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Colonic effects of TAK-637: A novel NK, receptor antagonist
1705 Endomorphin-1 Impairs Propulsion By Inhibiting Excitatory And Inhibitory Transmission In The Guinea Pig Isolated Colon Marcallo Tonini, Fabrizio De Ponti, Dept of Internal Medicine, Univ of Pavia, Pavia Italy; Giovanni Barbara, Roberto De Giorgio, Dept Internal Med & Gastroenterof, Univ of Bologna, Bologna Italy; Laura Anselmi, Barbara Balestra, Dept of Internal Medicine, Univ of Pavia, Pavia Italy; CaUaSternini, CUREand Dept Medicine and Neurobiology, UCLA, Los Angeles, CA Exogenous and endogenous opioids are potent inhibltors of intestinal transit. The recently discoveredendomorphins,which havethe highest affinity and selectivityfor/~-opioid receptor than any other endogenousopioids, have been proposed as the endogenousligands for this receptor. Endomorphinsdirectly activatethe/~-receptor and trigger its internalizationin enteric neurons, and act as full agonists. However, a pharmacologicat analysis of the effect of endomorphins on colonic propulsion has not been reported. AIM: To assessthe acute effect of endomorphin-1 on a) peristalsis in isolated segments of guinea pig distal colon, and on b) electrically evoked neurogenic contractions and relaxations in longitudinal and circular muscle colonic strips. METHODS:Peristalsiswas elicited by distending an intralominal mobile balloon with 0.05 ml of water.The velocity of anal balloondisplacement(mrrds) was considered as the main peristaltic parameter. Electrical field stimulation (EFS) was delivered as follows: 20-60 V, 0.5 ms pulse duration, 0.3-10 Hz for detection of neurogenic contractions or 0.3 Hz for detection of neurogenic non-adrenergic, non-cholinergic (NANC) relaxations in the presenceof hyoscine (1/~M) and guansthidine(3/~U). RESULTS:Control velocity of propulsion was 0.92_+0.1 mm/s. Endomorphin-1 (1-30 nM) concentration-dependentlyinhibited propulsion through a mechanism reverted by 100 nM naloxoneand 100 nM D-Phe-Cys-TyrD-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a highly selective/~-opioid receptor antagonist. The slowing of propulsion velocity was in the range of 10-60°/o. In longitudinal and circular muscle strips, neurogenic contractions induced by 0.3-10 Hz stimulation were inhibited by 1 endomorphin-t via a naloxone-and CTOP-sensitivemechanism. In muscle strips precontracted with 300 nM substance P, submaximal neurogenic NANC relaxations evoked by 0.3 Hz stimulation were markedly reduced (---70%) by 1 FM endomorphin-1. This inhibitory effect was counteracted by both opioid receptor antagonists at 100 nM. CONCLUSION: Colonic peristalsis was deceleratedby endomorphin-1 through a mechanisminvolving/~-opioid receptors. The antipropulsiveeffect of this endogenousopioid peptidewas mediatedby an inhibitory action on both ascending and descending pathways innervating longitudinal and circular muscle and regulating peristalsis. Supported by NIR grant DK54155, FAR 2000 & University of Pavia Special Research Project Funds, 1990.
