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Clinical Science (1992) 82, 169-173 (Printed in Great Britain) I69 location of enkephalinase and functional effects of [leu5]enkephalin and inhibition of enkephalinase in the feline main pancreatic and bile duct sphincters A. THUNE, L. JIVEGARD, H. POLLARD, J. MOREAU, J.C. SCHWARTZ and J. SVANVIK Department of Surgery, University of Goteborg, Sahlgrenska Hospital, Goteborg, Sweden, and Unit of Neurobiology, Centre Paul Broca de I’INSERM, Paris, France (Received I5 Aprill9 August I99 I; accepted 27 August I99 I ) 1. Morphological studies have demonstrated enkephalinergic nerve fibres in proximity to the sphincter of Oddi, and opiates are known to contract this sphincter. In this study, the flow resistances in the common bile duct and main pancreatic duct sphincters were studied simultaneously in anaesthetized cats using a perfusion technique. 2. Naloxone did not affect the activity of these sphincters under basal conditions, indicating that there is no basal enkephalinergic tone. 3. The response to [Leu’jenkephalin (0.015-15 pg/kg), morphine (1 mg/kg) and ketamine (10 mg/kg) was a naloxone-sensitive increased activity in the sphincters with a raised frequency of phasic contractions. The threshold dose for an effect of [Leu’lenkephalin on the sphincter of Oddi was 0.015 pg/kg and a maximal response was observed at 0.75 pg/kg. There were no differences in the response of the main pancreatic duct sphincter and the bile duct sphincter to the different drugs. 4. Immunoautoradiographic studies demonstrated enkephalinase in the spincter of Oddi. 5. Acetorphan (3 mg/kg intravenously), which inhibits endogenous enkephalinase both in the peripheral and the central nervous system when administered parenterally, caused a naloxone-sensitive contraction, whereas thiorphan (3-20 mg/kg), an enkephalinase inhibitor that does not easily penetrate the blood-brain barrier, had no effect on the sphincter of Oddi. 6. These results show that endogenous and exogenous opiates influence the function of the feline sphincter of Oddi and that enkephalins may be involved in the physiological control of this sphincter, although not under basal conditions. INTRODUCTION Previous research has suggested that the endogenous opiate [Leu’lenkephalin has both excitatory and inhibitory effects on the feline sphincter of Oddi (SO)[l], and morphological studies have demonstrated enkephalinergic nerve fibres in proximity to the smooth muscle fibres in this sphincter [2]. Endogenous enkephalins are inactivated partly by enkephalinase, a membrane metalloendopeptidase (EC 3.4.24.1 l ) , and this enzyme has been located in several peripheral organs, including those of the gatrointestinal tract [3]. Enkephalinase activity may be blocked with the drugs acetorphan and thiorphan [4]. The effects induced by these drugs on the gastrointestinal tract are blocked by naloxone [5, 61. Naloxone is an opiatereceptor antagonist with no agonistic effect at low concentrations [7]. Ketamine, a commonly used anaesthetic agent, has central and peripheral opiate agonist actions and anticholinergic effects [S, 91 besides its anaesthetic actions. The aim of this study was to study the role and mechanism of action of endogenous and intravenously administered [Leu’]enkephalin on the feline SO. Since ketamine has been used to induce or maintain anaesthesia in several previous studies on the SO, the effect on the feline SO of this drug was also tested. METHODS Experimental procedures Experiments were performed in cats anaesthetized with chloralose (60 mg/kg body weight intraperitoneally) and maintained with 10 mg/kg intravenously as needed. The effects of opiates were studied in both sphincteric segments of the SO by simultaneous perfusion of the main pancreatic duct sphincter (PDS) and the bile duct sphincter (BDS). The flow resistance (FR) exerted by these sphincteric segments was calculated from the pressure gradient between the distal common bile duct and the duodenum, the pancreatic duct and the duodenum, respectively, and the flow rate. The mean FR for each segment was estimated with the aid of integrators. The technical details of this method and the validity of the measurements are described in detail in previous reports [lo, 111. Key words: