Tachykinin-Dependent and -Independent Components of Peristalsis in the Guinea Pig Isolated Distal Colon

Transcription

1 GASTROENTEROLOGY 2001;120: Tachykinin-Dependent and -Independent Components of Peristalsis in the Guinea Pig Isolated Distal Colon MARCELLO TONINI,* VALERIA SPELTA,* FABRIZIO DE PONTI, ROBERTO DE GIORGIO, GIANLUIGI D AGOSTINO, VINCENZO STANGHELLINI, ROBERTO CORINALDESI, CATIA STERNINI, and FRANCESCA CREMA* *Department of Internal Medicine and Therapeutics, Division of Clinical and Experimental Pharmacology, University of Pavia, Italy; Departments of Pharmacology and Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy; Department of Experimental and Applied Pharmacology, School of Pharmacy, University of Pavia, Pavia, Italy; and CURE Digestive Diseases Research Center, Division of Digestive Diseases, Departments of Medicine and Neurobiology, UCLA and Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, California Background & Aims: In the intestine, tachykinins regulate motility by participating in neuromuscular and neuro-neuronal transmission. The aim of this study was to test the hypothesis that colonic propulsion is regulated by an interplay between tachykinergic and cholinergic transmission. Methods: Propulsion was elicited by intraluminal distention of a thin rubber balloon, which traveled from the oral to the anal end of guinea pig isolated distal colon segments. The overall contribution of endogenous tachykinins to colonic propulsion was examined by blocking NK 1,NK 2, and NK 3 receptors simultaneously. Results: NK 2 -receptor blockade by MEN inhibited propulsion, whereas blockade of NK 1 by SR or of NK 3 receptors by SR had minor effects on motility. Blockade of muscarinic or nicotinic receptors by hyoscine or hexamethonium decelerated peristalsis up to propulsion arrest. In the presence of partial muscarinic receptor blockade, the NK 1 - receptor antagonist SR and the NK 2 -receptor antagonist MEN markedly inhibited propulsion. Propulsion was also inhibited by the NK 3 -receptor antagonist SR in the presence of partial nicotinic receptor blockade. The simultaneous administration of the 3 tachykinin antagonists inhibited propulsion by 50%. Conclusions: This study demonstrates the existence of an interplay between tachykinergic and cholinergic pathways during peristalsis and the importance of endogenous tachykinins acting at multiple receptor sites in the control of colonic propulsion. In the guinea pig myenteric plexus, tachykinin-containing neurons comprise intrinsic primary afferent (sensory) neurons, ascending interneurons, and ascending motor neurons, which are part of circuits regulating excitatory reflexes and peristalsis. 1,2 Enteric tachykinins include substance P and neurokinin A, which are co-stored with acetylcholine (ACh) and act primarily as nonadrenergic, noncholinergic excitatory transmitters. 1,3,4 Three types of tachykinin receptors have been described so far: NK 1,NK 2, and NK 3 receptors. The NK 1 and NK 2 subtypes are located on smooth muscle cells, where they mediate contraction, and on neurons, 5 8 and NK 3 receptors have been identified on neurons. 9,10 Both NK 1 and NK 3 receptors are found on ascending and descending neuronal pathways, 11,12 where they mediate release of excitatory or inhibitory transmitters. 6,13 17 In addition, NK 1 receptors are located on the interstitial cells of Cajal 6,8 and NK 3 receptors on intrinsic sensory neurons. 12,16 Neuronal NK 2 receptors have also been reported on descending inhibitory pathways, at least in the guinea pig colon. 7,18 In the guinea pig ileum, NK 1 and NK 2 muscular receptors mediate the atropine-resistant component of the distention-evoked ascending reflex contraction of the circular muscle, 19 and NK 2 receptors have also been found to act synergistically with muscarinic receptors in the excitatory neuromuscular transmission during peristalsis in vitro. 20 In the guinea pig colon, NK 1,NK 2, and muscarinic receptors contribute to peristalsis in vitro 21 and to the ascending reflex contraction of the circular muscle in vivo. 22 This indicates that radial distention of the gut wall may co-release ACh and tachykinins. 21 Nevertheless, ganglionic and neuromuscular cholinergic transmissions are known to exert a primary role on guinea pig colonic propulsion because suppression of the cholinergic drive (either nicotinic or muscarinic) blocks peristalsis both in vitro 21,23 and in vivo. 24 The role of NK 3 receptors on colonic propulsion is still unknown. However, these receptors appear to play a Abbreviations used in this paper: ACh, acetylcholine; L-NOARG, N G -nitro-l-arginine by the American Gastroenterological Association /01/$35.00 doi: /gast

