Gut 1996; 39: 470-474

Inhibition of acetylcholine induced intestinalmotility by interleukin 1 i in the rat

A C Aube, H M Blottiere, C Scarpignato, C Cherbut, C Roze, J P Galmiche

AbstractBackground/Aims-The fact that raisedinterleukin 1j (IL1l,) concentrations havebeen found in the colonic mucosa of ratswith experimentally induced colitis and ofpatients with inflammatory bowel diseaseindicates that this cytokine may partici-pate in the disturbed intestinal motilityseen during inflammatory bowel disease.This study investigated whether IL13could change the contractility of (a) alongitudinal muscle-myenteric plexuspreparation from rat jejunum, ileum, andcolon and (b) isolated jejunal smoothmuscle cells.Methods-Isometric mechanical activityof intestinal segments was recorded usinga force transducer. Moreover, smoothmuscle cell length was measured by imageanalysis.Results-Although ILll did not affectjejunal, ileal, and colonic basal contrac-tility, it significantly reduced contractileresponse to acetylcholine (ACh). This sig-nificant inhibition was seen only after 90or 150 minutes of incubation with IL1 3.Pretreatment with cycloheximide blockedIL1 P induced inhibition of ACh stimu-lated jejunal contraction, suggesting that anewly synthesised protein was involved inthe effect. Nw-nitro-L-arginine (a nitricoxide synthase inhibitor) did not preventthe inhibition induced by IL1(. Blockingneural transmission with tetrodotoxinabolished the IL1(3 effect on jejunal con-tractile activity, whereas IL1( had noeffect on isolated and dispersed smoothmuscle cells.Conclusions-IL1(3 inhibits ACh inducedintestinal contraction and this inhibitoryeffect involves protein synthesis but isindependent ofnitric oxide synthesis. Thiseffect does not involve a myogenic mech-anism but is mediated through themyenteric plexus.(Gut 1996; 39: 470-474)

Keywords: interleukin 1(3, intestinal motility, entericnervous system, rat.

Interleukin 1P (ILl 3) is a pro-inflammatoryprotein produced by various cell types,including monocytes, platelets, chondrocytes,fibroblasts, keratinocytes, endothelial cells,and smooth muscle cells.1 It is one of the keymediators involved in inflammatory reactions.Significantly increased concentrations of IL1 ,B

have been found in the distal colonic mucosaof rats with experimentally induced colitis andin the intestine of patients with Crohn'sdisease.`A Intestinal inflammation in humansor animals is accompanied by motility changes,which may reflect alteration in the function ofsmooth muscle or the enteric nervous system,or both.5 IL 1 can also modulate the releaseof acetylcholine, norepinephrine, and sub-stance P, which are neuromediators located inthe rat myenteric plexus.68 Moreover, changein the colonic myoelectrical activity afterinduction of colitis with trinitrobenzene sul-phonic acid was significantly reduced by aninterleukin 1 receptor antagonist (ILl-ra).2These findings suggest a role for endogenousILl1P as a mediator of changed neural functionor motor activity in the inflamed intestine.Few data concerning the effects of ILl1P on

intestinal motility are available in publishedreports, and its mechanism of action is notcompletely understood. In vivo, the centralstimulatory action of endogenous IL,13 onintestinal motility seems to involve endogenousprostaglandins.9 Conversely, the action ofILlI3 on gastric motility is inhibitory.10 Indeed,both central and systemic administrations ofthis cytokine induce a longlasting delay in ratgastric emptying, an action that is mediatedthrough central IL1 receptors.'" The purposeof this study was to determine the effect ofIL 1P on rat intestinal contractility in vitro andto investigate its mechanism of action morethoroughly.

