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Nitric oxide pathways in circular muscle of the rat jejunum before and after small bowel transplantation
Nitric Oxide Pathways in Circular Muscle of the Rat Jejunum Before and After Small Bowel Transplantation Bruno M. Balsiger, M.D., Judith A. Duenes, B.A., Noriya Ohtani, M.D., Chikashi Shibata, M.D., Gianrico Farrugia, M.D., l/VilliamJ. Anding, B.A., Michael G. Sa~ M.D.
Previous studies suggest that nitric oxide synthase is upregulated after small bowel transplantation which may have implications in enteric dysfunction after small bowel transplantation. The aim of this study was to determine the role of nitric oxide in nonadrenergic, noncholinergic inhibitory function after small bowel transplantation in rat jejunal circular muscle. The following four groups of rats (n = --8 rats per group) were studied: Neurally intact control animals; 1 week after anesthesia and sham celiotomy, and either 1 week or 8 weeks after isogeneic, orthotopic small bowel transplantation. Full-thickness jejunal circular muscle strips were evaluated under isometric conditions for spontaneous contractile activity, response to electrical field stimulation, and effects of exogenous nitric oxide and nitric oxide antagonists. Spontaneous activity did not differ among groups. Electrical field stimulation inhibited activity similarly in all groups. Exogenous nitric oxide, NG-monomethyl L-arginine monoacetate salt (a nitric oxide synthase inhibitor), and methylene blue (cGMP antagonist) had no effect on spontaneous activity. Neither nitric oxide antagonist altered the inhibitory response to neural excitation by electrical field stimulation in any group. Nitric oxide, a known inhibitory neurotransmitter in other gut smooth muscle, has no apparent role in rat jejunal circular muscle before or after small bowel transplantation. (J GASTROINTEST SURG2000;4:86-92.) KEY WORDS: Motility, nitric oxide, smooth muscle, small bowel transplantation, inhibitory neurotransmitters
Although small bowel transplantation (SBT) is rapidly becoming a viable clinical option for gut failure, 1 little is known about changes in small bowel function after SBT. Understanding changes in enteric function is important because SBT is associated with multiple problems in the early and late postoperative periods including abnormalities in motility, diarrhea, and difficulty with enteral delivery of nutrition. 2 Indeed, changes in enteric function are not unexpected, because SBT obligates an extrinsic denervation of the graft, disruption of intrinsic (enteric) neural continuity of the graft with the still-innervated proximal and distal gut, an ischemia/reperfusion injury, and hostmediated immune responses to the graft.
Previous work from our laboratory has investigated the early and late effects of SBT on contractile activity of circular smooth muscle of the rat ileum. Although we found an impressive adrenergic hypersensitivity after SBT, spontaneous contractile activity remained unchanged. 3,4 W h e n examined by exogenous application of nitric oxide (NO) and N O antagonists and during intrinsic neural excitation by electrical field stimulation (EFS), inhibitory neurotransmission to ileal circular muscle appeared to be independent of N O . 5 In contrast, in jejunal circular muscle, SBT induced an increase in spontaneous contractile activity and augmented EFS-induced nonadrenergic, noncholinergic (NANC) inhibition. Because no changes
From the Department of Surgery and the GastroenterologyResearch Unit, Mayo Clinic and Foundation, Rochester,Minn. Supported by grant DK 39337 from the National Institutes of Health, United States Public Health Service, the Mayo Foundation, the SwissNational Foundation, and the Department of Visceral and Transplantation Surgery, Universityof Bern, Switzerland. Part of this study was presented at a poster session at the Fortieth Annual Meeting of The Societyfor Surgery of the AlimentaryTract, Orlando, Fla., May 16-19, 1999. An abstract of this work was published in Gastroenterology116:A1349, 1999. Correspondence: Michael G. Sarr, M.D., Professor of Surgery, Gastroenterology Research Unit, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. 86
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occurred in sensitivity to cholinergic or adrenergic agonists, 6 we wondered whether transplantation-related alterations in nitrergic nerves might explain these changes in contractile properties. Indeed, others have suggested that the number of nitrergic nerves is increased after extrinsic denervation and SBT, 7,8 and that NO synthase activity is upregulated by extrinsic denervation.9 Such changes in these presumably inhibitory nitrergic nerves may have marked effects on gut motor function. Therefore the aims of the present study were to determine the effect of N O in rat jejunal circular muscle and to identify the role of N O in EFS-induced changes in NANC inhibition before and after SBT. We hypothesized that exogenous NO would inhibit contractile activity and that changes in spontaneous contractile activity and EFS-induced NANC inhibition after SBT would be related to changes in a nitrergic mechanism with NO acting as the neurotransmitter. MATERIAL AND METHODS Preparation of Animals Procedures and animal care were performed according to the guidelines of the Animal Care and Use Committee of the Mayo Foundation in accordance with the guidelines of the National Institutes of Health and the Public Health Service Policy on the Human Use and Care of Laboratory Animals. Experimental Groups. Because we were interested in the physiologic effects of the SBT procedure and not rejection, immune suppression, or other immune phenomena, we specifically used syngeneic male Lewis rats (Harlan Sprague-Dawley, Indianapolis, Ind.) in all experiments to avoid confounding immune-related phenomena that occur after allotransplantation. After anesthesia was achieved by intraperitoneal sodium pentobarbital (Ampro Pharmacy, Arcadia, Calif.), orthotopic SBT was performed using standard microvascular techniques as described previously.6 In brief, the entire jejunoileum was removed from the donor rat after flushing the intestinal lumen and infusing the graft vasculature with chilled Ringer's lactate solution. The graft was revascularized by anastomosing donor aorta to recipient aorta (end-to-side) and donor portal vein to recipient inferior vena cava (end-to-side). After resecting the recipient jejunoileum, intestinal continuity was reestablished by end-to-end jejunojejunostomy and ileoileostomy. All rats were allowed free access to water and rat chow immediately postoperatively. Rats were studied 1 week and 8 weeks after SBT (SBT-1, n = 12; SBT8, n = 14). Naive rats without any surgical procedure
Nitric Oxide in Jejunal Motility After Transplantation