1706 Role For Calcitonin Gene-Related Peptido And Substance P In Hypersensitivity And AIIodynia In The Rat Colon Christophe Mazzia,Yvon Jule, S Lucchini, Dept of physiology, Faculty des Science Marseille-St Jerome, Marseiile France; Frederic Raymond, Laure Delafoy, Laurent Diop, Pfizer Global R&D, Fresnes France; Monique Henry, Dept of physiology, Faculty des Science Marseille-StJerome, Marseille France Backgroundand Aims: Pharmacologicalstudies performed in conscious rats demonstratedthe involvementof calcitonin gene-relatedpeptide(CGRP)in nociceptiveresponsesto distension in the rat colon. In the present work, using the trinitrobenzene sultonic acid (TNBS)-induced colonic allodynia, we investigated the morphological distribution of CGRP immunoreactive (CGRP-ir) nerves in the colon. Since it was shown that part of CGRP-containing primary sensory afferents is colocalizedwith substance P (SP), we analyzedthe distribution of single and double immunolabelled nerves to CGRPand SP. Methods: Proximal and distal colonic segments were removed one week after injection of TNBS in the proximal colon which produces a hypersensitivity and an allodynia in distal colon. Cryostat sections were studied by confocel microscopy after double immunofluorsscenca with antibodies raised against CGRPand SP. Quantitativeanalysis of immunoreactivitieswas determined in myenteric and submucosal plexuses; they were expressedas the percentagerelative to the surface area of the ganglia. Results: In non-treated rats (n=5), the density of CGRP-ir was higher in the submucosal than in the myenteric plexus (1.44 _+ 0.24% and 0.61 __ O.08%,respectivey; p
1707 Colonic Effects Of TAK-637: A Novel NK~ Receptor Antagonist. Kalina Venkova, Karl Tyler, Okla Fdn for Digest Research Basic Science Lab, Oklahoma City, OK; Debra Sotkowski-Markmann, TAP Pharm Products Inc, Deerfield, IL; Beverley Greenwood-Vanmeerveld, Okla Fdn for Digest Research Basic Science Lab, OklahomaCity, OK Background: Functional studies indicatethat tachykinin receptors are present in the intestine and are involved in peristaltic reflexes.NK~receptorsare found on muscle as well as inhibitory and excitatory motor neurons. The present study was designedto characterizethe contractile
A-331
activity in the colonic musculatureof a newly synthesizednon-peptideNK~receptorantagonist TAK-637. Methods: The antagonist effects of TAK-B37 on tachykinin-induced contractions and on neurally evoked or spontaneous contractile activity were studied in large intestinal muscle strips or segments isolated from guinea pigs. Results: The selective agonists of NK1 receptors [Sarg,Met(O)']-substance P (SP) or GR73632 induced contractions in colonic longitudinal muscle strips pretreatedwith atropine.TAK-B37(0.1-100 nM) shifted the concentration-responsecurves to the right without a significant reduction in maximal efficacy. Schild plot analysis revealedthat TAK-637 is a competitive antagonist of NK1 receptors with similar potency against contractions mediated by SP (Kb=2.4 nM, 95% CL 1.9-3.0 ) or GR73632 (Kb= 3.5 nM, 95% CL 1.7-7.1). The Kbvalues estimated by a single shift of the concentrationresponse curves to GR73632 induced by 100 nM TAK 637 were similar in muscles studied before or after chemical denervationwith T'rx (11.8 nM vs.14.5 nM). However,TAK-637 (100 nM) has no significant effect on the contractileresponseof colonic circular muscleto GR64349, a selective NK~ receptor agonist, or contractions of taenia coli induced by a selective NK3 agonist, senktide. TAK-637 (1 /.~vl) has no effect on cholinergic contractions induced by electrical field stimulation (EFS) in ileal longitudinal muscle-myenteric plexus strips (EFS: 0.5 ms, single pulse) or nonadrenergic,noncholinergic (NANC) responsesin colonic longitudinal muscle (EFS: 0.5 ms, 1-16 Hz). In contrast, TAK-637 (1 /LM) reduced spontaneousactivity and preventedthe developmentof capsaicin-inducedcontractions in isolated segmentsfrom the terminal ileum. Conclusion: Our study demonstratesthat TAK-637 is a competitiveantagonist of peripheral NK~ receptors in the intestinal tract. Supported by TAP Pharmaceutical Products inc.
