Neurotensin: dual effect on the motor activity of rat duodenum

l'.uropean Journal of Pharmacology, 212 (1992) 215-224

215

~: 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52311

Neurotensin: dual effect on the motor activity of rat d u o d e n u m F l a v i a Mul~:, A l c s s a n d r a P o s t o r i n o , A n n a G e r a c i a n d R o s a S c r i o Dipartimento di Biologia Cellulare e dello Sciluppo, Laboratorio di Fisiologia Generale, Unirersit~ di Palermo, Corso Tukory 129, 1-90134 Palermo, Raly

Received 4 April 1991, revised MS received 25 November 1991, accepted 3 December 1991

The cffccts of neurotensin on mechanical activity of rat duodenum were investigated using an isometric-isovolumic preparation. Neurotensin (1 pM to 10 nM) induced a concentration-dependent, tctrodotoxin (TTX)-inscnsitivc fall in both endoluminal pressure and isometric tension. At higher concentrations of neurotensin (1 nM to 1 ~M) the relaxation was followed by a concentration-dependent TTX-inscnsitivc contraction, detected only by an increase in endoluminal pressure. Different concentrations of neurotcnsin were required to desensitize the rclaxant and the contractile actions of the neuropeptide. The relaxation was antagonized by apamin, while the contractile response was blocked by nifcdipine. Neurotcnsin, when tested separately on longitudinal and circular muscular strips, caused relaxation of the longitudinal strips. Circular strips showed contractions in response to ncurotensin, following an inhibitory phase, if the strips wcrc spontaneously or pharmacologically activated. Thc results suggest the presence of two sets of neurotensin receptors with a differential localization between the two muscular layers in rat duodenum. Neurotensin; Neurotensin receptors; Non-adrenergic non-cholinergic (NANC) relaxation; Duodenum (rat); Neurotcnsin-induced rel,%xation; Neurotcnsin-induced contraction

1. Introduction

N e u r o t e n s i n is a tridecapeptide originally isolated and characterized from bovine hypothalamus (Carraway and L c c m a n , 1973); the peptidc was subsequently localized in a variety of brain nuclei where it may act as n e u r o t r a n s m i t t e r ( E m s o n et al., 1982). In the periphery, ncurotensin is largely c o n c e n t r a t e d in the gastrointestinal tract, particularly in the small intestine. Immunohistochemical studies have established its presence in the N-cells of the duodenal and ileal mucosa and to a lesser extent in the nervous elemcnts of the mycntcric plexus (Rcinecke et al., 1983; Schulzberg ct al., 1980; Sundler et al., 1977). Indeed, neurotensin possesses multiple actions on the gastrointestinal tract, y:elding effects that range from relaxant to contractile or biphasic responses, but its role as a paracrinc hormone, neurotransmitter or neurocrine h o r m o n e remains to be elucidated (Carraway and L c e m a n , 1973; H u i d o b r o - T o r o , 1983; H u i d o b r o - T o r o and Kullak, 1985; H u i d o b r o - T o r o and Way, 1982; H u i d o b r o - T o r o and Zhu, 1984; H u i d o b r o - T o r o et al., 1984; Kitabgi and Freychet, 1978, 1979; Kitabgi and Vincent, 1981;

Quirion et al., 1980). Little is known about the inhibitory effect and the mechanism of the inhibitory action of ncurotensin, and the link betwccn receptor activation and cellular response has never been investigated in the rat intestine. Such myorclaxant effccts could bc related to the inhibition of gastrointestinal motility observed in vivo following fat ingestion (AISaffar and Rosell, 11981; Hellstrom ct al., 1982; Keinke et al., 1986). A hypothesis has been advanced, concerning the role of neurotensin as a neuronal factor; it is suggested that ncurotensin or a related substance may act as m e d i a t o r of non-adrenergic non-cholincrgic ( N A N C ) inhibitory neurons supplying the circular muscle of the guinea-pig ileum ( G o e d e r t et at., 1984). T h e objectives of the present investigation werc: (1) to attempt to clarify w h e t h e r neurotensin interacts with specific receptors, and (2) to characterize the mechanism of the response to ncurotensin and its possible relation to the inhibitory effect induced by N A N C nerves in the rat d u o d e n u m .

2. Material and methods

C:)rrespo~dencc to: F. Mul~:, Dipartimcnto di Biologia Cellulare c dello Sviluppo, Corso "l'uko~" 129, 90134 Palermo, Italy.

