materials and methods Experiments were carried out on male (C57BL / 10SnJ) 6–10-week-old mice (Jackson Laboratory, Maine, USA) killed by cervical dislocation. The stomach was rapidly dissected from the abdomen and two strips were cut in the direction of the longitudinal muscle layer from each gastric fundus. One end of each strip was tied to a platinum rod while the other was connected to a force displacement transducer (Grass model FT03) by a silk thread for continuous recording of isometric tension. The transducer was coupled to a polygraph (Sanborn model 7700). Muscle strips were mounted in 5-ml double-jacketed organ baths containing Krebs–Henseleit solution, gassed with 95% O 2 –5% CO 2 mixture, of the following composition (mM): NaCl 118, KCl 4.7, MgSO 4 1.2, KH 2 PO 4 1.2, NaHCO 3 25, CaCl 2 2.5 and glucose 10 (pH 7.4). Prewarmed water (378C) was circulated through the outer jacket of the tissue bath via a constant-temperature circulator pump. The temperature of the Krebs–Henseleit solution in the organ bath was maintained within 60.58C. Electrical field stimulation (EFS) was applied via two platinum wire rings (2 mm diameter, 5 mm apart) through which the preparation was threaded. Electrical impulses (rectangular waves, 80 V, 4–16 Hz, 0.5 ms, for 15 s) were provided by a Grass model S8 stimulator. Strips were allowed to equilibrate for 1 h under an initial load of 0.8 g. During this period, repeated and prolonged washes of the preparations with Krebs–Henseleit solution were done to avoid accumulation of metabolites in the organ baths.
2.1. Drugs The following drugs were used: guanethidine sulphate, carbachol (CCh), N G -nitro-L-arginine ( L-NNA), 1H[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ), tetrodotoxin (TTX), apamin, a-chymotrypsin, vasoactive intestinal polypeptide (VIP), VIP 10-28, [D-p-ClPhe 6 ,Leu 17 ]-VIP, pituitary adenylate cyclase-activating peptide (PACAP-38), PACAP 6-38. All drugs were obtained from Sigma Chemical (St. Louis, MO), except for PACAP 6-38 which was obtained from Bachem (Bubendorf, Switzerland). The peptides were dissolved in a medium containing 0.1% BSA to avoid their absorption to glass and plastic apparatus. When contraction elicited by CCh reached a plateau, EFS or drugs were applied. The interval between two
Relaxant responses are expressed as percentage decrease relative to the muscular tension induced by 1 3 10 26 M CCh. Amplitude values of fast relaxations refer to the maximal peak obtained during the stimulation period. Amplitude values of sustained relaxations refer to the maximal peak, obtained following the stimulation period, with respect to prestimulus level. Wilcoxon test was applied for statistical analysis of data. Differences were considered significant at P , 0.05. Results are given as 6S.E. The number of muscle strip preparations is designated by n in the results.
3. Results As recently observed [20], EFS (4–16 Hz), applied during the plateau phase of the sustained increase in tension induced by 1 3 10 26 M CCh, in the presence of 1 3 10 26 M guanethidine, caused (n 5 26) relaxant responses whose amplitude increased by increasing stimulation frequency. These inhibitory responses consisted of a fast decrease in strip tension that reached the maximal peak (mean amplitude 30.863% at 4 Hz; 4764% at 8 Hz; 67.463% at 16 Hz) during the stimulation period (Figs. 1 and 2). At the lowest stimulation frequency employed (4 Hz) the fast relaxation was followed, at the end of the stimulation period, by a rapid return to the baseline. By increasing the stimulation frequency (8–16 Hz) strip tension no longer suddenly returned to the initial level at the end of the stimulation period but a further, sustained inhibitory component appeared (mean amplitude and duration: 12.163% for 3569.2 s at 8 Hz; 47.764% for 105618.8 s at 16 Hz) (Figs. 1 and 2). All relaxant responses to EFS were abolished by 1 3 10 26 M TTX. The NO synthesis inhibitor L-NNA (2 3 10 24 M) abolished (n 5 6), after 5–10 min of contact time, the EFS-induced fast relaxation without affecting the amplitude of the sustained relaxant responses. The same results were obtained in the presence of the guanylate cyclase inhibitor ODQ (1 3 10 26 M) (n 5 3). Addition of a-chymotrypsin (10 U / ml) to the bath medium (n 5 8) caused a slight increase (0.560.02%) of strip tension and after 20 min of contact time increased the amplitude of the EFS-induced fast relaxation at 8–16 Hz stimulation frequency (Fig. 3) and reduced by 97.162% the sustained one (Fig. 1). Apamin (1 3 10 26 M) did not statistically significantly
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Fig. 1. Effects of PACAP 6-38 on the EFS- and VIP-induced relaxant responses (upper records) and of VIP 10-28 on the relaxation to EFS and PACAP-38 (lower records) on CCh precontracted strips. PACAP 6-38 completely abolished the sustained response that follows the fast one at the highest stimulation frequency and enhances the amplitude of the fast inhibitory response. PACAP 6-38 also abolished the relaxant response to VIP. VIP 10-28 did not influence the amplitude of the fast or the slow component of the response to EFS nor influenced the relaxant response to PACAP-38.