2 March 2001 TACHYKINERGIC TRANSMISSION AND PROPULSION 939 marginal role on propulsive activity in the small intestine, 25,26 although electrophysiologic evidence shows their involvement in both ascending excitatory and descending inhibitory standing reflexes. 16,27 Although extensive data exist on the role and distribution of tachykinin receptors in the gut, only fragmentary information is available on the relative contribution of tachykinin-dependent and -independent pathways in subserving colonic peristalsis. In this study, we used the guinea pig isolated distal colon to determine whether there is an interaction between cholinergic and tachykinergic transmission at the neuro-neuronal and neuromuscular level. Specifically, our hypothesis was that NK 1 and NK 2 receptor mediated transmission could primarily interact with muscarinic mechanisms at the neuromuscular level, and NK 3 receptor mediated transmission could interact with nicotinic mechanisms at the ganglionic level. We also examined the overall contribution of endogenous tachykinins to propulsive activity by blocking NK 1,NK 2, and NK 3 receptors with selective antagonists (SR , MEN 11420, and SR ) Materials and Methods Animal care and procedures were in accordance with National Institutes of Health recommendations for the humane use of animals and with the European Union Directive 86/609 on the care and use of experimental animals. All experimental procedures were reviewed and approved by the Animal Use Committee of the institution where the experiments were performed. The number of animals used was also kept to the minimum necessary for a meaningful interpretation of the data. Male guinea pigs weighing g were stunned and bled. A 7 8-cm-long segment of distal colon was excised and placed in a 100-mL organ bath containing warmed (37 C) and oxygenated (95% O 2,5%CO 2 ) Tyrode solution to allow spontaneous evacuation of fecal pellets. After removal of the mesenteric attachment, the segment was set up horizontally with the oral end secured to a fixed holder and the free aboral end connected to an isotonic transducer under 1-g load. All experiments were started after at least 60 minutes of equilibration. Peristalsis Recording Propulsion was elicited by a thin rubber balloon inserted intraluminally 1 cm from the oral side and distended with ml of water from a syringe. The balloon was connected by a thread to an isotonic transducer loaded with 0.2 g. Peristalsis was elicited at intervals of at least 10 minutes. The velocity of propulsion (mm/s) was calculated by recording the time (seconds) required by the balloon to travel the entire length (millimeters) of the segment. 31 Balloon displacement was recorded by a chart recorder (Linseis L6512, Germany). The mean velocity calculated from at least 2 consecutive propulsions was taken as the control value. Experimental Protocol The involvement of NK 1,NK 2, and muscarinic receptors on colonic propulsion was investigated by constructing concentration response curves to SR ( nmol/ L), MEN ( nmol/l), and hyoscine ( nmol/l). To assess a potential interaction between cholinergic (muscarinic) and tachykinergic (NK 1 - and NK 2 -mediated) transmissions, curves to both tachykinin antagonists were repeated in the presence of 30 nmol/l hyoscine (a concentration causing 30% inhibition of propulsion velocity), while that of hyoscine was repeated in the presence of either NK 1 -ornk 2 - receptor antagonist (both at 300 nmol/l, a concentration inhibiting propulsion velocity by 10% 25%). The influence of NK 3 and nicotinic receptors on ganglionic transmission was investigated by constructing concentration response curves to SR ( nmol/l) and hexamethonium (3 100 mol/l). To assess a potential interaction between cholinergic (nicotinic) and tachykinergic (NK 3 -mediated) transmissions, the curve to hexamethonium was repeated