Adult male Wistar rats (250-350 g) were used. The rats were killed by cervical dislocation, the a b d o m e n

216 was rapidly opened by a midline incision and a duodenal segment of about 2 cm length was removed just distal to the pylorus.

2.1. Preparation of isolated duodenum The duodenal segment was placed in a 30-ml organ-bath, continuously pcrfuscd at a rate of 5 m l / m i n with Krebs solution at 37°C gassed with 95% 0 2 and 5% CO 2. The composition of the Krebs solution was the following (mM): NaCI 119, KCI 4.5, MgSO 4 2.5, NaHCO325 , K H 2 P O 4 1.2, CaCI 2 2.5, glucose 11.11. An isometric-isovolumic preparation was used. The distal end of the duodenal segment was connected via a T catheter to a syringe and to a pressure transducer (Statham P23). The proximal cnd was secured with surgical silk to a Grass FT03 isometric force-displacement transducer. The mechanical signals were detected as changes in both cndoluminal pressure (EP) and isometric tension (IT) and recorded on a Bcckman R411 polygraph. Isometric tension presumably measures the response of longitudinal muscle, while endoluminal pressure measures mostly circular muscle and some longitudinal muscle responses. The intestinal segment was distended with 0.3-0.5 ml physiological solution and subjected to an initial tension of 1.5-2 g. The preparation was allowed to equilibrate for at least 30 min before drug application. Electrical field stimulation (EFS) was applied in 5-s trains (0.5 ms, 30 Hz, supramaximal voltage) via platinum ring electrodes and was delivered by a Grass $88 stimulator. Agonists wcre added into the bath after switching off the perfusion for the time of testing. Occasionally, switching off the pcrfusion caused sligth relaxation, so a 5-min delay was allowed before addition of the drug into the bath. Perfusion was resumed after each addition of drugs. Carbachol (1/xM) or noradrenalinc (1 # M ) w e r e tested at the beginning of each experiment. The noradrenalinc-induced optimal effect was a relaxation of about 4.5-5 cm H 2 0 and 2.5-3 g; the carbachol-induced optimal effect was a contraction of about 15-16 cm H 2 0 and 3-3.5 g. The tissue was incubated with neurotensin for 2 min. Concentration-response curves to neurotcnsin wcrc obtained in the same preparation in a non-cumulative manner. Therefore, neurotensin was added to the bath as single addition at intervals of 30 min. The results arc expresscd as the percentagc of the maximal neurotensin response achieved. The conccntration of neurotcnsin required to cause a half-maximal relaxant effect (ICs0) or half-maximal contractile effect (ECs0) was calculated by interpolation from the rcspective concentration-response curves.

1983; Huidobro-Toro and Zhu, 1984; Huidobro-Toro and Kullak, 1985), was used to study receptor specificity. The tissues wcrc pretreated with a concentration of ncurotensin which was added to the perfusing solution. Different dcscnsitizing concentrations of the peptide ( 1 - 5 - 1 0 - 5 0 riM) were tested. At least 20 min elapsed (without washing the tissue) before determination of the effects of a neurotensin-desensitizing conccntration on the rcsponse to added ncurotcnsin. In fact, neurotcnsin, when added to the Krebs perfusing solution in order to desensitize the receptors, elicited repeated relaxations followed by contractions of duodenal smooth muscle. However, the resting conditions were gradually restored within 10-15 min. Each concentration-response curve was obtained in the same tissue before and after the neurotensin-induced desensitization. l'o explore the specificity of desensitization, the responses to carbachol (1 p.M) or noradrenaline (1 /~M) were tested before and after desensitization. Desensitization was reversible; the amplitude of the response to neuropcptide recovered afte~ extensive washout with normal Krcbs solution.

2.3. Preparation of duodenal circular and longitudinal strips Longitudinal and circular muscular strips (2 × 6 mm) were used in some experiments. Strips were cut parallel to the longitudinal or circular muscle axis, and were mounted vertically in 5-ml organ baths filled with Krcbs solution of the same composition as described earlier and bubbled with 5% CO2-95% 0 2 at 37°C. One end of each strip was secured by silk thread to a plcxiglass clip and the other end to a Grass FT03 force transducer. The strips were placed under an initial tension of 1 g and allowed to equilibrate for 1 h before the start of the experiment. Isometric tenskm was recorded on an ink-writing polygraph (Grass model 79D).