influence (n 5 7) either the fast or the sustained phase of the EFS-induced relaxations. Addition of 1 3 10 27 M VIP (n 5 8) or 1 3 10 27 M PACAP-38 (n 5 8) on the plateau phase elicited by CCh, caused TTX-insensitive sustained relaxant responses (mean amplitude 69.364.9% and 6565.2% for VIP and PACAP38, respectively) that were both abolished by a-chymotrypsin (10 U / ml) (Fig. 1). These direct smooth muscle responses were not influenced by either L-NNA (2 3 10 24 M), suggesting that nitric oxide is not involved in these relaxations, or by apamin (1 3 10 26 M). Addition to the bath medium of PACAP 6-38 (2 3 10 25 M) caused (n 5 8) a slight increase (0.460.03%) in strip tension. PACAP 6-38 enhanced, at the highest stimulation frequency employed (8–16 Hz), the amplitude of the EFS-induced fast relaxation (Figs. 2 and 3) and reduced by 96.563% the amplitude of the sustained one (Fig. 2). PACAP 6-38 (2 3 10 25 M) also abolished the relaxation caused by 1 3 10 27 M VIP (n 5 5) (Fig. 2) and by 1 3 10 27 M PACAP-38 (n 5 4). Addition to the bath medium of VIP 10-28 (2 3 10 25 M) (n 5 10) or [D-p-Cl-Phe 6 ,Leu 17 ]-VIP (2 3 10 25 M) (n 5 10), did not cause any effects on smooth muscle tension. Furthermore they did not influence the amplitude of either the fast (Fig. 3) or sustained inhibitory response
to EFS nor relaxation to 1 3 10 27 M PACAP-38 (Fig. 2) and 1 3 10 27 M VIP.
4. Discussion As previously observed [20] in CCh precontracted longitudinal muscle strips of the mouse gastric fundus and in the presence of guanethidine, EFS evokes fast relaxations during the stimulation period and sustained relaxant responses after the end of the stimulation period itself. These latter are fully manifested at the highest stimulation frequency employed. Therefore, the EFS-induced NANC inhibitory motility obtained in the longitudinal muscle strips from the mouse gastric fundus does not differ to that observed in other animal species either ‘in vitro’ or ‘in vivo’ [1,2,21–23] suggesting that more than one neurotransmitter appears to be involved in these responses. The abolition by the NO synthesis inhibitor, L-NNA, or by the guanylate cyclase inhibitor, ODQ, of the EFS-induced fast response indicates that NO is responsible for this phase of relaxation. In the present experiments VIP and PACAP both cause TTX-insensitive prolonged relaxations indicating a direct muscular relaxant effect. The lack of influence of the NO
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Fig. 2. Effects of a-chymotrypsin on inhibitory responses elicited by EFS (upper records), VIP (middle records) and PACAP-38 (bottom records) on CCh precontracted strips. a-Chymotrypsin (10 U / ml), after 20 min of contact time, completely abolished the sustained relaxation elicited by EFS at the highest stimulation frequency employed. Note also the increase in amplitude of the fast relaxant response elicited during EFS. a-Chymotrypsin (10 U / ml), after 20 min of contact time, also completely abolished the direct inhibitory responses caused by VIP and PACAP-38.