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served as neurally intact controls (NC, n = 4). Also, to determine the nonspecific effects of anesthesia and celiotomy, rats I week after celiotomy and intestinal manipulation were used as sham-operated controls (SC, n = 8). Recordingof Contractile Activity. A segment of proximal jejunum 7 to 10 cm distal to either the ligament of Treitz or the jejunojejunostomy after SBT was removed, immersed in chilled modified KrebsRinger's bicarbonate solution (concentrations in mmol/L: NaC1 116.4, KC14.7, CaCI2 2.5, MgSO4 1.2, K H 2 P O 4 1.2, NaHCO3 23.8, calcium disodium edetate 0.26, and glucose 11.1) and opened along the mesenteric border. Four to eight fifll-thickness muscle strips cut in the direction of the circular muscle layer were suspended vertically in 10 ml tissue chambers filled with modified Krebs-Ringer's bicarbonate solution; the chambers were maintained at 37.5 ° C and bubbled with 95% oxygen and 5% carbon dioxide (Puritan-Bennett Corp., Lenexa, Kan.). Because of the well-recognized variability between muscle strips in the same rat, identical experiments were performed on multiple muscle strips per rat. One end of the muscle strip was attached to a fixed hook, whereas the other end was connected to a noncompliant force transducer (Kulite Semiconductors Products, Inc., Leonia, N.J.) to measure isometric force. Experimental Protocol After an 80- to 90-minute equilibration period with intervening washout of the bath solution every 20 to 25 minutes, each strip was incrementally stretched at 12- to 15-minute intervals to its optimal length (Lo) beyond which further stretching did not increase the amplitude of spontaneous activity. 6 All subsequent experiments were performed at Lo; strips without spontaneous contractile activity or without any response to EFS were not used (less than 9% of strips). After measuring spontaneous basal contractile activity for a 5-minute interval at Lo, atropine (10 -7 tool/L), phentolamine (10 -5 mol/L), and propranolol (5 X 10 -6 tool/L) were added to the bath in eight chambers to induce NANC conditions, and spontaneous contractile activity under NANC conditions was measured for a 5-minute interval beginning 30 minutes later. NO in distilled water (3 x 10 -6 tO 3 X 10 -s tool/L) was applied directly into the chamber under NANC conditions to determine the effect of NO on spontaneous activity. NO was prepared by dissolving NO gas in distilled water previously degassed with helium according to the method reported previously. 3 Then, EFS was applied at increasing frequencies (1, 2, 3, 4, 5, 7, and 10 Hz) with a constant voltage (20 volts), pulse width (4 msec), and duration of
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Table I. Spontaneous contractile activity of rat jejunal circular smooth muscle* Group
Basal
NANC
L-NMMA
Methylene blue
SC
5.05 +_ 0.55
5.79 ___ 0.80
5.44 _+ 1.18
6.17 _ 1.28
SBT-1 SBT-8
4.47 -- 0.54 4.03 +- 0.40
4.89 +- 0.68 4.50 _ 0.54
4.28 --- 0.65 3.86 --- 0.52
4.19 -+ 0.87 4.89 --- 0.77
*Mean + SEN[, g • 5 rain/wet tissue; n = >- 8 rats per group.
stimulation (10 seconds) to obtain a frequencyresponse curve. T h e different frequency stimulations were separated by 5 minutes to allow spontaneous activity to recover before the next stimulation. T h e contractile response to EFS was quantitated only during the 10-second stimulation. NC-monomethyl-L arginine (L-NMMA, 10 -3 mol/L), a specific inhibitor of N O synthase, and methylene blue (10 -5 mol/L), a nonspecific inhibitor of soluble guanylate cyclase, were administered into each of four chambers. Contractile activity was then determined for a 5-minute interval beginning 3 0 minutes after drug administration. EFS was repeated under the same conditions in chambers with L - N M M A and methylene blue to determine the effect of these drugs on the response to EFS. In separate experiments in control rats (data not shown), EFS was investigated in the presence of 10 -6 mol/L tetrodotoxin which, by blocking sodium channels, inhibits almost all neural transmission; we confirmed that EFS-induced inhibition and contractile activity were tetrodotoxin sensitive and thus neurally mediated. At the conclusion of the experiments, each tissue was blotted and weighed.
Data Analysis We quantitated total contractile activity by measuring the integral of the force generated (g. 5 min area under the contractile curve) under each condition using specialized software (AcqKnowledge, Biopac Systems, Inc., Goleta, Calif.). Spontaneous basal activity at Lo (4 to 8 muscle strips per rat), and the effects of N A N C conditions (4 muscle strips per rat), L - N M M A (2 muscle strips per rat), and methylene blue (2 muscle strips per rat) on spontaneous activity were quantitated for 5-minute intervals. In EFS experiments (4 muscle strips per rat), we determined the response during the 10-second stimulation ("onresponse") and specifically excluded the response immediately after stopping stimulation (the so-called "off-response"). T h e integral of force generated during the 10-second EFS at each frequency was expressed as the percentage of spontaneous contractile activity for a mean interval of 10 seconds as calculated from the 5 minutes of contractile activity measured immediately before beginning EFS. Frequency-
response curves were generated. T h e effect of L - N M M A and methylene blue on EFS-induced inhibition was compared to the inhibition during EFS before administration of these agents in the same strip. All contractile data were standardized by milligrams of tissue wet weight. Analysis of variance combined with Student's t tests were used for comparisons among multiple groups (comparison of basal spontaneous activity across groups), and separate t tests for paired data were used to study the effect of various drugs on spontaneous activity and during EFS. A Bonferroni correction was made for multiple comparisons whenever appropriate. All data are presented as mean - standard error of the mean (SEND, and the n used for statistical comparisons was the number of rats (not muscle strips) per group. Since there were no differences between the N C and SC groups, we chose SC rats as controls for the SBT rats. Drugs DL-Propranolol hydrochloride, phentolamine hydrochloride, atropine sulfate, L-NMMA, and methylene blue were purchased from Sigma Chemical Company (St. Louis, Mo.). Nitric oxide gas was purchased from Matheson Gas Production, Inc. (Parsippany, N.J.). RESULTS
Spontaneous C o n t r a c t i l e Activity N o differences were noted in spontaneous basal contractile activity between groups. T h e effects of N A N C conditions, L-NMMA, and methylene blue on spontaneous activity are shown in Table I. Induction of N A N C conditions did not alter contractile activity significantly in any group (see Table I). Similarly, when evaluated under N A N C conditions, L - N M M A and methylene blue had little or no effect on spontaneous contractile activity (see Table I). Exogenous administration of N O (3 x 10 -6 tO 3 X 10 -5 mol/L) under N A N C conditions also had no inhibitory effect on spontaneous phasic activity, basal tension, or total contractile activity in any group (Fig. 1).
