A150 AGA ABSTRACTS GASTROENTEROLOGY Vol. 118, No.4

OFF responses toEFS: 5 Hz.SOV, trrs;antagonists= 1 ·1 0~; ·p<O.05vs CONT VEH;q,p<O<O.05vsCONTtreatment values are Nlcm2

non-cholinergic nerves. This increase is mediated, in part, by a loss off3-adrenergic and nitrergic inhibition

874

CHRONIC ILEAL INFLAMMATION LEADS TO TRANSMURALCHANGES IN THE INOS - NO SIGNALING PATHWAY.Fedias L. Christofi, Zacharias Suntres, Jun-Ge Yu, Uma Sundaram , TheOhio State Univ, Columbus, OH; Defense & Civil Institute of Environ­mental Medicine, Toronto, ON, Canada .

Alterations in mucosal production of NO and expression of iNOS areknown to occur in lBD . Changes in the iNOS-NO pathway during lBDwere further studied using a rabbit model of chronic ileitis. Chronicinflammation in pathogen free rabbits was produced by intragastric inoc­ulation with Eimeria magna oocytes . Experiments were done on ilealspecimens from 14 rabbits day 14 post-inoculation; means are averagevalues from 3-4 rabbits. NO concentration was measured by chemilumi­nescence. The production of NO in the mucosa of inflamed animalsincreased from 22.23±.04 ~M to 84.76=: .51~M (p=O.OOOI). The activityof mucosal NOS increased from 2.75=:.29 to 7.34:!:.45 nmoles/mgDNA/hr (p=0.0222). Inflammation increased blood NO from 12.39:!:.40 to19.36:!:.34 p.M (p=0.0153. No clear change in NO production occured insmooth muscle-myenteric plexus tissues (l2.95:!:.22p.M vs 15.35:!:.35p.M,p> .05). Labeling studies with a monoclonal anti-iNOS IgGI antibodywere used to analyze the cellular distribution of iNOS immunoreactivity(IR) . Laser confocal imaging showed that inflammation did not increase thenumber of iNOS-positive myenteric or submucous neurons (in­flamed,6.39:!:.95 myenteric neurons / ganglion, n=2,738 iNOS cellscounted vs normal,6.77 :!: 0.92 myenteric neurons / ganglion , n= 2,734iNOS cells counted, p> .05). Inflammation caused a 21% increase in iNOSintensity/expression in myenteric neurons (intensity increased from163.81:!:.70 to 197.87:!:.11 units. n=514 neurons , p< .OOOOI). Inflamma­tion caused a tiny 7:!:.02% increase in iNOS expression in submucousneurons (n=327 cells, p= .OO8). Inducible NOS was expressed in neuronswith diverse morphologies. Intense focal expression of iNOS was observedin inflamed villus and crypt epithelia near abscesses, but was marginallydetectable in non-inflamed villus epithelium. Diffuse staining occurred inthe cytoplasm of epithelia. MCID Imaging Analysis revealed a 4-5 foldincrease in iNOS IR / mm? proportional area. Intense iNOS stainingoccured at the apical and basolateral borders of the inflamed villus epithe ­lia, apical borders of some inflamed crypts , as well as in mononuclearleukocytes of the inflamed lamina propria. The results are consistent withmucosal alterations in the iNOS-NO pathway known to occur in IBD,suggesting that the rabbit enteritis model is a useful model to further studythe pathway. Chronic enteritis increases the expression of iNOS-NO pro­duction in epithelia, mononuclear leukocytes and myenteric neurons (seedfunds & NIH NCRR ISlO RRI1434).

875

CONSTITUTIVE PRODUCTION OF NITRIC OXIDE (NO) BY MY­ENTERIC NEURONS SUSTAINS INTRACELLULAR CYCLICGMP SYNTHESIS IN RAT COLONIC SMOOTH MUSCLE.Asensio A. Gonzalez , Sushil K. Sarna , Med Coll of WI & Zablocki VAMed Cntr, Dpts Surg & Physiol , Milwaukee , WI.

The rat colon is different from the human and the dog colon in the sensethat it generates spontaneous regularly occurring giant migrating contrac­tions (GMCs) in vivo and giant contractions (GCs) in vitro . AIM: The aimof this study was to determine whether the generation of spontaneous invitro GCs in the rat colon is modulated by constitutive release of NO orother neurotransmitters from the myenteric neurons . If so, is the modula­tion dependent on the synthesis of cGMP and activation of protein kinaseG (PKG)? METHODS: Circular muscle strips were prepared from the rattransverse colon . A standard 3 ml muscle bath was used to record con­tractions . RESULTS: Rat colonic muscle strips generated spontaneous GCs(0.21 :!: 0.02lmin and 3 :!: 0.6 g amplitude) which were unaffected byhexamethonium, atropine, VIP 10.28' and antagonists for serotoninergic(5-HT I 4 ) , histaminergic (HI and H2) and tachykininergic (NK 1•2) recep­tors (10 p.M each) . NK) receptor antagonist [Trp7, f3-Ala8-NKA4.IO) (10p.M) elevated tone and increased GC frequenc y to 0.51 :!: 0.05/min.Tetrodotoxin (TTX, I pM) or 0.5 p.M w-conotoxin (0.5 p.M) increased theGC frequency to 0.78 ± 0.05 and 0.65 ± 0.05/min and amplitude to 6.1 :!:0.1 and 5.7 ± 0.1 g respectively, A similar increase was obtained withN"'-nitro-L-arginine (L-NNA, 0.1 mM) (0.79 :!: O.04/min and 6.0 ± 0.1 g)and guanylate cyclase inhibitor ODQ I ~M (0.73 =: 0.06/min and 6.3 :!:0.2 g). However, L-NNA or ODQ did not affect GCs after neural blockade

