5-HT3 receptor mediated excitation of colonic afferent endings: Functional and anatomical correlates

28

serosa revealedthat the majority of distension-sensitivefibers were located in the cardia, up to 5 mm from the gastro-esophagealjunction. Baclofen decreasedboth basal (P<0.001) and distension-evoked activity of vagal afferent fibres, over the full range of volumes (2, 5 and 10 mls of air) and maximally at 10 mls by 40% (P<0.05, n = 7). 3-APPiAdose-dependently inhibited distension-evokedafferent firing frequency (P<0.001, n = 5). The effects of baclofen were reversedfollowing treatment with the GABAereceptor antagonist CGP 62349 (7 #M). Neither baclofen nor 3-APPiAtreatment significantly altered gastric complianceduring distension. Conclusion: These results demonstrate a direct inhibitory role for GABAereceptors on vagal afferent responsesto gastric distension. This may be a contributing mechanism to the efficacy of GABAereceptor agonists in their capacity to modulate TLESRsand reflux episodes. Supported by AstraZeneca.

5-HT3 Receptor Mediated Excitation of Colonic Afferent Endings: Functional and Anatomical Correlates Gareth A. Hicks, Glaxo Institute of Applied Pharmacology,Cambridge United Kingdom; Penny A. Lynn, Royal Adelaide Hosp, AdelaideAustralia; David Jenkins, Patrick P Humphrey, Glaxo Institute of Applied Pharmacology,Cambridge United Kingdom; L Ashley Blackshaw, Royal Adelaide Hosp, AdelaideAustralia Background: 5-HT3 receptor antagonists inhibit visceral pain from the colon by a mechanism thought to involve a reduction of excitation of spinal afferent fibres by endogenous 5-HT. However,this remainsto be proven, as nothing is known about the direct sensitivity of colonic afferent endings to 5-HT, or the receptors that may be involved. Here we report 5-HT3 and non 5-HT3-medlatedeffects of serotonergicagonists on rat colonic afferents and the presence of 5HT3 receptors on their cell bodies. Methods: Eiectrophysiolngy: Distal colon, inferior meserdericganglion and lumbar splanchnic nerve (LSN) were removedtogether. In a specialized organ bath,the colon was openedand pinnedflat mucosa-up.The mucosawas supertused with fatty acid.containing Krebs,and the serosa with normal Krebs.The LSN was led through a hole and 46 single units recordedfrom the LSN in an oil-filled chamber.3 classesof primary afferents were recorded as classified previously (Lynn & Blackshaw J.Physiol. 1999: 518: 271-282): 30 high threshold semsal (probing sensitive) afferents, 4 low threshold muscular (stretch sensitive) afferents, and 12 mucosal (stroking sensitive) afferents. Anatomy: Fast blue was injected into the wall of the distal colon following laparotomy under isoflurane anaesthetisia. Dorsal root ganglia (DRG) at Tll-$2 were removed 1-2 weeks after recovery and fixed overnight in 4% PFA. Sections (12p.m) were cut and mounted for observation of fluorescent label and 5-HT3 receptor immunohistochemistrywhich was performed in sections taken from the L1 DRG, a level containing cell bodies of the LSN. Resu/ts: 19/34 (56%) of all three classesof colonic afferents showed dose-dependentresponsesto 5-HT (10-1000p.M for 1 min). 5/29 (17%) respondedto the selective5-HT3agonist, 2 methyl 5-HT (100-1000,u.M). Responses were maintained throughout the period of exposure to the receptivefield. In 18 fibres, both agonists were tested; 3/18 responded to both, 8/18 responded to 5-HT alone; none respondedto 2 methyl 5-HTalone.Approximately26% of L1 DRG cell bodies retrogradely labelled from the colon displayed 5-HT3 receptor-like immunoreactlvity. Conc/usions:twothirds of the cells expressing 5-HT3 receptors in the DRG also express functional receptors at their peripheralterminals. 5-HT receptor agonists activate primary afferents via both 5-HT3 and non-5-HT3 receptor mechanisms. 5-HT3 receptor antibody was kindly provided by Dr Sarah Lummis, University of Cambridge, UK. LA.B. supported by NHMRC.