1708 Sustained Increase of the Pseudoaffective Visceromotor Response (VMR) to Colorectal Distention (CRR) in NIH/SWlSS Mice (NIH/SWISS) Following Trichinella Sldralis ( Tsp.) Enteritis Lu Wang, P A. Blennerhassett,Yk Mac, McMaster Univ, IDRP, Hamilton Canada;M Abrahamson, A Bayati, AstraZeneca,Molndal Sweden; S M. Collins, G Tougas, McMaster Univ, IDRP, Hamilton Canada Background: Visceral hypersensitivity (VH) and altered function are the central features seen in functional bowel disorders. These symptoms often appearfollowing resolution of an acute gastrointestinalinfection. In anaesthetizedN/H/SWISS, Tsp.inducedenteritis causespersistent VH with increasedcolonic afferent single fiber responseto CRD for up to 8 weeks, long after resolution of the acute enteritis (Gastro 11B:A701,2000). To assess if VH to CRD is also associatedwith somatic pseodoaffectivechanges, we examinedVMR to CRD following Tsp. enteritis, recording abdominal wall electromyographie(EMG) responseto CRD in conscious mice. Methods: In N/H/SW/SS males, 2 permanent EMG electrodes were placed within the oblique abdominal muscle. Following recoveryfrom surgery, the mice were trained to tolerate 2cm CRD balloon while placed in a Bollmano cage. VMR to CRD was recorded before, 4 and 6 weeks following Tsp. enteritis, with the distal end of the balloon located 2.5 cm from the anal verge. An increasing CRD paradigm (lO-30-40-60mmHg, lOsec) was applied with 5minute rest betweenstimuli. ~VMR is expressedas change in AUC of EMG during CRD over baseline EMG. Results: Mean_+SE(n) Summary and Conclusions: In uninfected mice, CRD produces a pressure-dependentincrease of the AVMR to CRD (0.07, 0.6, 0.7, 0.8 AUC for 10, 30, 40, 60mmHg, respectively, p < 0.05), which was significantly increased 4 and 6 weeks after Tsp.over control values (p< 0.05). These observations are in agreement with our previousfinding that Tsp.enteritis elicits a persistentcolonic visceral afferent hyperalgesic response to CRD in the absence of colitis and long after resolution of the acute enteritis. These findings demonstrating colonic hypersensitivityfollowing Tsp infection and the in vivo motility abnormalities seen in the same animals (Bercik,DOW 2001) make this an appropriate model of post-infective irritable bowel syndrome. AVMRvs. CRD Pressure(mmHg)(*p
4wkpostTsp 6v~ post Tsp
0.07±0.01(50) 0.6t-0.03(50) 0.07±0.07(18) 0.9i-0.21(18)* 0.03±0.07(5) 1.1±0.3(5)*
0.7+0.05(SO) 1.2.~0.21(1B)* 1.2~0.23 (5)*
0.8~c0.03(50) 1.3_+0.17(18)* 1.5-~0.38(5)*
1709 Expression of NMDA Receptor Subunits in Different Types of Primary Afferent Neurons Juan Carlos Marvizon, James A. McRoberts, HelenaS. Ennes, CURE/UCLANeuroenteric Disease Prog, UCLA Sch of Medicine, Los Angeles, CA; Thord Johansson, Brit Corneliussen, Lisa Jinton, AstreZenecaR&D, MOIndalSweden; Emeran A. Mayer, CURE/ UCLA Neuroenteric DiseaseProg, UCLA Sch of Medicine, Los Angeles, CA BACKGROUND: Previous research our laboratory indicated that extrinsic primary aBACKGROUND:Previous researchour laboratory indicatedthat extrinsic primary efferents innervating the rat colon expressNMDA receptors(NMDA-Rs) that are involved in visceratnociception. Functional NMOA-Rs must have at least one NR1 subunit and one NR2 subunit, of which there are four types, NR2A-D.The properties of NMDA-Rs are largely determined by the NR2 subunits that they contain. AIMS: To study the subunit composition of NMDA receptors in dorsal root ganglia (DRG). METHODS:NMDA receptor subunit-specific antibodies were used to perform Western blots of DRG extracts. DRG sections (25/~m) double-labeledwith these antibodies and with markers of different types of DRG neuronswere examinedusing confocal microscopy. RESULTS:Western blots of DRG extracts indicated the presence of the NR1, NR2B, NR2Cand NR2D subunits, but not the NR2A subunit. In DRG sections, practically all cells were stained with a NR1 subonlt antibody, and with an antibody recognizing both the NR2A and NR2B subun~ (NR2A/B), but not with an antibody recognizing only the NR2A subunit. In contrast, only small and medium size neurons were labeled with antibodies recognizingboth the NR2Cand NR2Dsubunits (NR2C/D). Co-stainingwith an antibody against neurofilamentof 200 Kda,a specific marker of A-fibers, revealedthat A-fibers stainedpositively for NR1 and NR2NB (albeit less intensely than C-fibers), but not for NR2C/D. Hence, NR2C/ D immunoreactivity (i.r.) was present exclusively in C-fibers. Co-staining with antibodies