Methods

MEASUREMENT OF MUSCLE CONTRACTION

Animals and apparatusMale Wistar rats (250-300 g) were killed bycervical dislocation. One to 1.5 cm longsegments of jejunum, ileum, and proximalcolon were quickly removed, opened along themesenteric border, and cleaned of intraluminalcontents in a Krebs-bicarbonate solution (pH7.4) composed of (mmol/l) 128 NaCI, 4.5 KC1,2.5 CaCl2, 1.18 MgS04, 1.18 KH2P04, 125NaHCO3, and 5-55 D-glucose. Longitudinalmuscle-myenteric plexus (LM-MP) prep-arations from jejunum, ileum, and colon werepeeled from the underlying circular muscle andthen suspended under 1 g (for jejunum andileum) or 2 g (for colon) of tension in a 10 mlorgan bath containing continuously oxygenated(5% CO2, 95% 02) Krebs-bicarbonatesolution. Preparations were then allowed to

Centre de Rechercheen Nutrition Humainede Nantes, EquipeINSERM 'Biologic dela Motricite Digestive',Nantes, FranceA C AubeC ScarpignatoJ P Galmiche

Laboratoire deTechnologie Appliquea la Nutrition, INRA,Nantes, FranceHM BlottiereC Cherbut

INSERM U410, Paris,FranceC RozeCorrespondence to:Professor J P Galmiche,Department ofHepato-Gastroenterology,Hopital Hotel Dieu,BP 1005, 44035 NantesCedex 01, France.Accepted for publication18 April 1996

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equilibrate for 60 minutes. The isometriclongitudinal mechanical activity of the seg-ments was recorded using a force transducer(Basile no 7005, Comerio, VA, Italy), aspreviously described.`2 At the beginning of eachexperiment, acetylcholine chloride (ACh, 10`M) was applied and served as a control. Acomplete dose response curve to ACh (10-` to10-3 M) was also established. Test substanceswere applied successively with repeatedwashings of 20 to 30 minutes between eachconcentration. The viability ofeach preparationwas checked at the end of each experiment bycontrolling spontaneous mechanical activityand the response to 10-5 M ACh. Results wereexpressed as tension (g) and normalised forcross sectional area (CS), which was deter-mined using the following equation13:

CS (mm2)=((tissue wet weight (mg)/tissue length (mm)) Xdensity (mg/mm')

A density of 1.05 was used according toVermillon et al. 13

Experimental designIn a first set of experiments, the effect of IL1was studied on spontaneous and ACh inducedcontractility. ACh (10` M) was applied on theLM-MP jejunal preparation five, 90 or 150minutes after incubation with IL13 (1-50ng/ml). A dose response curve to ACh (10-9 to10-3 M) was obtained in a cummulativemanner after 90 minutes incubation with ILl ,B(10 ng/ml). ACh (10-5 M) was also applied onLM-MP ileal and colonic preparations five or90 minutes after incubation with IL1 P (10 ng/ml). To exclude an effect caused by possibleendotoxin contamination, IL 130 (10 ng/ml) wasboiled for 20 minutes before being added to thetissue. In a second set of experiments, the effectof potential inhibitory agents on the LM-MPjejunal motor response to ACh (10-5 M) wasinvestigated before and after 90 minutesexposure to IL13 (10 ng/ml). The followingdrugs were used: cycloheximide (3.5X 10-4 M)to inhibit protein synthesis, Nw-nitro-L-arginine (L-NNA, 3 X 10-4 M) to inhibitnitric oxide synthase, and tetrodotoxin (TTX,10-6 M) to block nervous transmission.L-NNA and cycloheximide were applied 15and 20 minutes respectively before IL13; andTTX was added to the bath five minutes beforeapplication of IL1,B. In control experiments, avolume of Krebs-bicarbonated solution equalto the volume of the inhibitory agents used wasadded to the bath. All tested substances wereadministered in a volume that did not exceed1% of the whole bath volume.