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Nitric Oxide in Jejunal Motility After Transplantation
10-e M
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5 min Fig. 2. Effect of electrical field stimulation on spontaneous contractile activity of rat jejunal circular muscle in vitro under NANC conditions in the SBT-1 group. A, Characteristic inhibitory effect at all frequencies. Inset represents an expansion of the segment within the box. B, Net contractile response. This less common pattern was seen in 9%, 10%, 47%, and 18% of strips in the NC, SC, SBT-I, and SBT-8 groups, respectively. E f f e c t o f Electrical F i e l d S t i m u l a t i o n Fig. 2 shows two different overall patterns of responses to E F S - - a primarily inhibitory one (Fig. 2, A) and a procontractile one (Fig. 2, B). T h e most frequendy observed pattern (198 of 227 strips; Fig. 2, A) consisted of inhibition of phasic contractions and/or
reduction in basal tension (and therefore decreasing total force) during low-frequency (1 to 7 Hz) EFS; this inhibitory effect was not apparent at 10 Hz. This pattern was present in 91%, 90%, 53%, and 82% of the strips in the N C , SC, SBT-1, and SBT-8 groups, respectively. T h e second pattern (Fig. 2, B) consisted
90
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of EFS evoking no inhibition but rather stimulating contractile activity at all frequencies tested; this pattern occurred mostly in the SBT-1 group (47% of the strips); in 4 of the 12 rats all strips showed this response. Because we were interested in the inhibitory function, we focused on the former inhibitory responses. The frequency-response curves in all groups are shown in Fig. 3. In SBT-1 rats, the overall frequency-response pattern was similar to that of the SC group, but the inhibitory response at 7 Hz was smaller and the contraction at 10 Hz greater than in the SC and SBT-8 rats (see Table II). The effects of the N O antagonists L - N M M A and methylene blue on the EFS response curves in each group are shown in Table II; neither L - N M M A nor methylene blue altered the frequency-response curve to EFS in any group. DISCUSSION
Nitric oxide has been shown to be an important inhibitory neurotransmitter in gut smooth muscle in various species. 1° Our study was designed based on the hypothesis that N O has a modulatory role before and after isogeneic SBT in circular muscle of the rat jejunum. Neither exogenous application of N O , blocking N O synthase with L-NMMA, nor inhibiting soluble guanylate cyclase with methylene blue, the presumed intracellular pathway by which N O inhibits contractile activity, had any noticeable effect on spontaneous contractile activity in rat jejunal circular muscle; these observations imply that N O does not modulate spontaneous continuous phasic activity or basal tone in this tissue. Similarly, the N O antagonists LN M M A and methylene blue did not abrogate the EFS-induced inhibition in any group. These results suggest that under our experimental conditions, nitrergic pathways do not appear to play an important or major role in mediating long-term N A N C inhibition either in normally innervated rat circular jejunal muscle or a major role in extrinsically denervated jejunum after SBT. Inasmuch as N O synthase-positive nerve fibers are abundant in the rat circular muscle layer,s it is possible that the role of neuronally released N O is to inhibit muscle activity under stimulated conditions or to modulate release of other neurotransmitters. N O modulation of acetylcholine release has been reported in canine colonic circular smooth muscle. 11 T h e data in our study were obtained under N A N C conditions, which would have masked any effect of N O on acetylcholine release. The lack of any obvious role of N O as an important inhibitory neurotransmitter in rat jejunal circular muscle is in agreement with our observations in rat ileal circular muscle where N O also did not appear to mediate
Vol. 4, No. 1 2000
Nitric Oxide in Jejunal Motility After Transplantation
I tO
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150 Fig. 3. Frequency-response patterns to electrical field stimulation of rat jejunal circular muscle in vitro under N A N C conditions. Note only those muscle strips showing an inhibitory response are graphed; this includes 90%, 53%, and 82% of all muscle strips in the SC, SBT-1, and SBT-8 groups, respectively. N o differences were noted between groups.
an important NANC rat inhibition, s Similar findings occurred in our preliminary work in jejunal longitudinal muscle, where the tetrodotoxin-sensitive (neurally mediated) inhibition of contractile activity induced by EFS was blocked by L-NMMA only at 1 Hz, and methylene blue had no effect on the EFS-induced inhibition. 11 Based on these observations, we conclude that N O does not appear to mediate an important NANC inhibitory function in rat jejunal and ileal circular muscle, and that other NO-independent neurotransmitters play a more prominent role in inhibitory modulation of contractile activity in this tissue. In contrast, exogenous NO does markedly inhibit contractions in rat jejunal longitudinal muscle, and inhibition of NO synthase abrogates the EFS-induced inhibition at 1 Hz.12 These contrasting effects suggest a differing importance and distribution of nitrergic pathways in circular and longitudinal muscle layers in the rat jejunum. Increased EFS-induced inhibition after SBT in the longitudinal but not in the circular muscle may further support this concept, x3 Several groups have suggested that extrinsic denervation and SBT induce an increase in nitrergic nerves in the myenteric plexus and a concomitant increase in their inhibitory influence in NANC inhibition. Morphologic studies have suggested that after either extrinsic denervation or SBT the number of nitrergic neurons in small bowel is increased in the rat and the guinea pig. 7,s The increased inhibitory function in rats after extrinsic denervation appeared related to upregulation of NO synthase in the enteric nervous system.9 Our results suggest that such neural changes after SBT have more important effects in the longitudinal versus the circular muscle in the rat jejunum. The apparent lack of a prominent role of NO as an inhibitor in the circular jejunal muscle was surprising
and unexpected because of the prominent nitrergic innervation of rat circular muscle of the gut. Effects of other NO-like products cannot be excluded. Recently different redox states of NO have been demonstrated to affect membrane potential in a different manner and may possibly have different physiologic effects. 14 Similarly, in jejunal circular muscle from humans and from dogs, 1°,1s N O plays a quite prominent role in N A N C inhibitory function, although other nonnitrergic inhibitory neurotransmitters are implicated as well. In view of the reported findings, we believe that other potential candidate NANC inhibitory neurotransmitters, such as carbon monoxide,16 vasoactive intestinal polypeptide, 17 adenosine triphosphate, is or pituitary adenylate cyclase-activating polypeptide 19 should be evaluated in rat jejunal and ileal circular muscle. The apparent differences between the longitudinal and circular muscle layers is of considerable interest; such differences between muscle layers show the specialized function of different anatomic regions and muscle layers of the gut. In addition, the contractile patterns vary not only between muscle layers but also between anatomic regions. For instance, motor patterns in the ileum are considerably different from those in the jejunum.2°,21Thus findings in one muscle layer within a region cannot be applied directly to other layers of gut wall in the same or distant regions. These differences in specific NANC functions in the neurally intact jejunum suggest differences in enteric and extrinsic innervation to circular and longitudinal muscle of the rat jejunum. Our findings have potential clinical and physiologic importance. After SBT, gut function is impaired with diarrhea and abnormalities in absorptive function. 2 Previous experimental work suggested an increase in nitrergic nerves after models of extrinsic denerva-
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tion. 7-9 Our experiments were specifically designed to examine nitrergic (inhibitory) pathways as well as non-nitrergic, nonadrenergic, and noncholinergic pathways that might be altered after SBT; our goal was to define mechanisms of alteration in jejunal circular muscle contractility as mediated by enteric neural dysfunction. Combined with our work in the ileum, which showed abnormalities in response of circular muscle to adrenergic agonists, 4,s and a role of NO in jejunal longitudinal muscle, 12 the current experiments highlight differences not only in anatomic location (jejunum vs. ileum) but also in muscle layer (circular vs. longitudinal). To elucidate these physiologic phenomena and to more specifically address clinical complications related to SBT, further studies in rat and other models of isogeneic SBT are required.