2.4. Data analysis The amplitude of the relaxations induced by different experimental procedures was measured from the baseline (an ideal midline between the spontaneous changes of activity) to the lowest point reached. The contractile response was calculated as the increase in tension above thc baseline. All data arc given as means _+ S.E.; n indicates the number of different animal preparations. Statistical significance was estimated using the paired Student's t-test and a P value less than 0.05 was considered to be significant.

2.2. Desensitization protocol

2.5. Drugs

Desensitization, obtained according to the protocol dcscribed previously (Huidobro-Toro and Yoshimura,

The following drugs were uscd: neurotcnsin acetate salt (NT), carbamylcholine chloride (carbachol), at-

217

r~pinc sulphate, guanethidine sulphate, propranolol hydrochloride, tolazolinc hydrochloride, methyscrgide hydrogen maleate, noradrenaline hydrochloride, ranitidine hydrochloride, diphenhydramine hydrochloride, indomcthacin, tetrodotoxin (q*FX), apamin, nifedipine. All drugs were purchased from Sigma, except guancthidine which was a gift from Ciba, ranitidine, a gift from Glaxo and mcthysergide, a gift from Sandoz. Neuv,~tensin was tested in the presence of different antagonists, which were added to the pcrfusing solution at least 20 min before the addition of ncurotcnsin to the bath. Ncurotensin was also tested before and after 15-min pre-incubation with T I ' X , apamin and nifedipinc or 1 h after prc-incubation with indomethacin. Stock solutions were prepared using distilled water and were kept frozen. The working solutions were prepared frcsh on the day of the experiment by diluting the stock solutions in saline. Indomcthacin and nifedipinc were dissolved in absolute ethanol. Control tests using ethanol alone showed that neither resting activity nor basal tone were significantly modified. Care was taken to avoid exposure of noradrenaline and nifedipine to light. Some experiments were performed using a solution containing no calcium salt. The preparations were incubated for at least 20 rain with calcium-free Krebs solution.

3. Results

3_ 1. EJfects of neurotensin on the duodenal segment As previously described, rat duodenum preparations in vitro exibited spontaneous phasic contractions, made up of changes in both endoluminal pressure and isometric tension (from 2 to 10 cm H 2 0 ; from 0.2 to 1 g) (Postorino et al., 1990a; Serio et al., 1990). Neurotcnsin caused a complex muscular response in the rat duodenal segment (fig. 1). At the lowest concentrations of the neuropeptide (1 pM to 10 nM), there was an early, concentration-dependent relaxant effect characterized by a transient fall in both endoluminal pressure and isometric tension. Occasionally this initial relaxation was followed by a brief and short-lived rebound contraction (in 11 out of 37 preparations) but this effect was not concentration-related. The duration of the relaxation was quite constant (19.5 + 1.2 s; n = 22) irrespective of the concentration used. The maximal a:nplitude of the relaxant effect was 4 + (I.2 cm H 2 0 for endoluminal pressure and 2.1 ._-Z0.3 g for isometric tension, both reaching about 85%. of the effect caused by 1 /xM NA. In addition, at the highest concentrations, neurotensin (1 nM to 1 /xM) induced a delayed concentration-dependent contractile effect which faded slightly, even if the control level of muscular activity had not recovered within 2 min. This contractile effect

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Fi~. 1. ~ff~ct~ i n ~ u ~ ~y n ~ o t ~ n ~ i ~ (0.1 ~ M ) on ~ n d o l u m i ~ l pressure (upper trace) and on isometric tension (lower trace) of an isolated segment o f rat duodenum It shoul~ be noted that the relaxation was followed by a rebound contraction (marked by dotted lin~) ~nd hy a~ i ~ c ~ s ~ i~ W ~ u r e ton~ (t~r~ive co~tr~ctil~ ~ff~ct marked by horizontal line). Solid line wflh arrow indicates period of application o f peptidc.

was characterized by a significant increase in tone, frequency and amplitude of phasic mechanical activity. The 'rebound contraction' was distinguished from the contractile effect since it appeared as a 'phasic' contraction, of higher amplitude than the delayed neurotensin-induccd contraction, which, on the contra~,, looked like a 'tonic' cffcct. The delayed contractile effect was a peculiarity of the endoluminal pressure recordings. In fact, it was variably present on the isometric tension recordings and, if it was present, its amplitude stayed constant at the different ncurotensin concentrations tested. In the presence of sufficient q~FX (1 /~M) to block electrically stimulated nerve responses, neither the early relaxant nor the contractile response to neurotensin were significantly different from those under control conditions. Atropine (1 #M), tolazolinc (1 /~M) plus propranolol (1 g M ) , a mixture of diphenhydramine (10/~M) and ranitidine (10 ~M), mcthysergidc (10 /.~M) and indomcthacin (10 ~zM) failed to modify the effects induced by neurotensin (0.1 /~M). The results of these experiments arc summarized in table 1.