in the presence of 100 nmol/l SR (a concentration causing 25% inhibition of propulsion velocity), while the curve of SR was repeated in the presence of 3 mol/l hexamethonium (a concentration causing 15% inhibition of propulsion velocity). Finally, the overall influence of endogenous tachykinins on colonic propulsion was investigated by coincubating tissues with high concentrations of SR (1 mol/l), MEN (1 mol/l), and SR (100 nmol/l). To construct concentration response curves, antagonists were added cumulatively by using up to 3 concentrations. Because full antagonism is achieved after a long incubation time, a contact time of minutes was always allowed. 28,29 Any drug-induced change in the velocity of propulsion was calculated as percent change with respect to control velocity. Statistical Analysis The results are given as means SEM of n experiments, n being the number of colonic segments taken from different guinea pigs (total number of animals used was 92). Results were analyzed by use of the Student t test for paired or unpaired data or with 1-way ANOVA (with the Scheffé correction for multiple comparisons), as appropriate. A P value of 0.05 was regarded as significant. Drugs and Solutions The following drugs were used: SR , SR (Sanofi Research, Montpellier, France), MEN (Menarini Pharmaceuticals, Florence, Italy), ( )hyoscine hydrochloride, and hexamethonium chloride (Sigma-Aldrich, Milan, Italy). SR and SR were dissolved in dimethyl sulfoxide and then diluted in distilled water. Diluted dimethyl sulfoxide had no effect on colonic propulsion. All

3 940 TONINI ET AL. GASTROENTEROLOGY Vol. 120, No. 4 Figure 1. Effect of SR (n 5 10), MEN (n 5), and hyoscine (n 4 12) on propulsion velocity in the guinea pig colon. Values are means SEM and are expressed as percentages of control values. *P 0.05; **P 0.01 vs. control. other drugs were dissolved and diluted in distilled water. Antagonists were administered in volumes not exceeding 2% of the bath volume. The composition of the Tyrode solution was mmol/l NaCl, 2.7 mmol/l KCl, 1.8 mmol/l CaCl 2, 1.04 mmol/l MgCl 2, 0.4 mmol/l NaH 2 PO 4, 11.9 mmol/l NaHCO 3, and 5.5 mmol/l glucose (ph 7.3). Results Radial distention of the colonic wall by an intraluminally inflated balloon elicited a propulsive wave of contraction, which displaced the balloon anally at a mean velocity of mm/s (n 103). Effects of SR , MEN 11420, or Hyoscine Administration of the selective NK 1 -receptor antagonist SR in the 10 nmol/l to 1 mol/l concentration range (n 5 10) did not significantly affect the velocity of propulsion. However, the 2 lowest concentrations (10 and 30 nmol/l) showed a trend toward facilitation of propulsion velocity, whereas the highest concentrations (300 nmol/l, 1 mol/l) displayed a trend toward inhibition (Figure 1). The selective NK 2 -receptor antagonist MEN significantly (P 0.05) reduced the velocity of propulsion in the nmol/l concentration range (n 5), whereas no significant effects were observed in the presence of the highest (300 nmol/l and 1 mol/l) antagonist concentrations (n 5; Figure 1). Hyoscine (10 nmol/l to 1 mol/l, n 4 12) concentration-dependently inhibited the velocity of propulsion, achieving blockade of peristalsis at 1 mol/l (Figure 1). Effects of SR or MEN Combined With Hyoscine In the presence of 30 nmol/l hyoscine (a concentration causing 30% inhibition of the velocity of propulsion), SR (10 nmol/l to 1 mol/l; n 4) and MEN (10 nmol/l to 1 mol/l; n 5 6) inhibited propulsion velocity at all concentrations tested, achieving blockade at 100 nmol/l (P 0.05 or better; Figure 2A and B). The net effect resulting from the combination of hyoscine with NK 1 and NK 2 antagonists was significantly higher than the sum of inhibitory effects caused by each individual agent. This provides evidence for a synergistic interaction between muscarinic and NK 1 -NK 2 receptor mediated mechanisms. Like- Figure 2. Concentrationdependent inhibition by (A) SR and (B) MEN in the presence of a threshold concentration of hyoscine (30 nmol/l, per se inhibiting propulsion velocity by approximately 30%). Upper curves in each panel represent the same data as reported in Figure 1. Values are means SEM (n 4 10) and are expressed as percentages of control values. *P 0.05; **P 0.01 vs. SR or MEN alone.