MEASUREMENT OF CONTRACTILE RESPONSE IN

DISPERSED SMOOTH MUSCLE CELLS

Cell preparationSmooth muscle cells were isolated from LM-MPof rat jejunum using a modification of themethod of Bitar et al, 4 as previously described.15The LM-MP preparation was cut into smallpieces and incubated at 37°C for three successive30 minute periods in Ca2`-free phosphate buffer

solution (PBS) containing 2% bovine serumalbumin, 0.1% collagenase, and 0.1% soybeantrypsin inhibitor. The pieces were washed inenzyme free PBS, and the cells were allowed todisperse under gentle pipette trituration.

Measurement ofcontractile responseCells (101) were exposed to ACh (10-7 M) forthree minutes before being fixed with 3%glutaraldehyde. Cell length was measured byimage analysis using Image 1.49 software(NIH, Bethesda, MD, USA). The length of 50cells was measured in each experiment, and theresults expressed as mean (SEM). To test theeffect of IL13 on ACh induced contraction,cells were incubated for 90 minutes with PBS(control experiments) or ILl( (10 ngfml)before being exposed to ACh (10-7 M).

DATA ANALYSISAll data are presented as means (SEM). Signi-ficance among groups was tested by one wayanalysis of variance (ANOVA).

CHEMICALS

Drugs and chemicals were purchased fromSigma Chemical Co (L'Isle d'Abeau Chesnes,St Quentin Fallavier Cedex, France), andenzymes from ICN (Orsay, France). Humanrecombinant IL13P was obtained from PeproTech (Le Perray en Yvelines, Paris, France).

Results

EFFECTS OF ILI1P ON LM-MP PREPARATIONS

ILl 1 (1 to 50 ng/ml) applied to jejunal LM-MP preparations over a period of five, 90 or150 minutes did not affect the basal tone ofjejunal muscle. In the same way, IL13 (10 ng/ml) applied to ileal and colonic LM-MP prep-arations over five or 90 minutes did not changethe basal tone of intestinal muscle (data notshown).Addition of IL13 to the bath five minutes

before ACh did not modify the ACh evokedcontractile intestinal response. However,exposure ofthe jejunal preparation to ILl13 (10or 50 ng/ml) for 90 or 150 minutes resulted ina significant (p<0 05, n=8) reduction of AChinduced contraction. Inhibition after 90 and150 minutes incubation with 10 ng/ml of ILl 13was 23.1 (3)% and 30.1 (10)% respectively(Fig 1A and 1B). A dose response curve toACh was performed after 90 minutes exposureto IL 13P (10 ng/ml) showing that the inhibitoryeffect of IL1l 1 was maintained whatever theACh concentration used (Fig 2). Exposure ofileal and colonic segments to IL 13 (10 ng/ml)for 90 minutes significantly reduced AChinduced contraction (Fig 3A and 3B).

Preincubation of the LM-MP preparationswith 10 ng/ml of boiled IL113 did not affect theACh (10-5 M) induced jejunal response(Fig 1A).The inhibiting action of ILl 1 was com-

pletely abolished by cycloheximide (3-5 X1O

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Interleukin 1, and intestinal motility

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Figure 1: Effect of interleukin 1(3 (ILJ(:, 1, 10 and 50 ng/ml) added to the bath 90minutes (A) and 150 minutes (B) before 1j5M acetylcholine (ACh) induced contractionsin isolated longitudinal muscle-myenteric plexus preparation of ratjejunum. Effect of boiledIL13 on the ACh inducedjejunal response (A). The viability of the preparation wasassessed by their response toACh at the end ofeach experiment (A), (B). Each columnrepresents the mean (SEM) ofvalues obtainedfrom 12 strips. *p<0.05 versus ACh alone.

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Figure 2: Effect of interleukin 1(3 (ILI/3: 10 ng/ml) addedto the bath 90 minutes before acetykholine (ACh) doseresponse curve (1 O9M to 1 (3 M) in isolated longitudinalmuscle-myenteric plexus preparation of ratjejunum. Eachpoint represents the mean (SEM) of values obtainedfromeight strips. *p<0*05 versus Ach alone.