3.2. Relaxant effects Figure 2 shows the concentration-response curves for the relaxant effect induced by neurotensin on cndoluminal pressure and on isometric tension in the control and after various neurotcnsin concentrations desensitizing the neurotensin receptors. No difference was found in the potency or efficacy of neurotcnsin for inducing the relaxant effect on endoluminal pressure and on isometric tension. Preincubation of intestinal segments with 1 nM neurotensin did not significantly modify the concentration-response curves for the pep-

218 'I'AIH,E 1

TABLE 2

Lack of cffect of tetrodotoxin (TTX), atropinc (Atr), tolazolinc (Tol) and propranolol (Prop), indomelhacin (lnd), ranitidinc (Rd) and diphcnhydranfinc (Diphe), and methysergidc (IMeth) on tbc complcx rcsponse of rat dt, odenum induced by net, rotcnsin (0.1 "aM). The values arc given as means+S.E. No significant diffcrcnces werc found betwccn the responses in the absence and in the prcscncc of tbc w~rious antagonists.

Potency of neurotcnsin (NT) for inducing a relaxant effect (ICso) or a contractile effect (ECs0) on cndoluminal pressure (EP) and isometric tension (1"1')of rat duodenum. "llac values are given as means t- S.E. a p < 0.01 as compared to the 1C50 before desensitization, b p < 0.01 as compared to the EC.s0 before dcscnsitization.

n

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EP (cm H 2 0 )

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Contractile effects

Ncurotensin caused a c o n c e n t r a t i o n - d e p e n d e n t contractile effect on endoluminal pressure (fig. 3), with a maximum response of 9.2_+ 0.9 cm I I 2 0 , which was about 60% of the 1 btM carbachol contracture. The contractile effect on isometric tension was not taken into account since it appeared as an erratic response. It should be m e n t i o n e d that the concentration range, for inducing contractile effects was greater than that required to obtain relaxation. In fact, the relaxant re-

pressure

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ation was not significantly modified in the presence of neurotcnsin r c c c p t o r desensitization, and was 4.6 + 1.4 cm H 2 0 and 2.5 + 0.2 g before and 5.6.+_ 1.3 cm H 2 0 and 2.6 + 0.2 g after trcatment (P > 0.05; n = 5).

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tide. W h e n the desensitizing concentration of neum t e n s i n was increased to 5 nM, thc neurotcnsin conc e n t r a t i o n - r c s p o n s c curve was displaced to the right with a significant reduction of the maximal relaxant effect of the peptide (P < 0.01; n = 5). With 10 nM ncurotcnsin there was an almost c o m p l e t e loss of thc response to neurotcnsin and the curves were markedly tlattcncd, reaching a plateau at about 20% relaxation. The ICs0 valucs for the relaxant effects of n e u m t c n s i n before and after ncurotcnsin receptor desensitization arc shown in table 2. The noradrenalinc-indttced relax-

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- LOg MBLA:~ CONgt-XT4,~r10N Fig. 2. Concentration-response curves for the relaxant effect induced by ncurotensin on rat duodenum endoh, minal pressure and isometric tension, in thc control and after various desensitizing concentrations of the peptidc. The effects arc expressed as a percentage of the ncu,otensin maximal relaxant response. Symbols represent the mean of the values obtained from five preparations and bars show the S.E. Responses to the relaxanl action of ncurotensin were determined prior to ( • ) and following the pcrft, sion of desensitizing concentrations of 1 ( ~ ) . 5 (t3), 10 (,~) nM of neurotcnsin.

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Fig. 3. Concentration-response cu~cs for the contractile effect of neurotcnsin on rat duodenum cndoluminal pressure, in tbc control and aftcr various desensitizing concentrations of lhe pcptide. The effects are expressed as a perccntage of tbc neurotcnsin maximal contractile response. Symbols represent the means of the values obtained from 6 preparations and bars show the S.E. Responses to the contractile action of ncurotcnsin wcrc determincd prior to ( ~ ) and following thc pcrfusion of desensitizing concentrations of 5 ( ~ ) , 10 ( ~ ), 50 ( • ) nM of neurotcnsin.

