Naunyn-Schmiedeberg's

Naunyn-Schmiedeberg's Arch Pharmacol (1990) 341: 256- 261

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Pharmacology © Springer-Verlag 1990

Human isolated ileum: motor responses of the circular muscle to electrical field stimulation and exogenous neuropeptides Carlo Alberto Maggi, Riccardo Patacchini, Paolo Santicioli, Sandro Giuliani, Damiano Turini 1, Gabriele Barbanti ~, Antonio Giachetti, and Alberto Meli Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence, 1-5013t Italy 1 Department of Surgical Pathology and Urologic Clinic, University of Ferrara, Italy Received September 22, 1989/Accepted November 13, 1989

Summary. (1) Circularly-oriented muscle strips from the human ileum responded to electrical field stimulation ( 1 50 Hz) with frequency-related primary relaxation at low frequency and primary contractions at high frequencies of stimulation. Both responses were abolished or markedly reduced by tetrodotoxin (1 gM). (2)Atropine (3 laM) or omega conotoxin (0.1 gM) reduced but dit not abolish contraction to electrical field stimulation and enhanced the relaxation. Omega conotoxin (0.1 gM) did not affect carbachol-induced contraction nor isoprenaline-induced relaxation. (3)Neurokinin A and substance P (1 n M I gM) produced a concentration-dependent contraction. The N K - I receptor selective agonist, [Pro9]Sp sulfone and the NK-2 receptor selective agonist []~AlaS]NKA(4-10) produced a contraction superimposable to that of substance P and neurokinin A, respectively. On the other hand, [MePhe7]-neurokinin B, an NK-3 receptor selective agonist was ineffective up to I gM. The response to substance P or neurokinin A was unaffected by atropine (3gM). (4) Galanin, up to 0.1 gM, produced a weak and inconsistent contraction. (5) Vasoactive intestinal polypeptide (10 n M i gM) produced a concentration-dependent relaxation while human alpha calcitonin gene-related peptide exerted a weak and inconsistent relaxant effect. (6) These findings indicate that both cholinergic excitatory and non-cholinergic inhibitory nerves affect the motility of the circular muscle of the human small intestine. Transmitter release from excitatory nerves seems largely mediated by activation of omega conotoxin-sensitive (N-type) calcium channels. Tachykinins exert a potent contractile effect, independently ofcholinergic nerves, via N K - I and NK-2 receptors. Key words: Human - Isolated ileum - Circular muscle Tachykinins - Tachykinin receptors - Galanin - VIP CGRP

Introduction In the past 15 years (Pearse ans Polak 1975) much evidence has accumulated indicating that various peptides, released either from intrinsic neurons or extrinsic nerves play an important role in regulating motility of the intestine (see Costa and Furness 1982; Costa et al. 1987; Furness and Costa 1987; Barth6 and Holzer 1985 for reviews). Available evidence, based on pharmacological antagonism, receptor Send offprint requests to C. A. Maggi at the above address

desensitization by exogenously administered peptides and immunoblockade techniques indicates that several peptides such as tachykinins (substance P, SP and neurokinin A, NKA), galanin, calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) play a transmitter role in the mammalian gut (Angel et al. 1983; Grider et al. 1985; Costa et al. 1985; Barth6 and Holzer 1985; Maggi et al. 1986a; Muramatsu and Yanaihara 1988). With few exceptions (Holzer 1982) systematic comparative studies on species differences in the responses to peptides of the mammalian gut have not been performed, but it appears evident that marked species-related differences exist. These may involve different mechanism of action or differences in receptors mediating a particular effect. As an example, C G R P inhibits the motility of the small intenstine longitudinal muscle in both rats and guinea-pigs. However C G R P is a direct smooth muscle relaxant in the latter species while a fraction of the relaxation in the rat intestine is neurally mediated (Maggi et al. 1986 a; Barth6 et al. 1987). With regard to the action of tachykinins, these peptides exert a direct contractile effect on the longitudinal muscle of the rat small intestine which involves both NK-1 and NK-2 receptors (Maggi et al. 1986b). By contrast, the action of tachykinins in the longitudinal muscle of the guinea-pig ileum has at least two components, one direct on smooth muscle cells via N K - I receptors and one indirect, due to activation of intramural neurons, via NK-3 receptors (Barth6 and Holzer 1985; Laufer et al. 1985, 1988; Guard and Watson 1987). Anatomical studies have shown that several peptides (including substance P, galanin, C G R P and VIP) are present in nerve fibers and neurons of the human gut (Ferri et al. 1983, 1988; Brodin et al. 1983; Llewellyn-Smith et al. 1984; Bauer et al. 1986; Bishop et al. 1986; Wattchow et al. 1988) but functional studies on the motor responses of the human isolated small intestine are few (Bennett 1965; Whitney 1965; Shirazi et al. 1980; Maggi et al. 1988a). The existence of species-related differences in innervation and expression of peptide receptors makes it important to get knowledge about the functional properties of the human gut. We have described recently (Maggi et al. 1989a) the response of the longitudinal muscle from the human isolated small intestine to electrical field stimulation, tachykinins, galanin, VIP and CGRP. Here we present data about the response of the circular muscle from the human ileum to these stimuli.