enkephalin, enkephalinase, ketamine, opiate, sphincter of Oddi. Abbreviations: BDS, bile duct sphincter; FC, frequency of contractions; FR, flow resistance; mAb, monoclonal antibody; PBS, phosphate-bufferedsaline; PDS, pancreatic duct sphincter; SO, sphincter of Oddi; TTX, tetrodotoxin. Correspondence: D r ]oar Svanvik, Department of Surgery I, Sahlgrenska Hospital, S-413 45 Goteborg, Sweden. I70 A. Thune et al. Mean FR values were calculated during 5 rnin periods before and after administration of the drugs. When responses after blocking with naloxone were tested, the investigated drug was injected 5 rnin after naloxone. The left femoral artery was cannulated and was connected to a pressure transducer, and the left femoral vein was used for intravenous injections of all drugs except for the nerve-blocking agent tetrodotoxin (TTX), which was given as a bolus dose into the celiac artery via the splenic artery. Physiological saline (150 mmol/l NaCI), given by the same intra-arterial route, was used as a control. Sweden; [Leus]enkephalin was supplied by Fluka AG, Buchs, Switzerland; TTX was obtained from Sigma Chemicals, St Louis, MO, U.S.A. Statistical methods Statistical analyses were performed by using the paired or unpaired t-test and linear regression analysis. A P value of less than 0.05 was considered significant. All results are given as means t- SEM. RESULTS Immuno-autoradiographic studies Specimens, including the common bile duct and the duodenum, were taken from three cats and were immediately frozen in liquid nitrogen. The region of the SO was cut (20 p m thick) on a cryostat (Ames) at - 18"C, thawmounted on gelatin-coated glass slides and stored at -20°C until used. The sections were warmed to room temperature, dried for 30 rnin and washed twice for 30 min in 0.1 mol/l phosphate-buffered saline (PBS),pH 7.4, immediately before incubation for 3 h at 20°C with 0.2 ml of an 12sI-labelledmonoclonal antibody (mAb) known to recognize cat enkephalinase (mAb 135, 2.5 x 106 c.p.m./ ml) [GI dissolved in PBS containing 0.2'10 (w/v) gelatin and 0.1"/0 (v/v) Tween 20. The slides were rinsed twice for 3 rnin in the same supplemented buffer, followed by two 3 rnin rinses in pure PBS. Finally, they were dipped into distilled water, dried and apposed to H3-sensitive hyperfilm (Amersham) in X-ray cassettes and the films were developed 2-3 days later. Non-specific labelling was assessed by using a control, similarly "'I-iodinated, mAb (mAb 85 A2)known for its inability to recognize cat enkephalinase. Function of the SO during basal conditions and in response to TTX and naloxone During basal conditions, FR was 4.7 +- 0.7 cmH,O hml-l in the BDS and 5.2k0.7 cmH,O h-' ml-I in the PDS in 18 cats. Spontaneous synchronous contractions were registered in both the PDS and the BDS and no difference in the frequency of contractions (FC) was observed. FC was 4.9 k 0.6 min- under basal conditions. Administration of TTX (9 pg/kg body weight) in six animals significantly increased FR (change in FR: BDS, 6.19 k 1.27 cmH,O h- I ml- I, P < 0.01; PDS, 3.89 k 0.68 cmH,O h-I ml-l, P<O.01) and (change in FC: 4.2k0.9 min-I, P < 0.01). Injection of 2 ml of physiological saline into the celiac artery neither affected FC nor FR. Naloxone (1 mg/kg intravenously) given under basal conditions did not affect FR in either of the sphincters (change in F R BDS, 0.2OkO.18 cmH,O h - ' ml-'; PDS, 0.1 1 k0.12 cmH,O h - l ml-I). Neither did this drug cause any change in FC (change in FC: 0.0 k 0.3 min- I, 17 = 7). Effects of exogenous opiate agonists Drugs Acetorphan and thiorphan were provided by Laboratoire Bioproject, Paris, France; Nalonee (naloxone hydrochloride) was purchased from Du Pont, Wilmington, DE, U.S.A.; Ketalar (ketamine hydrochloride) was obtained from Parke Davis, Morris Plains, NJ, U.S.A.; Morfin (morphine chloride) was purchased from ACO, Solna, Intravenous injection of morphine (1 mg/kg) significantly increased FR in both sphincters (change in FR: BDS, 3.17k1.09 cmH,O h - ' m1-I; PDS, 2.35k0.58 cmH,O h-I ml-I). A similar response was noted in response to ketamine (10 mg/kg) and [Leu5]enkephalin (15 pg/kg) (Table 1).The response to the different drugs were of comparable magnitude. The increment in FR Table I. Change in FR calculated from the change in