219

sponse appeared, and reached saturation, at concentrations lower than those required for maximal contractions. Pretreatment of the tissue with 5 nM ncurotcnsin did not significantly alter the concentrationresponse curve for the contractile effect induced by ncurotensin. Increasing the neuropeptide concentration to 10 nM caused a shift of the curve to the right and downwards with a significant reduction of the maximal contractile effect (P <0.01; n = 6). After a 50-nM desensitizing concentration, the curve was markedly flattened, with a maximal cffcct at 30% of the control. Under such conditions, the 1 /xM carbachol-induccd contractile effect was not affected, being 16.5 _+ 2.2 cm H 2 0 before and 16.2 _+ 1.8 cm I t 2 0 alter desensitization. The ECs0 values for the contractile effect of neurotensin before and after desensitization are shown in table 2.

(Jenkinson, 1981). When apamin (10 nM) was added to the solution perfusing the duodenal segment, the spontaneous phasic activity, detected as changes of the endoluminal pressure, was enhanced in amplitude and frequency. There was no modification of the spontaneous changes in isometric tension in the presence of apamin. Prctreatment of the duodenum with 10 nM apamin caused an about 50% reduction in the relaxation induced by ncurotensin (0.1 p,M). Increasing the concentration of apamin to 100 nM abolished the neurotensin-induced r c l ~ a t i o n and thc rebound contraction, when it was present, whcreas it failed to affect the contractile effect (fig. 4). Apamin 10 nM caused a non-parallel shift to the right of the concentration-response curve for the ncurotcnsin relaxant effect, increasing thc ICs, from 0.23 +_ 0.08 to 5.5 + 1.2 nM for pressure and 0.140_+ 0.10 to 5.7_+ 1.7 nM for tension (fig. 4). Apamin 10 nM did not substantially alter the concentration-response curve for the contractile effect, failing to change the potency and the maximal effect of the neuropeptide. The calculated EC,~0 was 17 + 7 nM for the control and 14 = 3 nM after pretreatment with the bee venom. The noradrcnalinc-induccd relaxation was not significantly modified in the

3.4. Effect of apamin on neurotensin-induced response The response to neurotensin was tested in the presence of apamin, thc 19-amino acid bee venom toxin, which is known to prevent the opening of C a : - d e p e n dent K~-channcls operated by inhibitory stimuli

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Fig. 4. t';ffects of apamin (10 nM) on the complex response to ncnrotcnsin in a rat duodcnal segment. Concentration response c u ~ e s |o r thc relaxant (upper panels') or contractile (lower panel) effects induced by neurotensin on endoluminal pressure and isometric tension, in the control ( ~! ) and after apamin ( 10 nM) ( ~ ) . Effccts are expressed as a percentage of the ncurotensin maximal response. Symbols represent the means of the values obtained fiom five preparations and bars show the S.E. Lower panel on the right shows that the relaxant effect of the ncuropeptide (0.1 ,aM) was antagonized in the presence of apamin (10 nM), while the increase in the pressure tone remained unaffected.

220

CONTROL

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Fig. 5. Effect of lack of calcium in the extraceilular solution on the complex response induced by neurotensin (0.1 ~M) on rat duodenum endoluminal pressure and isometric tension. The rclaxant component was strongly reduced in Ca ~ ~-free solution but the contractile response was abolished.

presence of apamin (10 nM) being 5.9 N 2.4 cm H 2 0 and 1.9 + 0.8 g in the control and 6.5 N 2.7 cm H e O and 2.0 N 0.8 g after treatment (P > 0.05) (n = 4).

3.5. lzffects of Ca-'--free solution and nifedipine on neuroter~'in-induced responses The relaxant and contractile effects of neurotensin wcrc also tested in the presence of nifedipine or in the absence of Ca -~+ in the physiological solution. In a Ca2"-frcc Krebs the fall in endoluminal pressure and in isometric tension was markedly reduced in amplitude (about 3(1% of the control), whereas the increase in pressure tone was completely abolished (n = 5) (fig. 5). Nifedipine pretreatmcnt (1 riM) failed to modify the spontaneous mechanical activity and the complex response to 0.1 /aM neurotensin; increasing the concentration to 5 nM slightly reduced spontanous activity. Under such conditions, the relaxant component of the response to ncurotensin was unaffected, whereas the contractile effect was significantly decreased (P < 0.01;

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Fig. 6. Influence of nifedipine (5 nM) on both components of the complex response of rat duodenum to neurotensin (0.1 #M). The data obtained from four preparations are expressed as m e a n s + S.E. and reported as percentages of the maximal response (relaxant or contractile) of the control. * P < 0.05. Open columns: relaxant effect in the control. Closed column: contractile effect in the control. Striped columns: nifcdipine.