M), which however did not modify the con-

tractile effect ofACh when given alone (Fig 4).The nitric oxide synthase inhibitor L-NNA(3X 10A M) did not prevent the inhibitioncaused by ILI ,B but increased the AChresponse of smooth muscle when given alone(Fig 4).Blockade of neural transmission by TTX

(10 6 M) suppressed the inhibition of ACh

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Figure 3: Inhibitory effect of interleukin l(3 (IL1,8) addedto the bath 90 minutes before 10i M acetylcholine (ACh)induced contractions in isolated longitudinal muscle-myenteric plexus preparation of rat ileum (A) and colon(B). Each column represents the mean (SEM) ofvaluesobtainedfrom 12 strips. *p<0.05 versus ACh alone.

induced contraction caused by IL1 ,B whileTTX alone did not alter ACh induced jejunalcontraction.

EFFECT OF IL1PON CONTRACTION OF

DISPERSED JEJUNAL SMOOTH MUSCLE CELLS

Isolated cells of various lengths (range 26 to 83,um) were found in the unstimulated prep-aration. Exposure of smooth muscle cells toIL1P (10 ng/ml) for 90 minutes had no signifi-cant effect on their length (52.7 (1.9) versus

55.6 (2.3) ,um). Application of ACh (10-7 M)on jejunal smooth muscle cells after 90 minutesof incubation in PBS resulted in a significantcell shortening of 23.5 (6)% (p<OOOl). Whensmooth muscle cells were preincubated withILl,13 for 90 minutes, the contractile effect of

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7 MACh

EEFX WE03

Control Cyclo L-NNA TTXFigure 4: Effects of cycloheximide (Cyclo, 35X 1j4 M), NW-Nitro-L-Arginine (L-NNA,3X 10 4M), tetrodotoxin (ITX, 106 M) on iO-s M acetylcholine (ACh) inducedcontractile response after 90 minutes ofincubation with interleukin 1(3 (IL1,8, 10 ng/ml).Each column represents the mean (SEM) ofvalues obtainedfrom 12 strips. *p<0.05 versusACh alone.

ACh did not change significantly (40.3 (1-4)versus 42.3 (1.8) ,um).

DiscussionThe results of this investigation show that ILlis a potent inhibitor of rat jejunal, ileal, andcolonic smooth muscle contraction in responseto ACh. This effect was only apparent after a

certain delay (90 or 150 minutes) after initialexposure to IL1l(3. The effect on jejunum was

abolished by cycloheximide and TTX butpersisted after pretreatment with L-NNA. Thepotent inhibitory motor effect of IL1 seen inour experimental conditions was not caused bycontamination by endotoxin (for example,lipopolysaccharide) because it was heat sensi-tive. The ability of ILl to inhibit ACh in-duced jejunal contraction corroborates previousreports showing that IL1,B inhibits the in vitromotility of gastric and other smooth muscles,'0including vascular and airway ones.'6 17As inflammatory bowel disease occurs

mainly in terminal ileum and right colon, weevaluated the effect of ILl1 on ACh ileal andproximal colonic contractile response. Our invitro findings indicate that IL1 (3 (10 ng/ml), asseen for the jejunum, produced a similarinhibitory effect in rat ileum and colon. Arecent in vivo study found that IL1 affectedthe motility of various intestinal regions,suppressing postprandial motility at the jejunalsite and modulating caecocolonic motoractivity in conscious rats.9As reported in previous studies performed

on vascular and airway smooth muscle,'6 17 theinhibitory effect ofIL1 on jejunal motility wasfound to be concentration dependent. TheILl concentrations that were effective in our

study are similar to those that inhibited gastricmotilityl' and ACh release from rat longitudi-nal muscle preparation.8 This concentrationrange is similar to that measured in freshintestinal mucosa of patients with IBD.'8