257 Methods

The motor responses to electrical field stimulation and neuropeptides were investigated on 65 ileal strips from 22 patients (age 4 5 - 7 8 years) who underwent abdominal surgery for carcinoma of the bladder base (enterocystoplasty). No patient received radio- or chemotherapy before intervention. In all patients, pre-anesthetic medication was intramuscular atropine (ling) and diazepam (10rag). Anesthesia was induced by sodium thiopenthal (500 mg i.v.) and maintained with N20/O2 (1/2) and halothane ( 0 . 6 1%). The patients received pancuronium bromide (6 mg i.v.) during induction of anesthesia. All specimens appeared macroscopically normal without signs of tumor or inflammation. The tissues were placed in ice-cold Krebs solution within 2 - 3 rain after surgical removal. Composition of Krebs solution was as follows (mM): NaC1 119, NaHCO3 25, KH2PO4 1.2, MgSO4 1.5, KC1 4.7, CaCI2 2.5 and glucose 11. The specimens were pinned flat on a Petri dish containing Krebs solution and the mucosa was carefully dissected out. Small strips ( 0 . 5 0.8 cm long, 2 - 3 m m wide) of muscle were cut along the circular axis. The strips were stored overnight in ice-cold (4 ° C) continuously oxygenated (96% O2, 4% COz) Krebs solution, e.g. functional experiments started 1 6 - 2 0 h after surgery ("stored" strips) (cf. Maggi et al. 1989 a). The strips were suspended under a resting tension of 10 m N in organ baths (5 ml) containing oxygenated Krebs solution at 37 ° C. Mechanical activity was recorded by means of isotonic transducers and recorded on a Basile 7050 Unirecord. Some experiments involving the response to electrical field stimulation, tachykinins or galanin were conducted in parallel on paired strips excised from the same samples which were either: (a) cut along the circular and longitudinal axis (isotonic recording of activity from paired strips) or (b) cut along the circular axis and tension recorded by an isotonic and isometric transducer (load 10 mN) on paired strips. In these latter experiments (n = 4, data not shown) no qualitative difference between response to electrical or chemical (neuropeptides) stimulation was found in circular strips between isotonic and isometric recording. The strips were electrically field stimulated by means of two platinum wire electrodes placed at the top and the bottom of the organ bath by means of a GRASS $11 stimulator. All experiments commenced after an equilibration period of 90 - 120 min during which the bathing solution was renewed every 1 0 - 1 5 min. Contractile and relaxant responses were expressed as % of the maximal contractile response to carbachol (0.1 mM) or of the maximal relaxant response to isoprenaline (10 ~tM). These responses were assessed twice in each strip before studying the responses to electrical field stimulation or to neuropeptides. Frequency-response curves to electrical field stimulation (1 - 50 Hz, 0.5 ms pulse width, 60 V, train duration 5 s) were constructed as follows: trains of stimuli were manually delivered at 5 - 10 rain intervals with progressive increase in frequency of stimulation from I to 50 Hz. At this time the solution in the bath was changed by rinsing and the frequency-response curve repeated after 30 min. When reproducible responses were obtained (usually the third curve was superimposable to the second) atropine or omega conotoxin were added to the bath and a new curve was constructed 15 rain later. Control experiments (n = 3) showed that the

response to a fourth frequency response did not differ from the third. Omega conotoxin and atropine were investigated on different strips. In some experiments the effects of carbachol or isoprenaline were checked again, at the end of the frequency response curve, in presence of atropine or omega conotoxin. Concentration-response curves to peptides were constructed in a non-cumulative manner to avoid desensitization. In a few experiments, cumulative concentration-response curves to substance P and neurokinin A were obtained. For non-cumulative curves, increasing concentrations of peptides were added to the bath at 1 0 - 2 0 rain intervals and left in contact until maixmal responses developed. A through washing out was made for t h r e e - f o u r times at 2 rain intervals between doses. The effect ofpeptides was studied on strips different from those on which the response to electrical field stimulation was investigated. The effects of substance P and neurokinin A were studied on paired strips from the same samples. In some instances the response to tachykinins was investigated in the absence and presence of atropine.