30 s

EFS

EFS

Fig. 7. Inhibitor' effects of electrical field stimulation (EFS) (0.5 ms, 30 tlz, supramaximal w~ltagc, 5 s train) on endoluminal pressure (upper traces) and isometric tension (lower traces) of rat duodenum in the control and after 5 nM neurotcnsin, the concentration desensitizing the neurotcnsin inhibitory receptors.

n = 4) (fig. 6). Figure 6 shows that nifcdipinc failed to modify the maximal relaxant effect of ncurotensin on both endoluminal pressure and isometric tension, whereas it antagonized significantly the contractile effect of neurotensin (0.1 / a M ) o n cndoluminal pressure. We could not test the response to neurotensin in the presence of a greater concentration of nifedipine (10 nM), because this calcium channel antagonist caused non-specific effects, abolishing spontaneous mechanical activity and the duodenal rcsponsc to NA, carbachol and electrical field stimulation.

3. 6. Electrical fieM stimulation and neurotensin desensitization As previously described (Serio et al., 1990), electrical field stimulation, performed in the presence of atropine (1 /aM) and guanethidine (1 /aM), induced in rat duodenum a TTX-sensitive transient fall both in endoluminal pressure and in isomctric tension, followed by a rebound contraction. The electrical field stimulation-induced relaxation was completely abolished in the prescncc of apamin (100 nM). Neurotcnsin 10 nM, which desensitizes the reccptors responsible for the ncurotcnsin-induced relaxant effect failed to affect significantly the electrical field stimulation-induced relaxation, which was 12.6 + 3 cm H e O and 3.6 _+ 0.1 g in the control and 10.8_+ 2.5 cm H z O and 3.6_+ 0.1 after desensitization (P > 0.05; n = 5) (fig. 7).

3. 7. Effects of neurotensin on longitudinal and circular muscular strips We attempted to further differentiate the actions of neurotensin on the longitudinal and circular muscles of the rat duodenum, by testing the neurotcnsin effects on each muscle layer separately.

221

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] I!|1~

Fig. 8. Mechanical activity detected in circular (CS) and longitudinal (LS) strips and effects induced by ncurotensin (0.1 p.M). Arrows indicate ncurotensin application. In the experiment shown in the middle tracing ncurotcnsin was added after TTX pretreatmcnt.

Longitudinal strips always showed spontaneous phasic mechanical activity similar to that detected in the entire segment. Neurotcnsin (1-100 nM) induced either a TTX-inscnsitive reduction of spontaneous activity or, more frequently, relaxation by about 0.5 g (35% of 1 /xM NA-induccd relaxation) (n = 5). Prctrcatment for 3 min of the preparation with 100 nM apamin completely abolished the neurotcnsin-induccd relaxazion and the response was not converted into a contraction. Circular strips variably presented spontaneous mechanical activity. This phasic activity was characterized by irregular contraction of variable amplitude (0.2-1.2 g) and low frequency. T T X (1 > M ) pretrcatment resuited in the appearance of contractions. Neurotensin (100 nM) induced TTX-inscnsitive contractions (1.5-2 g) (n = 5) and a slight iucrease in tone in the quiescent strips. In spontaneously active or T T X - p r e t r c a t c d strips, ncurotensin caused a transient block of mechanical activity, followed by a delayed increase in the amplitude and frequency of contractions (fig. 8).

4. Discussion

The present results show that neurotcnsin not only has relaxant effects but also exerts contractile effects on the rat duodenum. Previous studies have shown that neurotensin induces relaxation of rat duodenum (Carrzway and Leeman, 1973; Donoso et al., 1986; Kitabgi, 1982; R6kaeus et al., 1977), whereas contractile re-