26 Hyperalgesia and Up-Regulation of Nocicoptive Genes in Sensmy Noirons in PancreaUtis Is Associated with Up-Regulation nt Nenm Gfowth Factor (NGF) and Requires the Activation of Tyrosine Kinace Receptor. Hiroki Toma, John H. Winston, Lei Zou, Maria-AdelaideMicci, Mohan Shenoy, PankajJ. Pasricha, Univ of Texas Medical Branch, Galveston,TX Background:.Although pain is a cardinal feature in pancreatitis,little is known about molecular mechanisms for the pathogenesisof pain. NGF and its tyrosine kinase receptor trkA appear to be key players in the developmentof inflammatory hyperalgesiain models of somatic pain. The aim of this study was to determine its role in the nociceptive response to pancreatitis. Methods:Acute pancreatitis was induced in Sprague-Dawleyrats by L-arginine as previously described. Pancreata were processed for the analysis of, 1) NGF mRNA level by RNase protection assay, 2) NGF protein level by ELISA, 3) tyrosine phosphorylationof ~ receptor by immunoprecipitationand Western blotting, 4) immunohistochemistry. Dorsal rOot ganglia (DRG) from T9 to It of both sides were collected from each rat and Northern blotting was done for expression of nociceptive genes, preprotachykinin (PPT), CGRP, vanilloid receptor 1 (VR1). NGF-trkA receptor signaling was inhibited using a tyrosine Idnase inhibitor, k252a (80/,~g/kgbw, injectedintraperitoneally,or vehicle,oncedaily for 6 days). In vivo pain responses were assessedby Von Frey Filaments (VFF)testingof somatic referral regions at 1 week postinduction. Resul~ NGFlevelsin the inflamed pancreasshoweda significant increasecompared to controls in mRNA expression (24-fold, P < 0.01) and protein content (6-fold, P < 0.05) from 3 to 7 days. On Western blotting, the intensity of phoshotyrosine bend was higher in rats with pancreatifiscomparedto controls. The immunoreactivityof tyrosine-phosphorylated trkA was colocalized with substance P, CGRP or VR1 immunoreactive nerve fibers in the inflamed pancreas.Northern blotting showed an up-regulationof PPT, CGRP,VRt expression in DRG of rats with pancreatifis as compared to controls (fold increase: PPT; 1.56_+0.55, CGRP; 2.37_+1.21, VR1; 1.43_+0.37, n = 13, P < 6.01). VFF thresholds for pain in rats with pancreatitis were significantly increased by k252a treatment ( %: 79.8_+14.9 (vehicle) vs 105.7_+14.0 (k252a), n = 6, P < 0.05), associatedwith suppressionof both nociceptivegenes response in DRG and phosphotyrosine in pancreasby k252a treatment. Conclusion~ These results indicate that the activation of NGF-trkA receptor system mediates pain processing in pancreatitis and show that NGF-trkA system can be a potential novel therapeutic target for patients with painful pancreatitis.