4 March 2001 TACHYKINERGIC TRANSMISSION AND PROPULSION 941 Effects of SR and Hexamethonium Either Alone or in Combination Administration of the selective NK 3 -receptor antagonist SR in the nmol/l concentration range (n 4 5) inhibited propulsion only at the highest concentration ( 20% inhibition; P 0.05; Figure 4A). Hexamethonium (3 100 mol/l; n 4 8) concentrationdependently decreased the velocity of propulsion, achieving blockade at 100 mol/l (Figure 4B). In the presence of 100 nmol/l SR , inhibition by hexamethonium (3 100 mol/l; n 4 8) was potentiated (P 0.05 or better) compared with hexamethonium alone (Figure 4B). The net effect resulting from the combination of hexamethonium with SR was similar to the sum of inhibitory effects caused by each individual agent. This provides evidence for an additive interaction between nicotinic and NK 3 receptor mediated mechanisms. Likewise, in the presence of 3 mol/l hexamethonium, the effect of SR ( nmol/l) on propulsion was enhanced in an additive manner (Figure 4A). Figure 3. Potentiation by SR or MEN (300 nmol/l each) of hyoscine-induced inhibition of propulsion velocity in the guinea pig colon. The upper curve reports the same data as reported in Figure 1. Values are means SEM (n 4 12) and are expressed as percentages of control values. *P 0.05 vs. hyoscine alone. wise, inhibition of colonic propulsion by hyoscine (10 nmol/l to 1 mol/l; n 4 6) was significantly potentiated (P 0.05) in the presence of threshold inhibitory concentrations of SR (300 nmol/l) and MEN (300 nmol/l), which caused 10% 25% inhibition per se (Figure 3). Effect of Combined SR , MEN 11420, and SR A combination of SR and MEN (each at 1 mol/l) reduced the velocity of propulsion by approximately 35%. However, this effect did not reach statistical significance (Figure 5). Coincubation of colonic segments with SR (1 mol/l), MEN (1 mol/l), and SR (100 nmol/l), each inhibiting propulsion by 10% 30% when used alone, reduced the velocity of propulsion to 54.2% 14.7% (P 0.05; n 6; Figure 5). A representative tracing showing the balloon progression in the absence and in the presence of NKreceptor blockade is illustrated in Figure 6. In the presence of NK 1 -, NK 2 -, and NK 3 -receptor blockade, either 1 mol/l hyoscine (n 3) or 100 mol/l hexamethonium (n 3) abolished propulsion (data not shown). Discussion This study suggests an interaction between cholinergic and tachykinergic transmission during propulsion in the guinea pig distal colon that appears Figure 4. (A) Effect of SR alone or combined with 3 mol/l hexamethonium and (B) effect of hexamethonium alone or combined with 100 nmol/l SR on propulsion velocity in the guinea pig colon. Values are means SEM (n 4 8) and are expressed as percentages of control values. Upper curves: *P 0.05, **P 0.01 vs. control; lower curves: *P 0.05, **P 0.01 vs. SR or hexamethonium alone.