sponses to ncurotcnsin have never been described, at least for the rat small intestine. It is difficult to reconcile our findings with previous observations but the disagreement could be due to different experimental conditions. In fact, in our experiments, a contractile effect was detected only for cndo[ttmina[ pressure and with isolated strips of circular muscle, while earlier workers had studied changes in isometric tension. Therefore, it is possible that ncurotensin induces contractile effects only in the circular muscle of rat duodenum. Moreover, duodenal smooth muscle, which we used, was not contracted by prior addition of acetylcholine. Consistent with our findings a biphasic response to neurotcnsin has been noted in other preparations, such as guinea pig ileum, canine corpus, cat i[eocecal sphincter region or mouse colon (Fontaine and I~ebrun, 1985; Kitabgi and Freychet, 1978; McLean and Fox, 1983; Rothstein and Ouyang, 1989). Reports on other gastrointestinal preparations have demonstrated that the inhibitory action of neurotensin is post-junctional, while contractile effects can be exerted directly on the smooth muscle or can be due to neuronal mobilization of acctylcholine (I Iuidobro-Toro and Ku[lak, 1985; Huidobro-Toro and Way, 1982; Iluidobro-Toro and Zhu, 1984; lluidobro-Toro ct al., 1984; Kitabgi and Frcychct, 1978, 1979; Sanders c t a l . , 1982) or to histamine release from mast cells (McLean and Fox, 1983). It is likely that the relaxant and the contractile actions of neurotcnsin on rat duodenum are due to direct effects of the ncuropeptide on receptors located on the smooth muscle membrane. In fact, the early ncurotensin-induccd muscle relaxation and the delayed contractile effect were not antagonized by atropine, adrenoccptor blocking agents, mcthysergidc or histamine antagonists, suggesting that neither the release of these endogenous neurotransmitters nor the occupation of their receptor sites is involved in this complex response. In addition, neither the relaxant nor the contractile components of the response to ncurotcnsin were antagonized by q-TX, which indicated that this complex response was not of neuronal origin. It has also been demonstrated that ncurotensin-induccd contractions may be due to the release of a mctabolitc of arachidonic acid (Fontainc and Lebrun, 1985; Fox ct al., 1987). Since incubation of our preparation with indomethacin did not block the contractile response, it seems likely that prostaglandin synthesis is not involved in the increase of muscle tone due to exogenous neurotcnsin in rat duodenum. Differences in EC.so values, in sensitivity to desensitization and in r e c e p t o r - e f f e c t o r coupling mechanism for the relaxant and contractile effects were found in our preparation. (1) Neurotensin had a different potency for inducing relaxant or contractile effects, since the ICs0 and ECs0 values were significantly different. In fact, the

222 relaxant response appeared and reached saturation at concentrations lower than those required for a maximal contractile effect. It can be excluded that the maximal relaxant effect was constrained by the intervention of the contractile response, since neurotensin potency and efficacy were the same for pressure and tension. (2) A general criterion used to characterize the ncurotcnsin receptors is desensitization. In fact, duc the lack of a specific ncurotcnsin receptor blocking agent, desensitization can represent an useful pharmacological tool in the study of receptors, although the mechanism of homologous desensitization remains largely unknown (Huidobro-Toro and Zhu, 1984). In rat duodenum, the two components of the ncurotcnsin effects on muscle showed different kinetics of desensitization. The concentration of neuropeptidc required for desensitization to the relaxant effect of ncurotensin was different from the desensitizing concentration for thc contractile action, the ncurotcnsin relaxant action being more sensitive to desensitization than the contractile action. In our preparation, desensitization to the inhibitory and excitatory actions of neurotensin is a selective process, failing to affect noradrenaline-induced relaxation or the contraction in response to carbachol. (3) There appear to be different intracellular transduction mechanisms mediating relaxation and contraction. In fact, the relaxant but not the contractile actions of ncurotcnsin were blocked non-competitiveIy by apamin. Therefore, it could be suggested that ncurotcnsin-induced relaxation is related to the activation of K ~ channels, Ca 2 ~-dependent and apamin-sensitivc. Similar blockade by apamin of the ncurotensin-induced inhibition was observed in cxperiments with rabbit ileum and mouse or guinea-pig colon (Fontaine and Lebrun, 1985; tluidobro-Toro and Yoshimura, 1983; Kitabgi and Vincent, 1981). In contrast to the relaxant action, the neurotcnsin-induced contractions were reduced by nifedipinc, suggesting that the contractile component in the rat duodenum is linked to diydropyridine-sensitive calcium channels. The additional experiments using a calcium-free Krcbs solution indicated that, in rat duodenum, the relaxant effect is less dependent on extracellular calcium than the contractile one. The importance of Ca z- entry in both inhibitory and excitatory postjunctional effects has been demonstrated for other gastrointestinal preparations (Fontaine and Lcbrun, 1985; Fox e t a l . , 1987; HuidobroToro and Kullak, 1985; Kitabgi and Vincent, 1981). These arguments might provide evidence tk)r the existence of two different types of ncurotensin receptors, one mediating the relaxant response and another, coupled to a contractile mechanism. Consistent with our hypothesis, evidence has been provided for the coexistence of inhibitory and excitatory, receptors in