We thank Deborah L Frank for her assistance in the preparation of this manuscript. REFERENCES 1. Koeoshis SA, Reyes J, Todo S, Starzl TE. Small intestinal transplantation for irreversible intestinal failure in children. Dig Dis SCI 1997;42:1997-2008. 2. Asfar S, Atldson P, Ghent C, DuffJ, Wall W, Williams S, Seidman E, Grant D. Small bowel transplantation: A life-saving option for selected patients with intestinal failure. Dig Dis Sci 1996;41:875-883. 3. Shibata C, Balsiger MN, Anding WJ, Sarr MG. Adrenergie denervation hypersensitivity in ileal circular smooth muscle after small bowel transplantation in rats. Dig Dis Sci 1997; 42:2213-2221. 4. Shibata C, Murr MM, Balsiger B, Anding WJ, Sarr MG. Contractile activity of circular smooth muscle in rats 1 year after small bowel transplantation: Differing adaptive response of the jejunum and ileum to denervation. J GASTROINTESTSURG 1998;2:463-472. 5. Shibata C, Balsiger BM, Anding WJ, Duenes JA, Miller VM, Sarr MG. Functional changes in non-adrenergic non-cholinergic inhibitory neurons in ileal circular smooth muscle after small bowel transplantation in rats. Dig Dis Sci 1998;43:24462454. 6. Murr MM, Miller ViM, Sarr MG. Contractile properties of enteric smooth muscle after small bowel transplantation in rats. AmJ Surg 1996;171:212-218. 7. Yunker AM, Galligan JJ. Extrinsic denervation increases NADPH diaphorase staining in myenteric nerves of guinea pig ileum. Neurosci Lett 1994;167:51-54.
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8. Stadelman AM, Fink JG, Mustin E, Walgenbach-Telford S, Telford GL, Koch TR. Non-adrenergic non-cholinergic (NANC) inhibitory nerves are altered in rat small intestine following transplantation [abstr]. Gastroenterology 1994; 106:A843. 9. Nakao K, Takahashi "IT, Utsunomiya J, Owyang C. Extrinsic neural control of nitric oxide synthase expression in the myenteric plexus of rat jejunum. J Physiol 1998;507(part 2):549-560. 10. Sander KM, Ward JM. Nitric oxide as a mediator of nonadrenergic non-cholinergic neurotransmission. Am J Physiol 1992;262:G379-G392. 11. Rae MG, Khoyi MA, Keef KD. Modulation of cholinergic neuromuscular transmission by nitric oxide in canine colonic circular smooth muscle. Am J Physiol 1998;275:G1324G1332. 12. Balsiger BM, Ohtani N, Shibata C, Sarr MG. Non-adrenergic non-cholinergic (NANC) inhibition in longitudinal smooth muscle of rat jejunum: Minor role of nitric oxide (NO) [abstr]. Gastroenterology 1998;114:A716. 13. Balsiger BM, Ohtani N, Shibata C, Murr MM, Anding WJ, Duenes JA, Sarr MG. Small bowel transplantation (SBT) alters non-adrenergic non-cholinergic (NANC) inhibition in longitudinal smooth muscle of the rat jejunum [abstr]. Gastroenterology 1998;114:A1377. 14. Goyal RK, He XD. Evidence of NO redox form of nitric oxide as nitrergic inhibitory neurotransmitrer in gut. Am J Physiol 1998;275:G1185-G1192. 15. Stark ME, Bauer AJ, Sarr MG, SzurszewskiJH. Nitric oxide mediates inhibitory nerve input in human and canine jejunum. Gastroenterology 1993;104:398-409. 16. Farrugia G, Irons WA, Rae JL, Sarr MG, Szurszewski JH. Activation of whole cell currents in isolated human jejunal circular smooth muscle cells by carbon monoxide. Am J Physiol 1993;264:G1184-G1189. 17. Suzuki M, Noldhara K, Maruse S, Kobayashi S. Vasoactive intestinal polypeptides as a chemical messenger in the neural control of the pacemaker activities of the circular muscle coat in the canine proximal colon. Ann N Y Acad Sci 1996;805: 692-696. 18. Crist JR, He XD, Goyal RK. Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle. J Physiol 1992;447:119-131. 19. Parkman HP, Pagano AP, Ryan JE Dual effects of PACAP on guinea pig gallbladder muscle via PACAP-preferring and VIP/PACAP-preferring receptors. Am J Physiol 1997;272: G1433-G1438. 20. Quigley EMM, Borody TJ, Phillips SF, Wienbeck M, Tucker RL, Haddad A. Motility of the terminal ileum and ileocecal sphincter in healthy humans. Gastroenterology 1984;87:857866. 21. Ehrlein H-J, Schemann M, Siegle M-L. Motor patterns of small intestine determined by closely spaced extraluminal transducers and videofluoroscopy. Am J Physiol 1987;253(3 part 1):G259-267.