5 942 TONINI ET AL. GASTROENTEROLOGY Vol. 120, No. 4 Figure 5. Effect of combined blockade of NK 1,NK 2, and NK 3 receptors by SR (1 mol/l), MEN (1 mol/l), and SR (100 nmol/l) on propulsion velocity in the guinea pig colon. Values are means SEM (n 5 8) and are expressed as percentages of control values. *P 0.05 vs. control. synergistic at the neuromuscular level and additive at the neuro-neuronal level. The marked inhibition (up to blockade) by hyoscine or hexamethonium confirms the primary role of muscarinic and nicotinic transmission in the regulation of colonic propulsion in this species. 21,23 Nevertheless, endogenous tachykinins substantially contribute to propulsion; simultaneous blockade of NK 1, NK 2, and NK 3 receptors inhibited propulsion velocity by 50%, indicating the presence of a tachykinin-dependent and a tachykinin-independent component of peristalsis. The influence of NK-receptor antagonists per se on colonic propulsion deserves some comments. With the exception of the NK 2 -receptor antagonist MEN 11420, which significantly inhibited propulsion at nanomolar concentrations, blockade of either NK 1 or NK 3 receptors did not have a major impact on propulsive activity. Effect of NK 1 -Receptor Antagonism Low concentrations ( nmol/l) of the selective NK 1 -receptor antagonist SR showed a trend toward stimulation of peristalsis (maximal acceleration 20%), although this effect did not reach statistical significance. Of note, 100 nmol/l is a concentration 10 times as high as the pk B value (i.e., the affinity value) of this antagonist at excitatory NK 1 receptors in the guinea pig intestinal smooth muscle. 28 Postjunctional NK 1 receptors mediate noncholinergic excitatory junction potentials in colonic circular muscle 32 partially underlying the atropine-resistant component of the ascending reflex contraction. 22 However, it is unlikely that blockade of postjunctional NK 1 receptors facilitates peristalsis. This effect is also unlikely to result from blockade of NK 1 receptors located on interstitial cells of Cajal, 33 which are pacemaker cells coordinating intestinal motility. 34 Two other locations of NK 1 receptors have been described in the guinea pig intestine: prejunctional NK 1 receptors operating a feedback inhibition of transmitter release from excitatory motor neurons 9,35 and NK 1 receptors located on descending inhibitory pathways, 6,16,17 where they stimulate nitric oxide (NO) release. 17,36 Blockade of the latter subset of NK 1 receptors may facilitate peristalsis, as shown in the guinea pig small intestine, 20 where NK 1 antagonists are thought to partly inhibit NO release from descending pathways. 36,37 This facilitatory effect is similar to that caused by the NO synthase inhibitor N G -nitro-l-arginine (L-NOARG), which decreases the threshold volume required to trigger the emptying phase of ileal peristalsis 38 by inhibiting the accommodation of the circular muscle (an NO-mediated event) to intraluminal fluid delivery. 39 However, unlike findings on ileal peristalsis, colonic propulsion in the guinea pig is markedly inhibited by L-NOARG through impairment of descending circular muscle relaxation in front of the advancing bolus. 21 Thus, blockade of NK 1 receptors on descending pathways should reduce NO release and consequently inhibit rather than facilitate colonic propulsion in the guinea pig. Therefore, by exclusion, we ascribe the slight facilitatory effect observed with low SR concentrations to blockade of prejunctional NK 1 receptors, which exert a negative modulation of ACh release on excitatory motor neurons. 35,40 High concentrations of SR ( nmol/l) slightly reduced the velocity of propulsion, suggesting blockade of postjunctional NK 1 receptors, which Figure 6. Representative tracing showing the anal progression of an intraluminally distended balloon in an isolated distal colon segment in the (A) absence and (B) presence of SR (1 mol/l), MEN (1 mol/l), and SR (100 nmol/l). The velocity of propulsion in control conditions was 1.28 mm/s, whereas after NKreceptor blockade it was reduced to 0.55 mm/s. Dots indicate the inflation of the balloon.