3.3±1.6 17.4±2.S-20.7±3.9q. 19.h2.3

L·NNA

12.8±O S­19.1 +2.3

PR+PHPHENTPROP

15.63.2­10.1±3.5

ATR

7.2±1.2139±3.3$

6.9±1.1lS.hl .3-

VEH

CONTTNBS

872

ACTIONS OF GLIAL DERIVED NEUROTROPHIC FACTOR(GDNF) ON ELECTRICAL AND SYNAPTIC BEHAVIOR OF EN­TERIC NEURONS IN GUINEA-PIG SMALL INTESTINE.Jackie D. Wood, Yun Xia, Hong-Zhen Hu, Jun Ren, Sumei Liu, Ohio StateVniv Col1 of Medicine and Public Health, Columbus, OH.

Conventi onal intracel1ular microelectrode recording methods were used tostudy the actions of GDNF on enteric neuronal electrical and synapticbehav ior. Results were obtained from 36 myenteric and 44 submucousneurons in preparations from 27 guinea-pigs. Exposure to GDNF bypressure microejection had excitatory effects consisting of membranedepolarization and enhanced discharge of action potentials in 86% ofAH-type and 80% of S-type myenteric neurons . In the submucous plexus ,al1 AH-type and 92% of S-type neurons were stimulated by GDNF. Theresponses occurred in one of three patterns . One was a rapidly activatingdepolarization associated with decrea sed input resistance . The second wasan initial rapidly activating depolarization followed by slowly developingrepolarization, after which occurred a slowly activating and sustaineddepolarization. The rapid depolarization was associated with decreasedinput resistance. Input resistance increased during the repolarization withlittle change during the subsequent depolarization. The third kind ofresponse was a single slowly-activating depolarization associated withincreased input resistance. Tetrodotoxin blocked GDNF-evoked spike dis­charge , but not the membrane depolarization. Stimulus-evoked noradren­ergic inhibitory postsynaptic potentials (lPSPs)in submucous neurons werereversibly suppressed or abolished by GDNF in all of 9 neurons. Hyper­polarizing responses to norepinephrine were unaffected by GDNF. Thissuggested that the site of action of GDNF was at presynaptic inhibitoryreceptors on sympathetic nerve terminals, and that suppression of the IPSPsresulted from inhibition of norepinephrine release . Stimulus-evoked fastnicotinic postsynaptic potentials (fEPSPs)and slow synaptic excitation(sEPSPs) were suppressed in the myenteric and submucous plexus byGDNF. Depolarizing responses to acetylcholine, substance P or serotoninwere unaffected by GDNF. This suggested that the the site of GDNF actionwas at presynaptic inhibitory receptors on intrinsic cholinergic nerveterminals and at release sites for the unidentified sEPSP neurotransmitter.In addition to direct excitation of enteric neurons, the results suggest thatpresence of GDNF will release the sympathetic brake from secretomotorneurons to the intestinal crypts . This together with excitation of thesecretomotor neurons may lead to neurogenic secretory diarrhea .

873

INFLAMMATION INDUCED ALTERATIONS IN ADRENERGICCONTROL OF SMOOTH MUSCLE IN ACUTE COLITIS IN RATS.Aiping Zhao, Julie Nealon, Victor Pinerio-Carrerro, Terez Shea-Donohue,Vsuhs, Bethesda , MD.

Colitis causes progressive changes in the cholinergic, noncholinergic, andnitrergic control of smooth muscle contractility. However, there is lessinformation on inflammation-induced alterations in adrenergic innervation.Inflammation is also associated with a persistent elevation in LTD4 syn­thesis. LTD4 enhances the sensitivity of nerves to stimulation. Aim:todetermine the role of adrenergic nerves in colitis-induced alterations insmooth muscle function. Methods.Sprague-Dawley rats received intrarec­tal saline(CONT)or TNBS (100 mglkg in 50%ETOH) and were studied 4hrs later. Mucosa-free segments of distal colon were mounted in organbaths in their circular axis and stretched to Lo and subjected to electric fieldstimulation (EFS) in the absence (VEH) and presence of specific blockers :propanolol (PROP, f3-adrenergic), phentolamine (PHENT, a-adreneric,L-NNA (nitric oxide synthase) , atropine (ATR, muscarinic ) or Wy48,252(Wy, LTD4). Concentration-dependent response curves to carbachol werealso constructed . Results: Responses to carbachol were similar in bothgroups (26.6 :!: 0.5 vs 23.9 :!: 3.4 N/cm2). EFS evoked a frequency­dependent increase in the OFF response . Colitis enhanced the OFF re­sponse (table) to EFS and increased the sensitivity l.5-fold. Colit is alsoincreased the OFF responses in the presence of ATR with a 4-fold increasein sensitivity indicating an effect on non-cholinergic excitatory nerves. Incontrol rats, f3-adrenergic and nitrergic nerves suppressed EFS-stimulatedcontractions. In colitis , there was no difference in EFS responses betweenVEH and any antagonist. Wy completely blocked EFS response in bothcontrol and acute. Conclusions: In control s, OFF contractions to EFS arenon-cholinergic, dependent upon LTD4, and regulated by f3-adrenergic andnitrergic nerves. Acute colitis enhance s the response to stimulation of