Significantly more IL1 is produced spon-taneously by mononuclear cells from inflamedthan normal colonic mucosa. 19 As colonicmotor abnormalities observed in experimentalmodels of colitis were reduced by admin-istration of ILl-ra,2 it is probable that ILlacts as a modulator of motility in both healthyand diseased intestine.IL1 induced inhibition of jejunal contrac-

tility occurred only 90 or 150 minutes after

exposure to the cytokine, which is in agreementwith reports showing that the effect of ILl ( onvascular and airway smooth muscle is timedependent.'6 17 The delayed action of ILl (suggests a mechanism involving the synthesisof a new molecule, which may well be aprotein. As in the study of Main et al8 con-cerning the effect of IL1 (3 on ACh release, wefound that cycloheximide blocked the IL1(effect on intestinal motility completely,suggesting that a newly synthetised protein wasinvolved.

Nitric oxide synthase could be the inter-mediate protein involved in the IL1( effect.This enzyme has been found in vascularsmooth muscle20 and in myenteric plexus.2' Itis involved in the synthesis of nitric oxide,which is a potent vasorelaxant mediator and aninhibitory enteric neurotransmitter in thegastrointestinal tract. Beasley et al22 found thatthe IL1( induced relaxant effect on vascularcontraction in the rat was blocked by a potentinhibitor of nitric oxide synthase (NGmonomethyl-L-arginine). However, in ourstudy, application of L-NNA failed to affectthe inhibitory action of ILl1 P. Our findings areconsistent with other findings in the rat inwhich nitric oxide synthase blockade did notchange IL1 induced relaxation of the proxi-mal stomach.'0 It is noteworthy that pretreat-ment with L-NNA alone significantly in-creased the ACh induced contractile response.This finding corroborates earlier findingsindicating that inhibition of nitric oxidesynthase, through blockage of the synthesis ofthe relaxant nitric oxide, increases electricallyinduced cholinergic contractions of guinea pigileum and taenia coli.23 24The fact that the contractile inhibitory effect

of IL1P was abrogated by prior administrationof TTIX strongly suggests the existence of aneural mediation pathway. Such mediation hasalso been suggested in ILlB induced colonichypersecretion in the rat.28 Moreover, it hasbeen shown that exogenous IL1 ,B inhibits bothACh8 and norepinephrine6 release from ratmyenteric nerves. The notion that ILl P recep-tors exist on the neurons of the myentericplexus is indirectly supported by autoradio-graphic studies showing that ILl 3 receptorsoccur on neurons of the central nervoussystem.29 Other studies have shown that ILI (can modulate invertebrate neuron functions,30and it has been postulated that this cytokineacts directly on the mammalian brain.3'Finally, myenteric plexus would seem to be thetarget of ILl ( as this cytokine had no effect onisolated dispersed smooth muscle cells in ourstudy or in airway smooth muscle.'7Although the nature of the protein potentially

involved with 1L11(3 has not been determined,IL 1 might release other cytokines, includinginterleukin 6 (IL6) and tumour necrosisfactor,25 26 which could in turn affect gastro-intestinal motility.'0 In fact, it has been shownthat ILl1( induces IL6 expression in rat intestinalsmooth muscle cells.27 Another peptide medi-ating IL1 induced inhibition of contractionmay be the vasoactive intestinal peptide, amajor non-cholinergic non-adrenergic inhibitory

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474 Interleukin 13 and intestinal motility

transmitter of intestinal contraction. We arecurrently testing this hypothesis by using a vaso-active intestinal peptide receptor antagonist.We can exclude the possibility that the