Previous studies suggest that nitric oxide synthase is upregulated after small bowel transplantation which may have implications in enteric dysfunction after small bowel transplantation. The aim of this study was to determine the role of nitric oxide in nonadrenergic, noncholinergic inhibitory function after small bowel transplantation in rat jejunal circular muscle. The following four groups of rats (n = --8 rats per group) were studied: Neurally intact control animals; 1 week after anesthesia and sham celiotomy, and either 1 week or 8 weeks after isogeneic, orthotopic small bowel transplantation. Full-thickness jejunal circular muscle strips were evaluated under isometric conditions for spontaneous contractile activity, response to electrical field stimulation, and effects of exogenous nitric oxide and nitric oxide antagonists. Spontaneous activity did not differ among groups. Electrical field stimulation inhibited activity similarly in all groups. Exogenous nitric oxide, NG-monomethyl L-arginine monoacetate salt (a nitric oxide synthase inhibitor), and methylene blue (cGMP antagonist) had no effect on spontaneous activity. Neither nitric oxide antagonist altered the inhibitory response to neural excitation by electrical field stimulation in any group. Nitric oxide, a known inhibitory neurotransmitter in other gut smooth muscle, has no apparent role in rat jejunal circular muscle before or after small bowel transplantation. (J GASTROINTEST SURG2000;4:86-92.) KEY WORDS: Motility, nitric oxide, smooth muscle, small bowel transplantation, inhibitory neurotransmitters
Although small bowel transplantation (SBT) is rapidly becoming a viable clinical option for gut failure, 1 little is known about changes in small bowel function after SBT. Understanding changes in enteric function is important because SBT is associated with multiple problems in the early and late postoperative periods including abnormalities in motility, diarrhea, and difficulty with enteral delivery of nutrition. 2 Indeed, changes in enteric function are not unexpected, because SBT obligates an extrinsic denervation of the graft, disruption of intrinsic (enteric) neural continuity of the graft with the still-innervated proximal and distal gut, an ischemia/reperfusion injury, and hostmediated immune responses to the graft.
Previous work from our laboratory has investigated the early and late effects of SBT on contractile activity of circular smooth muscle of the rat ileum. Although we found an impressive adrenergic hypersensitivity after SBT, spontaneous contractile activity remained unchanged. 3,4 W h e n examined by exogenous application of nitric oxide (NO) and N O antagonists and during intrinsic neural excitation by electrical field stimulation (EFS), inhibitory neurotransmission to ileal circular muscle appeared to be independent of N O . 5 In contrast, in jejunal circular muscle, SBT induced an increase in spontaneous contractile activity and augmented EFS-induced nonadrenergic, noncholinergic (NANC) inhibition. Because no changes
From the Department of Surgery and the GastroenterologyResearch Unit, Mayo Clinic and Foundation, Rochester,Minn. Supported by grant DK 39337 from the National Institutes of Health, United States Public Health Service, the Mayo Foundation, the SwissNational Foundation, and the Department of Visceral and Transplantation Surgery, Universityof Bern, Switzerland. Part of this study was presented at a poster session at the Fortieth Annual Meeting of The Societyfor Surgery of the AlimentaryTract, Orlando, Fla., May 16-19, 1999. An abstract of this work was published in Gastroenterology116:A1349, 1999. Correspondence: Michael G. Sarr, M.D., Professor of Surgery, Gastroenterology Research Unit, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. 86
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occurred in sensitivity to cholinergic or adrenergic agonists, 6 we wondered whether transplantation-related alterations in nitrergic nerves might explain these changes in contractile properties. Indeed, others have suggested that the number of nitrergic nerves is increased after extrinsic denervation and SBT, 7,8 and that NO synthase activity is upregulated by extrinsic denervation.9 Such changes in these presumably inhibitory nitrergic nerves may have marked effects on gut motor function. Therefore the aims of the present study were to determine the effect of N O in rat jejunal circular muscle and to identify the role of N O in EFS-induced changes in NANC inhibition before and after SBT. We hypothesized that exogenous NO would inhibit contractile activity and that changes in spontaneous contractile activity and EFS-induced NANC inhibition after SBT would be related to changes in a nitrergic mechanism with NO acting as the neurotransmitter. MATERIAL AND METHODS Preparation of Animals Procedures and animal care were performed according to the guidelines of the Animal Care and Use Committee of the Mayo Foundation in accordance with the guidelines of the National Institutes of Health and the Public Health Service Policy on the Human Use and Care of Laboratory Animals. Experimental Groups. Because we were interested in the physiologic effects of the SBT procedure and not rejection, immune suppression, or other immune phenomena, we specifically used syngeneic male Lewis rats (Harlan Sprague-Dawley, Indianapolis, Ind.) in all experiments to avoid confounding immune-related phenomena that occur after allotransplantation. After anesthesia was achieved by intraperitoneal sodium pentobarbital (Ampro Pharmacy, Arcadia, Calif.), orthotopic SBT was performed using standard microvascular techniques as described previously.6 In brief, the entire jejunoileum was removed from the donor rat after flushing the intestinal lumen and infusing the graft vasculature with chilled Ringer's lactate solution. The graft was revascularized by anastomosing donor aorta to recipient aorta (end-to-side) and donor portal vein to recipient inferior vena cava (end-to-side). After resecting the recipient jejunoileum, intestinal continuity was reestablished by end-to-end jejunojejunostomy and ileoileostomy. All rats were allowed free access to water and rat chow immediately postoperatively. Rats were studied 1 week and 8 weeks after SBT (SBT-1, n = 12; SBT8, n = 14). Naive rats without any surgical procedure
Nitric Oxide in Jejunal Motility After Transplantation
87