synthesis inhibitor, L-NNA, on direct relaxation elicited either by VIP or PACAP-38 suggests that, in the longitudinal muscle strips from the gastric fundus of the mouse, the action of these peptides is not mediated by the release of NO at the effector level. Contrasting results on the influence of NO synthesis inhibitors on the relaxation induced by both VIP and PACAP in the gastrointestinal smooth muscle preparations have been reported [24–27] and they might be referred to the experimental method employed [28]. In the longitudinal muscle strips from the mouse gastric fundus, the abolition of the EFS-induced delayed relaxant response by a-chymotrypsin suggests that peptides are responsible for this kind of relaxant response and that their release occurs at the highest stimulation frequency employed. An inhibitory neurotransmitter role and a fre-
quency-dependent release have been reported for both VIP and PACAP-38 [11,29]. In the present experiments the abolition of the EFS-induced sustained relaxations by PACAP 6-38, compared with the lack of effects of VIP 10-28 and [D-p-Cl-Phe 6 -Leu 17 ]-VIP, might suggest that PACAP-38 is responsible in the longitudinal muscle strips from the mouse gastric fundus for the EFS-induced sustained relaxations. However, the lack of effects of apamin, an appropriate tool to distinguish PACAP-specific actions compared to VIP [30], on either the EFS or PACAP-38 induced sustained relaxation does not support a role for PACAP-38 itself in the EFS-induced sustained relaxation. The observation that PACAP 6-38 blocks both relaxation to VIP and PACAP-38 may indicate that, in the mouse gastric fundus, PACAP-38 interfere with VIP by acting on VIP receptors responsible for sustained relaxant
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Fig. 3. Effects of a-chymotrypsin (10 U / ml), PACAP 6-38 (2 3 10 25 M), VIP 10-28 (2 3 10 25 M) and [D-p-Cl-Phe 6 ,Leu 17 ]-VIP (2 3 10 25 M) on EFS-induced fast relaxations. Amplitude values represent percentage decreases relative to the tension induced by 1 3 10 26 M CCh. Values are means6S.E.; n 5 6–8 muscle strip preparations. *P , 0.05, significant compared with controls.
responses. The inefficacy of VIP 10-28 and [D-p-Cl-Phe 6 Leu 17 ]-VIP on relaxation to VIP confirms that they have not significant antagonistic properties in response to VIP, itself. The lack of effects of the NO synthesis inhibitor L-NNA, or the guanylate cyclase inhibitor ODQ, on the EFS-induced sustained responses suggests that NO is not involved in this phase of relaxation. A modulatory role for both VIP and PACAP has been reported [17–19,29]: in the present experiments, carried out on CCh precontracted strips, the increase in amplitude of the EFS-induced fast relaxation by a-chymotrypsin and PACAP 6-38 might indicate the presence of prejunctional peptidergic receptors modulating the release of the neurotransmitter responsible for the fast inhibitory response, that appears to be NO. The presence of PACAP receptors on nitrergic neurons has been recently reported in the rat colon [27]. However, since in our experiments PACAP 6-38 seems to interfere, at the muscular level, with VIP receptors we cannot exclude that the modulatory action is exerted by VIP. The inhibitory action of peptides on the release of the neurotransmitter responsible for the fast response may also account for the reduced amplitude of the EFS-induced fast relaxation observed in the dystrophic mice [20] in which sustained responses are well developed and a derangement of neurotransmitter release in favour of a peptidergic one has been suggested. In the guinea-pig gastric circular muscle strips PACAP 6-38 decreases rather than increases the amplitude of the relaxation to EFS [11]. The discrepancy observed may be related to different muscular layers and animal species employed. In fact, in the guinea-pig it appears that only one type of relaxant response is obtained that appears to be mediated by the concomitant release of more than one neurotransmitter since it is reduced but not abolished by L-NNA as well as by PACAP 6-38. In the rat distal colon
PACAP 6-38 and VIP 10-28 have been shown to reduce the amplitude of the EFS-induced fast response without affecting the sustained one [24]. In the mouse gastric fundus PACAP 6-38 not only abolished the slow, sustained relaxation but also increased the amplitude of the fast one at the highest stimulation frequency (i.e. when the peptide is released). Thus, as reported in the literature the variability of effects and the relative potencies of VIP and PACAP differ not only between species but also between various regions of the gut probably reflecting different sets of receptors [27] that differ in structure, ligand specificity and tissue distribution [31]: PAC1 receptors that exhibit high affinity for PACAP and low affinity for VIP and VPAC1 and VPAC2 that show high affinity for both VIP and PACAP. The present results suggest that in the mouse gastric fundus neuropeptides, other than being involved in the EFS-induced sustained relaxation, also modulate the release of the neurotransmitter responsible for the fast relaxation that appears to be NO. This may represent an important physiological mechanism in the control of gastric relaxation: when sustained relaxations are required, NANC fibres discharge at high frequency thus inducing peptide release and a slow, sustained relaxant response. Concomitantly, the release of the neurotransmitter, responsible for the fast short-lasting relaxation, characteristic of peristaltic movements, is inhibited by the peptides themselves.
Acknowledgements The financial support of Telethon-Italy (Grant no. 906 to F.C.) is gratefully acknowledged. This study was also supported by grants from the Ministero dell’Universita` e
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della Ricerca Scientifica e Tecnologica of Italy. The authors thank Alessandro Aiazzi, Salvatore Cammarata and Mario Dolfi for their skilled technical assistance and Adrio Vannucchi for the preparation of Figs. 1 and 2.
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