29 Oral PTopbalin Reduces Repeated Coloractal Distention (CRO)-InducedVisceral Pain And Lnmbocecrai Spinal Cord Neuronal Activation in Rats. Mulugeta Million, Lixin Wang, Timothy Maryanov, Santosh Coutinho, Emeran Mayer, Yvette Tache, UCLA, Los Angeles, CA BACKGROUND:The anticonvulsant agent, pregabalin,blocks thermal, surgery and chemicalinduced pain (JPET 295:162-67,2000). However, there are limited studies on whether this new class of agent is effective against pains of visceral origin. AIM: To study the effects of oral pregabalinon CRD-inducedabdominalcontractions (AC) and lumbo-sacral (L6-$1) spinal cord neuronal activation in awake rats. METHODS: Adult male SD rats (5-15/group) were implantedor not with electrodeson the externalabdominalmuscle.Two weekslater, pregabalin (30 mg/kg) or water was given orally to rats, inserted with a latex balloon through the anus into the rectocolic segment. CRD at 60 mmHg (10 min on/off, 2X) was conducted and the number of ACs/IO min or area under the curve of contractions (AUC)/IO min were measured. Lumbo-sacral spinal cord neuronal activation was studied 60 min after the end of the CRDs using Fos immunoltistochemistry. RESULTS:The control AC response/lO rain to the 1st and 2nd CRDswere 23.6 -+ 1.5 and 29.5 _+2.4 (p
27 5-HT, Receptor Agonists Stimulate Small intestinal Transit but Do Not Have Direct Visceral Antieocicoptive Effects in the Rat. Gareth A. Hicks, Glaxo Institute of Applied Pharmacology,Cambridge United Kingdom; Nick M. Clayton, Pam J. Gaskin, Glaxo Weilcome Inc, StevenageUnited Kingdom; Anthony J. Kirkup, Univ of Sheffield, Sheffield United Kingdom; Xin Su, Shailen Joshi, Ann Friedrich, Univ of iowa, iowa City, IA; Helen E. Connor, Brian Cox, Glaxo Wellcome Inc, StevenageUnited Kingdom; David Grundy, Univ of Sheffield, Sheffield United Kingdom; Gerald F. Gebhert, Univ of iowa, Iowa City, IA; Patrick Pa Humphrey, Glaxo institute of Applied Pharmacology, CambridgeUnited Kingdom Background: 5-HT4 receptor agonists are in clinical development for disorders involving constipation, including irritable bowel syndrome, in which pain is a major symptom. Although pain relief may be afforded by relief of constipation, it has been suggestedthat these agents have direct antinociceptlve actions (Schikowski et al, '98 Gastroenterol. 116, A643; Cnelho et al, '99 Gastroentero/. 118, A835). We therefore investigatedthe actions of two selective 5-HT4 receptoragonists upon neuronal activity and abdominalcontractionselicited by noxious intestinal distentions, at doses that stimulated small intestinal transit, in rats. Methods & Results: Small intestinal transit of "~Sn-radiolabelledmicmspheres was measured in male Brown Norway rats (180-220g, n =4-6 per dose) treated with p-aminodonidioe (10 pg/kg) as described previously (Clayton et al, '99, Neurogastro. & Motil.11,207-217). Tegaserod (0.1-1 mg/kg, i.p.) and prucalopride (1 & 3 mg/kg, i.p.) produced dose-dependentincreases in transit rate, tegaserod being approximately lO-fold more potent. This effect was prevented by prior administration of the selective5-HT4receptor antagonist, GR125487 (100 pg/kg, i.p. vs 0.3 mg/kg tegaserod, n=4; Gale et al '94, Brit. J. Pharmacol.113, 119P). To investigate potential direct neuronal effects of the agonists, extracellular recordings were made from anaesthetised male Wistar rat (330-400g) jejunal mesentaric afferent bundles (Kirkup et al., '98, Br. J. Pharmaco/., 125, 1352) and from single pelvic nervefibres in male SpregueDawley (350 550g) rats (Sengupta & Gebhart '94, J. Neurophyisio/., 71, 2046-60). In jejunal afferent bundles, neither baseline activity, nor that resulting from high pressure distention (ramp O60 mmHg, saline infusion lml/min), were affected by togasernd (1 or 3mg/kg i.v., n=4) or prucalopride(1 or 3 mg/kg i.v., n = 3 or 4). Similarly, activity in pelvic nervefibers in response to noxious colorectal distention (CRD; 80mmHg) was unaffected by prusalopride (1 & 3mg/ kg i.v., n=4). Furthermore, abdominal contractions induced in conscious rats by similar noxious CRD were also unaffected by procalopride (1 or 3 mg/kg i.p., n=4). Conclusion: Our studies suggest that doses of 5-HT4 agonists, which stimulate small intestinal transit, are unlikely to have direct visceral antinocicetive action in rats. We speculatethat any relief of pain observed in constipated IBS patients is likely to be secondaryto relief of the constipation itself.

Effect of oral pregabalinon repeatedCRDs-inducedneuronalactivationin the L6-$1 spinal segment : LaminaI-II, Ill-VII and IML (intermediolateralcolumn). Mean±SE, * p
Vehide+CRDs Prngabelin+CRDs

A.-6

Fos positive cells/section kll IML

IIkVII

90.4±2.8 74.6±2.6*

223.5_+5.6 164.9-/-_7.7*

31.6+1.2 25.1_+1.1*