served as neurally intact controls (NC, n = 4). Also, to determine the nonspecific effects of anesthesia and celiotomy, rats I week after celiotomy and intestinal manipulation were used as sham-operated controls (SC, n = 8). Recordingof Contractile Activity. A segment of proximal jejunum 7 to 10 cm distal to either the ligament of Treitz or the jejunojejunostomy after SBT was removed, immersed in chilled modified KrebsRinger's bicarbonate solution (concentrations in mmol/L: NaC1 116.4, KC14.7, CaCI2 2.5, MgSO4 1.2, K H 2 P O 4 1.2, NaHCO3 23.8, calcium disodium edetate 0.26, and glucose 11.1) and opened along the mesenteric border. Four to eight fifll-thickness muscle strips cut in the direction of the circular muscle layer were suspended vertically in 10 ml tissue chambers filled with modified Krebs-Ringer's bicarbonate solution; the chambers were maintained at 37.5 ° C and bubbled with 95% oxygen and 5% carbon dioxide (Puritan-Bennett Corp., Lenexa, Kan.). Because of the well-recognized variability between muscle strips in the same rat, identical experiments were performed on multiple muscle strips per rat. One end of the muscle strip was attached to a fixed hook, whereas the other end was connected to a noncompliant force transducer (Kulite Semiconductors Products, Inc., Leonia, N.J.) to measure isometric force. Experimental Protocol After an 80- to 90-minute equilibration period with intervening washout of the bath solution every 20 to 25 minutes, each strip was incrementally stretched at 12- to 15-minute intervals to its optimal length (Lo) beyond which further stretching did not increase the amplitude of spontaneous activity. 6 All subsequent experiments were performed at Lo; strips without spontaneous contractile activity or without any response to EFS were not used (less than 9% of strips). After measuring spontaneous basal contractile activity for a 5-minute interval at Lo, atropine (10 -7 tool/L), phentolamine (10 -5 mol/L), and propranolol (5 X 10 -6 tool/L) were added to the bath in eight chambers to induce NANC conditions, and spontaneous contractile activity under NANC conditions was measured for a 5-minute interval beginning 30 minutes later. NO in distilled water (3 x 10 -6 tO 3 X 10 -s tool/L) was applied directly into the chamber under NANC conditions to determine the effect of NO on spontaneous activity. NO was prepared by dissolving NO gas in distilled water previously degassed with helium according to the method reported previously. 3 Then, EFS was applied at increasing frequencies (1, 2, 3, 4, 5, 7, and 10 Hz) with a constant voltage (20 volts), pulse width (4 msec), and duration of
88
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Table I. Spontaneous contractile activity of rat jejunal circular smooth muscle* Group
Basal
NANC
L-NMMA
Methylene blue
SC
5.05 +_ 0.55
5.79 ___ 0.80
5.44 _+ 1.18
6.17 _ 1.28
SBT-1 SBT-8
4.47 -- 0.54 4.03 +- 0.40
4.89 +- 0.68 4.50 _ 0.54
4.28 --- 0.65 3.86 --- 0.52
4.19 -+ 0.87 4.89 --- 0.77
*Mean + SEN[, g • 5 rain/wet tissue; n = >- 8 rats per group.
stimulation (10 seconds) to obtain a frequencyresponse curve. T h e different frequency stimulations were separated by 5 minutes to allow spontaneous activity to recover before the next stimulation. T h e contractile response to EFS was quantitated only during the 10-second stimulation. NC-monomethyl-L arginine (L-NMMA, 10 -3 mol/L), a specific inhibitor of N O synthase, and methylene blue (10 -5 mol/L), a nonspecific inhibitor of soluble guanylate cyclase, were administered into each of four chambers. Contractile activity was then determined for a 5-minute interval beginning 3 0 minutes after drug administration. EFS was repeated under the same conditions in chambers with L - N M M A and methylene blue to determine the effect of these drugs on the response to EFS. In separate experiments in control rats (data not shown), EFS was investigated in the presence of 10 -6 mol/L tetrodotoxin which, by blocking sodium channels, inhibits almost all neural transmission; we confirmed that EFS-induced inhibition and contractile activity were tetrodotoxin sensitive and thus neurally mediated. At the conclusion of the experiments, each tissue was blotted and weighed.
Data Analysis We quantitated total contractile activity by measuring the integral of the force generated (g. 5 min area under the contractile curve) under each condition using specialized software (AcqKnowledge, Biopac Systems, Inc., Goleta, Calif.). Spontaneous basal activity at Lo (4 to 8 muscle strips per rat), and the effects of N A N C conditions (4 muscle strips per rat), L - N M M A (2 muscle strips per rat), and methylene blue (2 muscle strips per rat) on spontaneous activity were quantitated for 5-minute intervals. In EFS experiments (4 muscle strips per rat), we determined the response during the 10-second stimulation ("onresponse") and specifically excluded the response immediately after stopping stimulation (the so-called "off-response"). T h e integral of force generated during the 10-second EFS at each frequency was expressed as the percentage of spontaneous contractile activity for a mean interval of 10 seconds as calculated from the 5 minutes of contractile activity measured immediately before beginning EFS. Frequency-
response curves were generated. T h e effect of L - N M M A and methylene blue on EFS-induced inhibition was compared to the inhibition during EFS before administration of these agents in the same strip. All contractile data were standardized by milligrams of tissue wet weight. Analysis of variance combined with Student's t tests were used for comparisons among multiple groups (comparison of basal spontaneous activity across groups), and separate t tests for paired data were used to study the effect of various drugs on spontaneous activity and during EFS. A Bonferroni correction was made for multiple comparisons whenever appropriate. All data are presented as mean - standard error of the mean (SEND, and the n used for statistical comparisons was the number of rats (not muscle strips) per group. Since there were no differences between the N C and SC groups, we chose SC rats as controls for the SBT rats. Drugs DL-Propranolol hydrochloride, phentolamine hydrochloride, atropine sulfate, L-NMMA, and methylene blue were purchased from Sigma Chemical Company (St. Louis, Mo.). Nitric oxide gas was purchased from Matheson Gas Production, Inc. (Parsippany, N.J.). RESULTS
Spontaneous C o n t r a c t i l e Activity N o differences were noted in spontaneous basal contractile activity between groups. T h e effects of N A N C conditions, L-NMMA, and methylene blue on spontaneous activity are shown in Table I. Induction of N A N C conditions did not alter contractile activity significantly in any group (see Table I). Similarly, when evaluated under N A N C conditions, L - N M M A and methylene blue had little or no effect on spontaneous contractile activity (see Table I). Exogenous administration of N O (3 x 10 -6 tO 3 X 10 -5 mol/L) under N A N C conditions also had no inhibitory effect on spontaneous phasic activity, basal tension, or total contractile activity in any group (Fig. 1).
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Nitric Oxide in Jejunal Motility After Transplantation
10-e M
3xl 0"6M
Y
10-s M
V
89
3x10 -5 M
V
1 0.5g
5 min Fig. 1. Lack of effectof exogenousNO on spontaneouscontractile activityin vitro under NANC conditions in rat jejunal circular musclein the SC group. Similar lack of effectas seen in the NC and SBT groups as well.