6 March 2001 TACHYKINERGIC TRANSMISSION AND PROPULSION 943 are involved in excitatory neuromuscular transmission. 5,14 A concomitant increase in ACh release caused by blockade of prejunctional inhibitory NK 1 receptors 35,40 may oppose the postjunctional inhibitory effect of SR , thus leading to a low level of peristalsis inhibition. Therefore, the lack of statistical significance with any concentration of SR may be viewed as the final outcome of a balance between postjunctional and prejunctional (or presynaptic) inhibitory/excitatory effects. Alternatively, the lack of significant effects of SR on colonic propulsion may reflect a condition in which propulsive efficiency, in the presence of NK 1 - receptor blockade, is maintained unaltered by endogenous tachykinins acting at other NK receptors 9 and by an intact cholinergic drive. Our data differ from previously published results 21 that showed marked reduction of colonic propulsion by the NK 1 -receptor antagonist GR This apparent discrepancy may be attributable to the different methodology and the higher concentrations of antagonist used. Effect of NK 2 -Receptor Antagonism Low concentrations ( nmol/l) of the selective NK 2 antagonist MEN significantly inhibited the velocity of propulsion, an effect that gradually decreased (up to nonsignificant values) after cumulative application of higher antagonist concentrations (300 nmol/l and 1 mol/l). In the guinea pig gastrointestinal tract, NK 2 receptors are located on effector cells, where they mediate contraction 5,41 and participate in excitatory reflex responses of the circular muscle, 22 and on descending neurons, where they promote NO release and smooth muscle relaxation. 7,18 It is possible that the effect of low concentrations represents an action on inhibitory pathways, whereas the effect of high concentrations is attributable to blockade of muscular NK 2 receptors. Similarly to the blockade of NK 1 receptors on descending neurons, peristalsis inhibition after neuronal NK 2 -receptor blockade may result partly from reduction of NO release, leading to impairment of descending relaxation. This effect, together with blockade of postjunctional excitatory NK 2 receptors, 5,41 may underlie the overall impairment of propulsion caused by MEN By contrast, the NK 2 antagonists MEN and MEN were found to facilitate guinea pig colonic propulsion in vivo, probably as a result of inhibition (blockade) of NK 2 receptors on descending interneurons containing NO. 24 However, it is unlikely that the facilitatory effect observed in vivo may arise from blockade of NK 2 receptors located on descending nitrergic interneurons because blockade of NO production/release by L-NOARG in the guinea pig colon in vitro is associated with inhibition of propulsion. 21 The parallelism between the effects on colonic propulsion caused by blockade of NK 2 receptors on nitrergic pathways and blockade of NO production is further substantiated by recent observations in the rabbit isolated distal colon. 42 In this latter preparation, in which L-NOARG was previously found to accelerate propulsion by reducing the compliance of the circular muscle, 43 nanomolar concentrations of the 2 NK 2 antagonists, MEN and SR 48968, markedly enhanced velocity of propulsion through suppression of NOmediated inhibitory mechanisms. 42 Under our experimental conditions, cumulatively applied concentrations of MEN showed an inverse concentration response relationship. This may be attributable to the occurrence of adaptive changes in the function of other neuronal or postjunctional tachykinin receptors (or even of other transmitter receptors) to overcome the loss of function of blocked NK 2 receptors. Effects of NK 1 - and NK 2 -Receptor Blockade in Combination With Hyoscine Although blockade of NK 1 -NK 2 receptors, either individually or together, did not seem to have a major impact on propulsion, the simultaneous impairment of muscarinic transmission by a low concentration of hyoscine increased the effect of this receptor blockade. Like atropine, 21,23 hyoscine markedly inhibited propulsion up to its blockade. In the presence of 30 nmol/l hyoscine (a concentration inhibiting the velocity of propulsion by 30%), both SR and MEN markedly inhibited propulsion, up to propulsion blockade at high concentrations. The effect resulting from the combination of hyoscine with NK 1 and NK 2 antagonists was significantly more pronounced than the sum of inhibitory effects caused by each individual agent, providing evidence for a synergistic interaction between muscarinic and NK 1 -NK 2 receptors in controlling propulsion. This is consistent with the observation that a significant tachykinergic contribution to excitatory neuromuscular transmission is unmasked when postjunctional muscarinic receptors are partly blocked. 