1706 Role For Calcitonin Gene-Related Peptido And Substance P In Hypersensitivity And AIIodynia In The Rat Colon Christophe Mazzia,Yvon Jule, S Lucchini, Dept of physiology, Faculty des Science Marseille-St Jerome, Marseiile France; Frederic Raymond, Laure Delafoy, Laurent Diop, Pfizer Global R&D, Fresnes France; Monique Henry, Dept of physiology, Faculty des Science Marseille-StJerome, Marseille France Backgroundand Aims: Pharmacologicalstudies performed in conscious rats demonstratedthe involvementof calcitonin gene-relatedpeptide(CGRP)in nociceptiveresponsesto distension in the rat colon. In the present work, using the trinitrobenzene sultonic acid (TNBS)-induced colonic allodynia, we investigated the morphological distribution of CGRP immunoreactive (CGRP-ir) nerves in the colon. Since it was shown that part of CGRP-containing primary sensory afferents is colocalizedwith substance P (SP), we analyzedthe distribution of single and double immunolabelled nerves to CGRPand SP. Methods: Proximal and distal colonic segments were removed one week after injection of TNBS in the proximal colon which produces a hypersensitivity and an allodynia in distal colon. Cryostat sections were studied by confocel microscopy after double immunofluorsscenca with antibodies raised against CGRPand SP. Quantitativeanalysis of immunoreactivitieswas determined in myenteric and submucosal plexuses; they were expressedas the percentagerelative to the surface area of the ganglia. Results: In non-treated rats (n=5), the density of CGRP-ir was higher in the submucosal than in the myenteric plexus (1.44 _+ 0.24% and 0.61 __ O.08%,respectivey; p
1707 Colonic Effects Of TAK-637: A Novel NK~ Receptor Antagonist. Kalina Venkova, Karl Tyler, Okla Fdn for Digest Research Basic Science Lab, Oklahoma City, OK; Debra Sotkowski-Markmann, TAP Pharm Products Inc, Deerfield, IL; Beverley Greenwood-Vanmeerveld, Okla Fdn for Digest Research Basic Science Lab, OklahomaCity, OK Background: Functional studies indicatethat tachykinin receptors are present in the intestine and are involved in peristaltic reflexes.NK~receptorsare found on muscle as well as inhibitory and excitatory motor neurons. The present study was designedto characterizethe contractile
A-331
activity in the colonic musculatureof a newly synthesizednon-peptideNK~receptorantagonist TAK-637. Methods: The antagonist effects of TAK-B37 on tachykinin-induced contractions and on neurally evoked or spontaneous contractile activity were studied in large intestinal muscle strips or segments isolated from guinea pigs. Results: The selective agonists of NK1 receptors [Sarg,Met(O)']-substance P (SP) or GR73632 induced contractions in colonic longitudinal muscle strips pretreatedwith atropine.TAK-B37(0.1-100 nM) shifted the concentration-responsecurves to the right without a significant reduction in maximal efficacy. Schild plot analysis revealedthat TAK-637 is a competitive antagonist of NK1 receptors with similar potency against contractions mediated by SP (Kb=2.4 nM, 95% CL 1.9-3.0 ) or GR73632 (Kb= 3.5 nM, 95% CL 1.7-7.1). The Kbvalues estimated by a single shift of the concentrationresponse curves to GR73632 induced by 100 nM TAK 637 were similar in muscles studied before or after chemical denervationwith T'rx (11.8 nM vs.14.5 nM). However,TAK-637 (100 nM) has no significant effect on the contractileresponseof colonic circular muscleto GR64349, a selective NK~ receptor agonist, or contractions of taenia coli induced by a selective NK3 agonist, senktide. TAK-637 (1 /.~vl) has no effect on cholinergic contractions induced by electrical field stimulation (EFS) in ileal longitudinal muscle-myenteric plexus strips (EFS: 0.5 ms, single pulse) or nonadrenergic,noncholinergic (NANC) responsesin colonic longitudinal muscle (EFS: 0.5 ms, 1-16 Hz). In contrast, TAK-637 (1 /LM) reduced spontaneousactivity and preventedthe developmentof capsaicin-inducedcontractions in isolated segmentsfrom the terminal ileum. Conclusion: Our study demonstratesthat TAK-637 is a competitiveantagonist of peripheral NK~ receptors in the intestinal tract. Supported by TAP Pharmaceutical Products inc.