0.5 g EFS, 20 V, 4 ms A
u
u
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5 min Fig. 2. Effect of electrical field stimulation on spontaneous contractile activity of rat jejunal circular muscle in vitro under NANC conditions in the SBT-1 group. A, Characteristic inhibitory effect at all frequencies. Inset represents an expansion of the segment within the box. B, Net contractile response. This less common pattern was seen in 9%, 10%, 47%, and 18% of strips in the NC, SC, SBT-I, and SBT-8 groups, respectively. E f f e c t o f Electrical F i e l d S t i m u l a t i o n Fig. 2 shows two different overall patterns of responses to E F S - - a primarily inhibitory one (Fig. 2, A) and a procontractile one (Fig. 2, B). T h e most frequendy observed pattern (198 of 227 strips; Fig. 2, A) consisted of inhibition of phasic contractions and/or
reduction in basal tension (and therefore decreasing total force) during low-frequency (1 to 7 Hz) EFS; this inhibitory effect was not apparent at 10 Hz. This pattern was present in 91%, 90%, 53%, and 82% of the strips in the N C , SC, SBT-1, and SBT-8 groups, respectively. T h e second pattern (Fig. 2, B) consisted
90
Journal of Gastrointestinal Surgery
Balsiger et al.
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of EFS evoking no inhibition but rather stimulating contractile activity at all frequencies tested; this pattern occurred mostly in the SBT-1 group (47% of the strips); in 4 of the 12 rats all strips showed this response. Because we were interested in the inhibitory function, we focused on the former inhibitory responses. The frequency-response curves in all groups are shown in Fig. 3. In SBT-1 rats, the overall frequency-response pattern was similar to that of the SC group, but the inhibitory response at 7 Hz was smaller and the contraction at 10 Hz greater than in the SC and SBT-8 rats (see Table II). The effects of the N O antagonists L - N M M A and methylene blue on the EFS response curves in each group are shown in Table II; neither L - N M M A nor methylene blue altered the frequency-response curve to EFS in any group. DISCUSSION
Nitric oxide has been shown to be an important inhibitory neurotransmitter in gut smooth muscle in various species. 1° Our study was designed based on the hypothesis that N O has a modulatory role before and after isogeneic SBT in circular muscle of the rat jejunum. Neither exogenous application of N O , blocking N O synthase with L-NMMA, nor inhibiting soluble guanylate cyclase with methylene blue, the presumed intracellular pathway by which N O inhibits contractile activity, had any noticeable effect on spontaneous contractile activity in rat jejunal circular muscle; these observations imply that N O does not modulate spontaneous continuous phasic activity or basal tone in this tissue. Similarly, the N O antagonists LN M M A and methylene blue did not abrogate the EFS-induced inhibition in any group. These results suggest that under our experimental conditions, nitrergic pathways do not appear to play an important or major role in mediating long-term N A N C inhibition either in normally innervated rat circular jejunal muscle or a major role in extrinsically denervated jejunum after SBT. Inasmuch as N O synthase-positive nerve fibers are abundant in the rat circular muscle layer,s it is possible that the role of neuronally released N O is to inhibit muscle activity under stimulated conditions or to modulate release of other neurotransmitters. N O modulation of acetylcholine release has been reported in canine colonic circular smooth muscle. 11 T h e data in our study were obtained under N A N C conditions, which would have masked any effect of N O on acetylcholine release. The lack of any obvious role of N O as an important inhibitory neurotransmitter in rat jejunal circular muscle is in agreement with our observations in rat ileal circular muscle where N O also did not appear to mediate
Vol. 4, No. 1 2000
Nitric Oxide in Jejunal Motility After Transplantation
I tO
2
3
4
5
7
10
91
Hz
100 •
SC
•
SBT-lW
so tO
~
0
t-
50
[ ] SBT-8W
0
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150 Fig. 3. Frequency-response patterns to electrical field stimulation of rat jejunal circular muscle in vitro under N A N C conditions. Note only those muscle strips showing an inhibitory response are graphed; this includes 90%, 53%, and 82% of all muscle strips in the SC, SBT-1, and SBT-8 groups, respectively. N o differences were noted between groups.
an important NANC rat inhibition, s Similar findings occurred in our preliminary work in jejunal longitudinal muscle, where the tetrodotoxin-sensitive (neurally mediated) inhibition of contractile activity induced by EFS was blocked by L-NMMA only at 1 Hz, and methylene blue had no effect on the EFS-induced inhibition. 11 Based on these observations, we conclude that N O does not appear to mediate an important NANC inhibitory function in rat jejunal and ileal circular muscle, and that other NO-independent neurotransmitters play a more prominent role in inhibitory modulation of contractile activity in this tissue. In contrast, exogenous NO does markedly inhibit contractions in rat jejunal longitudinal muscle, and inhibition of NO synthase abrogates the EFS-induced inhibition at 1 Hz.12 These contrasting effects suggest a differing importance and distribution of nitrergic pathways in circular and longitudinal muscle layers in the rat jejunum. Increased EFS-induced inhibition after SBT in the longitudinal but not in the circular muscle may further support this concept, x3 Several groups have suggested that extrinsic denervation and SBT induce an increase in nitrergic nerves in the myenteric plexus and a concomitant increase in their inhibitory influence in NANC inhibition. Morphologic studies have suggested that after either extrinsic denervation or SBT the number of nitrergic neurons in small bowel is increased in the rat and the guinea pig. 7,s The increased inhibitory function in rats after extrinsic denervation appeared related to upregulation of NO synthase in the enteric nervous system.9 Our results suggest that such neural changes after SBT have more important effects in the longitudinal versus the circular muscle in the rat jejunum. The apparent lack of a prominent role of NO as an inhibitor in the circular jejunal muscle was surprising
and unexpected because of the prominent nitrergic innervation of rat circular muscle of the gut. Effects of other NO-like products cannot be excluded. Recently different redox states of NO have been demonstrated to affect membrane potential in a different manner and may possibly have different physiologic effects. 14 Similarly, in jejunal circular muscle from humans and from dogs, 1°,1s N O plays a quite prominent role in N A N C inhibitory function, although other nonnitrergic inhibitory neurotransmitters are implicated as well. In view of the reported findings, we believe that other potential candidate NANC inhibitory neurotransmitters, such as carbon monoxide,16 vasoactive intestinal polypeptide, 17 adenosine triphosphate, is or pituitary adenylate cyclase-activating polypeptide 19 should be evaluated in rat jejunal and ileal circular muscle. The apparent differences between the longitudinal and circular muscle layers is of considerable interest; such differences between muscle layers show the specialized function of different anatomic regions and muscle layers of the gut. In addition, the contractile patterns vary not only between muscle layers but also between anatomic regions. For instance, motor patterns in the ileum are considerably different from those in the jejunum.2°,21Thus findings in one muscle layer within a region cannot be applied directly to other layers of gut wall in the same or distant regions. These differences in specific NANC functions in the neurally intact jejunum suggest differences in enteric and extrinsic innervation to circular and longitudinal muscle of the rat jejunum. Our findings have potential clinical and physiologic importance. After SBT, gut function is impaired with diarrhea and abnormalities in absorptive function. 2 Previous experimental work suggested an increase in nitrergic nerves after models of extrinsic denerva-
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tion. 7-9 Our experiments were specifically designed to examine nitrergic (inhibitory) pathways as well as non-nitrergic, nonadrenergic, and noncholinergic pathways that might be altered after SBT; our goal was to define mechanisms of alteration in jejunal circular muscle contractility as mediated by enteric neural dysfunction. Combined with our work in the ileum, which showed abnormalities in response of circular muscle to adrenergic agonists, 4,s and a role of NO in jejunal longitudinal muscle, 12 the current experiments highlight differences not only in anatomic location (jejunum vs. ileum) but also in muscle layer (circular vs. longitudinal). To elucidate these physiologic phenomena and to more specifically address clinical complications related to SBT, further studies in rat and other models of isogeneic SBT are required.