22 In addition, the tachykinergic contribution to colonic propulsion might become more evident after blockade of prejunctional (or presynaptic) muscarinic inhibitory receptors, which induces increased release of tachykinins. 44,45 Circumstantial evidence suggesting a synergistic interaction between muscarinic and NK 2 receptors in the gastrointestinal tract was previously obtained by study of guinea pig ileal peristalsis 20 and rabbit colonic propulsion, in which the slight inhibitory effect caused by micromolar concentrations of MEN was markedly enhanced by threshold concentrations of atropine. 42

7 944 TONINI ET AL. GASTROENTEROLOGY Vol. 120, No. 4 Effect of NK 3 -Receptor Antagonism Neuro-neuronal tachykinergic transmission via NK 3 receptors has been documented in the enteric nervous system in both ascending and descending pathways, at least in the guinea pig small intestine. 13,16,27,46 In our hands, the selective NK 3 antagonist SR , applied at concentrations (up to 100 nmol/l) maintaining selectivity over NK 1 and NK 2 receptors, 29,47 only slightly inhibited the velocity of colonic propulsion. This suggests a minor contribution of NK 3 receptors under baseline conditions, i.e., with intact nicotinic transmission. When nicotinic synapses, which play a pivotal role in intestinal propulsion in the guinea pig, 23,48 were partially (15%) antagonized by a submaximal concentration of hexamethonium (3 mol/l), the addition of SR caused an inhibitory effect, which was the sum of the effects produced by each individual agent. This suggests an additive interaction at the neuro-neuronal level between the 2 classes of receptors, especially along the ascending excitatory pathways, where nicotinic synapses are much more abundant than in the descending pathways. 49 Evidence for the involvement of NK 3 receptors in the hexamethonium-resistant ganglionic transmission has been documented in the rat 50 and rabbit colon (Onori and Tonini, unpublished). Effects of NK 1 -, NK 2 -, and NK 3 -Receptor Antagonism on Colonic Propulsion From the above data, it might be concluded that tachykinins play only a minor role in colonic propulsion in baseline conditions. However, the experiments with simultaneous blockade of NK 1,NK 2, and NK 3 receptors revealed a substantial tachykinindependent component of peristalsis, with the remaining component (i.e., the tachykinin-independent component) completely antagonized by either hyoscine or hexamethonium. Simultaneous blockade of all neurokinin receptor subtypes is indeed the prerequisite to avoidance of an interaction of endogenous tachykinins with any of the 3 receptor subtypes and assessment of the tachykinin-dependent and -independent components of peristalsis. Therefore, we can conclude that endogenous tachykinins play an important role in subserving propulsive activity in the guinea pig colon by acting at multiple receptor sites. This role becomes especially apparent when cholinergic (muscarinic/nicotinic) transmission is partially antagonized and after suppression of the overall influence of endogenous tachykinins on motility, via NK 1 -, NK 2 -, and NK 3 - receptor blockade. References 1. Costa M, Brookes SJH, Steele PA, Gibbins I, Burcher E, Kandiah CJ. Neurochemical classification of myenteric neurons in the guinea-pig ileum. Neuroscience 1996;75: Kunze WAA, Furness JB. The enteric nervous system and regulation of intestinal motility. Annu Rev Physiol 1999;61: Grider JR. 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In vivo evidence for the involvement of tachykinin NK 3 receptors in the hexamethonium-resistant inhibitory transmission in the rat colon. Naunyn Schmiedebergs Arch Pharmacol 1996; 353: Received March 14, Accepted October 11, Address requests for reprints to: Marcello Tonini, Ph.D., Division of Clinical and Experimental Pharmacology, Department of Internal Medicine and Therapeutics, University of Pavia, Piazza Botta 10, I Pavia, Italy. tonini@unipv.it; fax: (39) Supported by funds from the University of Pavia (FAR 1998) and CARIPLO Foundation (to M.T.), Italy, and Morphology/Imaging Core of CURE Digestive Diseases Research Center grants DK and (to C.S.). The authors thank Dr. P. Baiardi for her advice on statistical analysis.