inhibitory effect of IL1P reflect changes in theacetylcholinesterase activity (enzyme whichdegrades ACh) and therefore in the bio-availability ofACh in presence ofILl P. In fact,application of neostigmine, an acetylcholin-esterase inhibitor, failed to affect the inhibitoryaction of ILl1P (data not shown). Our findingcorroborates earlier findings indicating thatIL1P suppresses ACh release in LM-MPjejunal preparations superfusated with Krebs'buffer solution containing physostigmine, anacetylcholinesterase inhibitor, suggesting thatthe ILl1P inhibitor effect could remain despitethe inhibition of acetylcholinesterase.8 More-over, the inhibitory effect of ILl P was main-tained even at high concentration. Finally,Palmer and Koch32 have recently shown thatjejunal acute inflammation was associated witha decrease in acetylcholinesterase activity. Ourstudy showed that ILl P inhibits the contractileresponse of rat jejunal, ileal, and colonicsmooth muscle to ACh. Inhibition of jejunalcontractility does not involve a myogenicmechanism but is mediated through the entericnervous system. It is independent of nitricoxide synthesis but involves a newlysynthesised protein. A previous study showedthat the effect of IL1 ,B on intestinal motility ismainly attributable to a central action. Ourstudy suggests that a peripheral action may alsobe involved in the genesis of the intestinalmotor alteration observed during inflammatorybowel disease.

1 Dinarello CA. Interleukin-l. Dig Dis Sci 1989; 33: 25-35S.2 Morteau 0, More J, Pons L, Bueno L. Platelet-activating

factor and interleukin 1 are involved in colonic dysmotilityin experimental colitis in rats. Gastroenterology 1993; 104:47-56.

3 Nakamura M, Saito H, Kasanuki J, Tamura Y, Yoshida S.Cytokine production in patients with inflammatory boweldisease. Gut 1992; 33: 933-7.

4 WoywodtA, Neustock P, Kruse A, Schwartling K, Ludwig D,Stange EF, et al. Cytokine expression in intestinal mucosabiopsies. In situ hybridisation of the mRNA forinterleukin-1 13, interleukin-6 and tumor necrosis factor-ain inflammatory bowel disease. Eur Cytokine Netw 1994;5: 387-95.

5 Verrnillon DL, Huizinga JD, Riddell RH, Collins SM.Altered small intestinal smooth muscle function inCrohn's Disease. Gastroenterology 1993; 104: 1692-9.

6 Hurst S, Collins SM. Interleukin-l,B modulation of nore-pinephrine release from rat myenteric nerves. AmJPhysiol1993; 264: G30-5.

7 Hurst SM, Stanisz AM, Sharkey KA, Collins SM. Inter-leukin 113-induced increase in substance P in ratmyenteric plexus. Gastroenterology 1993; 105: 1754-60.

8 Main C, Blennerhassett P, Collins SM. Human re-combinant interleukin 113 suppresses acetylcholine releasefrom rat myenteric plexus. Gastroenterology 1993; 104:1648-54.

9 Fargeas MF, Fioramonti J, Bueno L. Central action ofinterleukin 113 on intestinal motility in rats: mediation bytwo mechanisms. Gastroenterology 1993; 104: 377-83.

10 Montuschi P, Tringali G, Curro D, Ciabattoni G, Parente L,Preziosi P, et al. Evidence that interleukin-1 1 and tumornecrosis factor inhibit gastric fundus motility via the5-lipoxygenase pathway. Eur J Pharmacol 1994; 252:253-60.

11 Suto G, Kiraly A, Tache Y. Interleukin 11B inhibits gastricemptying in rats: mediation through prostaglandin andcorticotropin-releasing factor. Gastroenterology 1994; 106:1568-75.

12 Scarpignato C, Cartella A, Zappia L. Effect of cimetropiumbromide and other antispasmodic compounds on in vitroguinea-pig gallbladder. Methods Find Exp Clin Pharmacol1989; 11: 323-9.

13 Vermillon DL, Ernst PB, Collins SM. T-lymphocytemodulation of intestinal muscle function in thetrichinella-infected rat. Gastroenterology 1991; 101: 31-8.

14 Bitar KN, Zfass AM, Makhlouf GM. Interaction ofacetylcholine and cholecystokinin with dispersed smoothmuscle cells. AmJ Physiol 1979; 237: E172-6.