We thank Deborah L Frank for her assistance in the preparation of this manuscript. REFERENCES 1. Koeoshis SA, Reyes J, Todo S, Starzl TE. Small intestinal transplantation for irreversible intestinal failure in children. Dig Dis SCI 1997;42:1997-2008. 2. Asfar S, Atldson P, Ghent C, DuffJ, Wall W, Williams S, Seidman E, Grant D. Small bowel transplantation: A life-saving option for selected patients with intestinal failure. Dig Dis Sci 1996;41:875-883. 3. Shibata C, Balsiger MN, Anding WJ, Sarr MG. Adrenergie denervation hypersensitivity in ileal circular smooth muscle after small bowel transplantation in rats. Dig Dis Sci 1997; 42:2213-2221. 4. Shibata C, Murr MM, Balsiger B, Anding WJ, Sarr MG. Contractile activity of circular smooth muscle in rats 1 year after small bowel transplantation: Differing adaptive response of the jejunum and ileum to denervation. J GASTROINTESTSURG 1998;2:463-472. 5. Shibata C, Balsiger BM, Anding WJ, Duenes JA, Miller VM, Sarr MG. Functional changes in non-adrenergic non-cholinergic inhibitory neurons in ileal circular smooth muscle after small bowel transplantation in rats. Dig Dis Sci 1998;43:24462454. 6. Murr MM, Miller ViM, Sarr MG. Contractile properties of enteric smooth muscle after small bowel transplantation in rats. AmJ Surg 1996;171:212-218. 7. Yunker AM, Galligan JJ. Extrinsic denervation increases NADPH diaphorase staining in myenteric nerves of guinea pig ileum. Neurosci Lett 1994;167:51-54.
Journal of Gastrointestinal Surgery
8. Stadelman AM, Fink JG, Mustin E, Walgenbach-Telford S, Telford GL, Koch TR. Non-adrenergic non-cholinergic (NANC) inhibitory nerves are altered in rat small intestine following transplantation [abstr]. Gastroenterology 1994; 106:A843. 9. Nakao K, Takahashi "IT, Utsunomiya J, Owyang C. Extrinsic neural control of nitric oxide synthase expression in the myenteric plexus of rat jejunum. J Physiol 1998;507(part 2):549-560. 10. Sander KM, Ward JM. Nitric oxide as a mediator of nonadrenergic non-cholinergic neurotransmission. Am J Physiol 1992;262:G379-G392. 11. Rae MG, Khoyi MA, Keef KD. Modulation of cholinergic neuromuscular transmission by nitric oxide in canine colonic circular smooth muscle. Am J Physiol 1998;275:G1324G1332. 12. Balsiger BM, Ohtani N, Shibata C, Sarr MG. Non-adrenergic non-cholinergic (NANC) inhibition in longitudinal smooth muscle of rat jejunum: Minor role of nitric oxide (NO) [abstr]. Gastroenterology 1998;114:A716. 13. Balsiger BM, Ohtani N, Shibata C, Murr MM, Anding WJ, Duenes JA, Sarr MG. Small bowel transplantation (SBT) alters non-adrenergic non-cholinergic (NANC) inhibition in longitudinal smooth muscle of the rat jejunum [abstr]. Gastroenterology 1998;114:A1377. 14. Goyal RK, He XD. Evidence of NO redox form of nitric oxide as nitrergic inhibitory neurotransmitrer in gut. Am J Physiol 1998;275:G1185-G1192. 15. Stark ME, Bauer AJ, Sarr MG, SzurszewskiJH. Nitric oxide mediates inhibitory nerve input in human and canine jejunum. Gastroenterology 1993;104:398-409. 16. Farrugia G, Irons WA, Rae JL, Sarr MG, Szurszewski JH. Activation of whole cell currents in isolated human jejunal circular smooth muscle cells by carbon monoxide. Am J Physiol 1993;264:G1184-G1189. 17. Suzuki M, Noldhara K, Maruse S, Kobayashi S. Vasoactive intestinal polypeptides as a chemical messenger in the neural control of the pacemaker activities of the circular muscle coat in the canine proximal colon. Ann N Y Acad Sci 1996;805: 692-696. 18. Crist JR, He XD, Goyal RK. Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle. J Physiol 1992;447:119-131. 19. Parkman HP, Pagano AP, Ryan JE Dual effects of PACAP on guinea pig gallbladder muscle via PACAP-preferring and VIP/PACAP-preferring receptors. Am J Physiol 1997;272: G1433-G1438. 20. Quigley EMM, Borody TJ, Phillips SF, Wienbeck M, Tucker RL, Haddad A. Motility of the terminal ileum and ileocecal sphincter in healthy humans. Gastroenterology 1984;87:857866. 21. Ehrlein H-J, Schemann M, Siegle M-L. Motor patterns of small intestine determined by closely spaced extraluminal transducers and videofluoroscopy. Am J Physiol 1987;253(3 part 1):G259-267.