1708 Sustained Increase of the Pseudoaffective Visceromotor Response (VMR) to Colorectal Distention (CRR) in NIH/SWlSS Mice (NIH/SWISS) Following Trichinella Sldralis ( Tsp.) Enteritis Lu Wang, P A. Blennerhassett,Yk Mac, McMaster Univ, IDRP, Hamilton Canada;M Abrahamson, A Bayati, AstraZeneca,Molndal Sweden; S M. Collins, G Tougas, McMaster Univ, IDRP, Hamilton Canada Background: Visceral hypersensitivity (VH) and altered function are the central features seen in functional bowel disorders. These symptoms often appearfollowing resolution of an acute gastrointestinalinfection. In anaesthetizedN/H/SWISS, Tsp.inducedenteritis causespersistent VH with increasedcolonic afferent single fiber responseto CRD for up to 8 weeks, long after resolution of the acute enteritis (Gastro 11B:A701,2000). To assess if VH to CRD is also associatedwith somatic pseodoaffectivechanges, we examinedVMR to CRD following Tsp. enteritis, recording abdominal wall electromyographie(EMG) responseto CRD in conscious mice. Methods: In N/H/SW/SS males, 2 permanent EMG electrodes were placed within the oblique abdominal muscle. Following recoveryfrom surgery, the mice were trained to tolerate 2cm CRD balloon while placed in a Bollmano cage. VMR to CRD was recorded before, 4 and 6 weeks following Tsp. enteritis, with the distal end of the balloon located 2.5 cm from the anal verge. An increasing CRD paradigm (lO-30-40-60mmHg, lOsec) was applied with 5minute rest betweenstimuli. ~VMR is expressedas change in AUC of EMG during CRD over baseline EMG. Results: Mean_+SE(n) Summary and Conclusions: In uninfected mice, CRD produces a pressure-dependentincrease of the AVMR to CRD (0.07, 0.6, 0.7, 0.8 AUC for 10, 30, 40, 60mmHg, respectively, p < 0.05), which was significantly increased 4 and 6 weeks after Tsp.over control values (p< 0.05). These observations are in agreement with our previousfinding that Tsp.enteritis elicits a persistentcolonic visceral afferent hyperalgesic response to CRD in the absence of colitis and long after resolution of the acute enteritis. These findings demonstrating colonic hypersensitivityfollowing Tsp infection and the in vivo motility abnormalities seen in the same animals (Bercik,DOW 2001) make this an appropriate model of post-infective irritable bowel syndrome. AVMRvs. CRD Pressure(mmHg)(*p
4wkpostTsp 6v~ post Tsp
0.07±0.01(50) 0.6t-0.03(50) 0.07±0.07(18) 0.9i-0.21(18)* 0.03±0.07(5) 1.1±0.3(5)*
0.7+0.05(SO) 1.2.~0.21(1B)* 1.2~0.23 (5)*
0.8~c0.03(50) 1.3_+0.17(18)* 1.5-~0.38(5)*
1709 Expression of NMDA Receptor Subunits in Different Types of Primary Afferent Neurons Juan Carlos Marvizon, James A. McRoberts, HelenaS. Ennes, CURE/UCLANeuroenteric Disease Prog, UCLA Sch of Medicine, Los Angeles, CA; Thord Johansson, Brit Corneliussen, Lisa Jinton, AstreZenecaR&D, MOIndalSweden; Emeran A. Mayer, CURE/ UCLA Neuroenteric DiseaseProg, UCLA Sch of Medicine, Los Angeles, CA BACKGROUND: Previous research our laboratory indicated that extrinsic primary aBACKGROUND:Previous researchour laboratory indicatedthat extrinsic primary efferents innervating the rat colon expressNMDA receptors(NMDA-Rs) that are involved in visceratnociception. Functional NMOA-Rs must have at least one NR1 subunit and one NR2 subunit, of which there are four types, NR2A-D.The properties of NMDA-Rs are largely determined by the NR2 subunits that they contain. AIMS: To study the subunit composition of NMDA receptors in dorsal root ganglia (DRG). METHODS:NMDA receptor subunit-specific antibodies were used to perform Western blots of DRG extracts. DRG sections (25/~m) double-labeledwith these antibodies and with markers of different types of DRG neuronswere examinedusing confocal microscopy. RESULTS:Western blots of DRG extracts indicated the presence of the NR1, NR2B, NR2Cand NR2D subunits, but not the NR2A subunit. In DRG sections, practically all cells were stained with a NR1 subonlt antibody, and with an antibody recognizing both the NR2A and NR2B subun~ (NR2A/B), but not with an antibody recognizing only the NR2A subunit. In contrast, only small and medium size neurons were labeled with antibodies recognizingboth the NR2Cand NR2Dsubunits (NR2C/D). Co-stainingwith an antibody against neurofilamentof 200 Kda,a specific marker of A-fibers, revealedthat A-fibers stainedpositively for NR1 and NR2NB (albeit less intensely than C-fibers), but not for NR2C/D. Hence, NR2C/ D immunoreactivity (i.r.) was present exclusively in C-fibers. Co-staining with antibodies