15 CherbutC,AubeAC,BlottiereHM,PacaudP,Scarpignato C,Galmiche JP. In vitro contractile effects of short-chainfatty acids in the rat terminal ileum. Gut 1996; 38: 53-8.

16 Beasley D, Cohen RA, Levinsky NG. Interleukin 1 inhibitscontraction ofvascular smooth muscle. J Clin Invest 1989;83: 331-5.

17 Tamaoki J, Yamawaki I, Takeyama K, Chiyotani A,Yamauchi F, Konno K. Interleukin-l,B inhibits airwaysmooth muscle contraction via epithelium-dependentmechanism. Am Y Respir Crit Care Med 1994; 149:134-7.

18 Ligumsky M, Simon PL, Karmeli F, Rachmilewitz D. Roleof interleukin 1 in inflammatory bowel disease-enhancedproduction during active disease. Gut 1990; 31: 686-9.

19 Mahida YR, Wu K, Jewell DP. Enhanced production ofinterleukin-l-beta by mononuclear cells isolated frommucosa with active ulcerative colitis of Crohn's disease.Gut 1989; 30: 835-8.

20 Fleming I, Gray GA, Julou-Schaeffer G, Parratt JR,Stoclet JC. Incubation with endotoxin activates theL-arginine pathway in vascular tissue. Bioch Biophys ResCommun 1990; 171: 562-8.

21 Bredt DS, Hwang PM, Snyder SH. Localization of nitricoxide synthase indicating a neural role for nitric oxide.Nature 1990; 347: 768-70.

22 Beasley D, Schwartz JH, Brenner BM. Interleukin 1 inducesprolonged L-arginine-dependent cyclic guanosine mono-phosphate and nitrite production in rat vascular smoothmuscle cells. Y Clin Invest 1991; 87: 602-8.

23 Knudsen MA, Tottrup A. A possible role of the L-arginine-nitric oxide pathway in the modulation in the guinea-pigtaenia coli. BrJPharmnacol 1992; 107: 837-41.

24 Wiklund CU, Olgart C, Wiklung NP, Gustafsson LE.Modulation of neuroeffector transmission by endogenousnitric oxide - a role for acetylcholine receptor-activatednitric oxide formation, as indicated by measurements ofnitric oxide/nitrite release. Eur Jf Pharmacol 1993; 240:235-42.

25 Ikejima T, Okusawa S, Ghezzi P, van der Meer JW,Dinarello CA. Interleukin-1 induces tumor necrosisfactor (TNF) in human peripheral blood mononuclearcells in vitro and a circulating TNF-like activity in rabbits._' Infect Dis 1990; 162: 215-23.

26 Loppnow H, Libby P. Proliferating or interleukin1-activated human vascular smooth cells secrete copiousinterleukin 6. YClin Invest 1990; 85: 731-8.

27 Khan I, Blennerhassett MG, Kataeva GV, Collins SM.Interleukin 113 induces the expression of interleukin 6 inrat intestinal smooth muscle cells. Gastroenterology 1995;108: 1720-8.

28 Theodorou V, Eutamene H, Fioramonti J, Junien JL,Bueno L. Interleukin 1 induces a neurally mediatedcolonic secretion in rats: involvement of mast cells andprostaglandins. Gastroenterology 1994; 106: 1493-500.

29 Xin L, Blatteis CM. Blockade by interleukin-1 receptorantagonist of IL-1 beta-induced neuronal activity inguinea-pig preoptic area slices. Brain Res 1992; 569:348-52.

30 Sawada M, Hara N, Maeno T. Ionic mechanism of theoutward current induced by extracellular ejection ofinterleukin- 1 onto identified neurons of aplysia. Brain Res1991; 545: 248-56.

31 Shibata M, Blatteis CM. Differential effects of cytokines onthermosensitive neurons in guinea pig preoptic area slices.AmJ Physiol 1991; 261: R1096-103.

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