Statistical analysis. All data in the text are mean _+ standard error (SE) of the mean. Statistical analysis was performed by means of the Student's t-test for paired or unpaired data or by means of the analysis of variance, when applicable. Drugs. Atropine HC1 and isoprenaline HC1 (Serva, Heidelberg, FRG), guanethidine sulphate (ICFI, Milan Italy) tetrodotoxin (Sankyo, Tokyo, Japan), substance P, neurokinin A (NKA), galanin, vasoactive intestinal polypeptide, human alpha CGRP, omega conotoxin fraction GVIA (all from Peninsula, San Carlos, CA, USA). [Pro9]-SP sulfone and [MePheT]-neurokinin B, two selective agonists of the NK-1 and NK-3 receptors, respectively (Drapeau et al. 1987), were kind gifts of Prof. D. Regoli, Dept. of Pharmacology and Physiology, Sherbrooke University, Canada. [/~AlaS]-NKA(4-10) a newly developed selective NK-2 receptor agonist (Rovero et al. 1989) was synthesized by Dr. P. Rovero, Chemistry Department, Menarini Parmaceuticals, by conventional solid-phase methods.

Results

Response to electrical field stimulation About 70% of the strips (47 out of 68) showed spontaneous activity the amplitude of which was < 5 - 1 0 % of the maximal response to carbachol (0.1 raM). In some instances spontaneous activty was present for some hours at the beginning of the experiment and then disappeared. Isoprenaline (10 gM) induced relaxation and suppression of spontaneous activity in all instances. The response to carbachol was prevented by atropine (3 gM, contact time 15 rain). In the experiments in which isometric recording was made the contractile response carbachol ranged between 15 and 32 m N and the relaxant response to isoprenaline between 2.5 and 5 raN. A complete frequency-response curve to electrical field stimulation was determined in 11 strips from different patients. Three patterns of response were observed: (a) primary relaxation at low frequencies and primary contractions at

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Fig. 3. Concentration-dependent contractile response of circularly oriented muscle strips from the human ileum to substance P (SP), neurokinin A (NKA) in the absence or presence of atropine (3 gM) (left panel) or to synthetic, receptor-selective tachykinin agonists. Each value is mean _+ SE of at least 4 experiments

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Fig. 2. Effect of atropine (3 I~M, upperpanels) or omega conotoxin (CTX, 0.1 gM, lowerpanels) on the contractile and relaxant response of circularly oriented muscle strips from the human ileum to electrical field stimulation. The contraction and/or relaxation effect observed at each frequency of stimulation is expressed as percent of the maixmal response obtained in each strip. Each value is mean _+ SE of 4 - 6 experiments. * Significantly different from control, P < 0.05

Atropine (3 gM) had no effect on spontaneous activity but produced a small reduction in tone in 4 out of 6 cases and abolished or greatly reduced ( > 90%) the contraction to carbachol (0.1 raM). In the presence of atropine (contact time 15 min, n = 6), a primary relaxation in response to electrical field stimulation was evident at all frequencies tested. Further, the amplitude of the primary relaxation was significantly increased as compared to controls (Fig. 2). In contrast, the amplitude of the contraction to electrical field stimulation was significantly reduced, although not abolished, at all frequencies tested (Fig. 2). In a few cases (n = 3 strips from different patients) the response to field stimulation was also assessed in the presence of atropine plus guanethidine (3 gM each, contact time 60 rain). In these experiments, the amplitude of the nervemediated relaxation was not significantly different from that observed with atropine alone (data not shown).

Effect of omega conotoxin high frequencies (3 cases); (b)primary relaxation and rebound contractions at low frequencies and primary contraction at high frequencies (6 cases) (Fig. 1) or (c) frequencydependent primary contractions (2 cases). Overall, a primary relaxation to electrical field stimulation was observed in 9 out of 11 strips. The primary relaxation was observed at 1 5 H z in 3 cases, at 1 to 1 0 - 2 0 Hz in 6 cases. The contraction to field stimulation was frequency-dependent (left panels in Fig. 2). The maximal response was observed at 50 Hz in 9 out of 11 cases and at 20 Hz in the remainder. The contraction to field stimulation at 50 Hz averaged 45 _+ 4% (n = 11) of the maximal contraction to carbachol (0.1 mM) in the same strips. The primary relaxation induced by field stimulation was maximal at 2 - 5 Hz (right panels in Fig. 2) and averaged 42 + 3% of the maximal relaxation produced by isoprenaline (10 laM).

Omega conotoxin (CTX, 0.1 gM, n = 4) had no effect on tone in 2 cases and produced a small contraction in 2 strips. In the presence of CTX (contact time 15 min) a primary relaxation to field stimulation became evident at all frequencies tested (Fig. 2); the contraction to field stimulation was abolished up to 10 Hz while a residual response, significantly smaller than that obtained during the control period, was produced at 2 0 - 5 0 Hz (Fig. 2). CTX did not affect the contraction to carbachol (n = 3), nor the relaxation to isoprenaline (n = 3).

Effect of tachykinins Both SP and N K A (1 n M - - 1 gM) produced a concentration-dependent contraction (Fig. 3, n = 6). As shown in Figs. 3 and 4, N K A was more effective than SP. The response

259 Discussion

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Human isolated ileum: motor responses of the circular muscle to electrical field stimulation and exogenous neuropeptides.

(1) Circularly-oriented muscle strips from the human ileum responded to electrical field stimulation (1-50 Hz) with frequency-related primary relaxati...
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