guinea-pig ileum, using the desensitization as a criterion to characterize ncurotensin receptors (HuidobroToro and Zhu, 1984). Our results could have been predicted if the difference lies in the efficiency of r e c e p t o r - e f f e c t o r coupling rather than in the receptors themselves, Therefore, a common receptor could be coupled to two different transduction mcchamisms. Si~ce we have never seen contractile effects on Iongitudinal muscle, the ncurotcnsin receptor present on this muscular layer seems not to be linked to the metaboIic pathway responsible for contraction. However, pharmacological or molccolar studies arc needed to establish the existence of different types of receptors. Our results from experiments using longitudinal and circular muscle strips indicate that the two mcchanicaI recordings in the present work correspond to the activities of the two muscular layers and, interestingly, point out the differential effects of neurotcnsin on the muscular layers. In fact, only relaxation, which was TF, Xresistant and apamin-sensitive, was observed in longitudinal strips, whereas a TTX-insensitive inhibitor, effect followed by a contractile one was detected in circular strips. A similar pattern of response is consistent with the reports by Karaus and Sarna (1985) about differential effect of ncurotensin in the canine ileum, but it is in contrast with the results obtained with guinea-pig ileum. There arc several reports that neurotensin relaxes circular muscle of guinea-pig ileum (Goedert et al., 1984; Yamanaka et al., 1987), while it contracts or has a biphasic effect on longitudinal muscle (Kitabgi and Freychet, 1978, 1979; Yamanaka et al., 1987). Species differences may explain these conflicting data. In fact, although the predominant action of neurotensin on the dog, rat and rabbit small intestine seems to be inhibitory, exogenous neurotensin may excite, inhibit or have biphasic effects on gastrointestinal' motility, depending on the species, segment or muscle type examined. The experimental conditions also have to be taken into account; the circular muscle of the canine smaIl intestine has been shown to relax in response to injecti~)ns of neurotensin in vivo, although ncurotensin causes excitation when studied in vitro (Fox ct al., 1987; Sakai e t a l . , 1984). The differential effects on the two muscular layers in our preparation could perhaps be interpreted as indicating that neurotensin may play a role in the modulating the peristaltic activity of the gut or more probably, can act as a complex peripheral humoral factor. Goedcrt et al. (1984) have postulated that neurotensin is a transmitter of N A N C inhibitory nerves in the circular muscle of guinea-pig ileum, since the nerve stimulation-induced relaxation was inhibited by preincubation with the ncurotensin antiserum. Since the presence of a N A N C innervat:ion in rat duodenum has been amply demonstrated (Maggi etal., 1984; Manzini et al., 1985; Postorino et al., 1990a; 1990b) and involve-

223

mcnt of purines in N A N C ncurotransmission has been ruled out (Scrio et al., 1990), we attempted to verify whether the inhibitory receptors, located on the smooth cells, could mediate the relaxing response due to the activation of N A N C nerves. Indeed, in rat duodenum, as wcll as in guinea-pig ileum (Goedcrt ct al., 1984), aF,amin antagonizes both ncurotensin- and nerve stimulation-induced muscle relaxation. However, consistent with the findings of Yamanaka et al. (1987), in our experiments ncurotcnsin did not act as a putative transmitter of N A N C inhibitory nerves, as desensitization of the inhibitory receptors failed to modify significantly the relaxation induced by NAN(; inhibitor), nerves. In conclusion, smooth num,

ncurotcnsin,

acting

directly

on

the

m u s c l e , i n d u c e s a d u a l e f f e c t in t h e r a t d u o d e relaxation

and

contraction.

It p r o b a b l y

acts on

two diffcrcnt receptors or alternatively activates two diffcrcnt biochemical mcchanisms, onc mediating relaxation and the other coupled

to a contractile

mecha-

nism.

Acknowledgements Thc authors are particularly greatful to Prof. G. Burnstock for his heipful suggestions and to F. Bonvissuto for his technical assistance. This work was supported by a grant from Ministcro della Pubblica lstruzione, Italia.

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