mean pressure during a 5 min period before and after an intravenous injection of [Leu']enkephalin, acetorphan or ketamine. The increase in FR was significant (P <0.05. paired r-test. n = 5 for each drug) for all three drugs. After pretreatment with naloxone the responses were all significantly reduced (P<O.O5. unpaired t-test, n = 5 for each drug). Values are meanstsm. Change in FR (cmH,O h-l m1-I) Acetorphan (3 mglkg) (15 yglkg) [Le~~lenkephalin Ketamine (10 mglkg) Alone After pretreatment with naloxone Alone After pretreatment with naloxone Alone After pretreatment with naloxone BDS 3.51k0.81 1.481l.lI 2.7610.76 0.39f0.43 2.22k0.70 0.42t0.19 PDS 4.37 k0.75 I .39 f I .O 2.77 f0.93 0.3410.35 1.29k0.29 0.51 t O . 1 I [Leus]enkephalin and t h e sphincter of O d d i induced by [Leus]enkephalin or ketamine was of short duration (10-20 min). After pretreatment with naloxone, the response to [Leu5]enkephalin and ketamine were significantly reduced (Table 1). Morphine (change in FC: 3.5f1.0 min-I, PcO.05, n = 5 ) and ketamine (change in FC: 2.4k0.6 min-I, P<O.O5, n = 5 ) significantly increased FC in the SO. F R correlated significantly ( P < 0.001) with the increase in FC (Fig. 1). 171 doses of [Leu'lenkephalin above 0.75 ,ug/kg, a tonic contraction lasting several minutes was recorded and FC could not be determined. Pretreatment with TTX (9 pg/kg) significantly reduced the increment in FR caused by an injection of [Leus]enkephalin (15 pg/kg) (change in F R PDS, 0.88 k0.75 cmH,O h-' ml-I, P<O.O5; BDS, 0.26f0.75 cmH,O h-' ml-', P < 0.05) in five experiments. Localization of enkephalinase in the feline SO Dose-response analysis and mode of action of [Leus]enkephalin [Leu5]enkephalin was administered in doses ranging from 1.5 X to 15 pg/kg. FR increased progressively from 15 x ,ug/kg, and a maximal response was observed at a dose of 0.75 pug/kg (Fig. 2). [Leus]enkephalin (0.15 pglkg) significantly increased FC in the SO (change in FC: 2.41.0.5 min-', P<0.05). At Autoradiographs generated with '251-labelledmAb 135 demonstrated a distribution of autoradiographic grains located in the smooth muscle surrounding the distal common bile duct and the distal main pancreatic duct (Fig. 3). On the same transverse section, labelling of the epithelium of the small intestinal mucosa is also observed. Non-specific labelling obtained using '251-labelled mAb 85 A2 under the same conditions was negligible (Fig. 3). Effects of inhibition of enkephalinase by acetorphan and thiorphan Injection of acetorphan ( 3 mg/kg) caused a naloxonesensitive, significant increase in FR in the SO (Table 1)as well as a significant increase in the FC (change in F C 3.3 f 0.9 min- I , P < 0.05, n = 5). The effects of thiorphan were analysed at doses from 3 to 20 mg/kg in seven animals. No change in FR ( r< 0.01, linear regression dose-response analysis) or in FC ( r < 0.01, linear regression dose-response analysis) was observed. / 0 0 0% 0 , I -2 . I 0 0. 0 . I . 2 I - 4 I - I 8 6 Change in FC (min-') Fig. I. Correlation between the change in FC and the change in FR obtained by use of linear regression analysis. n=43, r=0.70, P<O.OOI. 1 . 5 ~ 1 0 - ~ 1 . 5 ~ 110.-5~~ 1 0 -0.15 ~ 0.75 1.5 15 [Leu'lenkephalin (pglkg) Fig. 2. Dose-response analysis for [Leu'lenkephalin in the PDS (a) and the BDS (0).The change in FR was calculated from the change in mean pressure during a 5 min period before and after an intravenous injection. The difference between the response t o doses of 0.75 pglkg and higher was significant (P <0.05) compared with the response to the lowest dose (1.5 X pglkg). Values are means with bars indicating SEN. DISCUSSION The results of this study confirm previous findings of opiates having excitatory properties on the feline SO [ 1, 121. The opiate agonists morphine, [Leus]enkephalin and ketamine thus induced a contraction of the PDS and the BDS. The response to [Leus]enkephalin was dosedependent and was significantly inhibited by pretreatment with TTX as well as by pretreatment with naloxone. There were no significant differences in the responses of the main PDS and BDS to the opiate agonists, indicating a common sphincteric complex with the same actions on FR and FC in both systems. These results concord with those obtained in a recent study [lo]. TTX blocks nerve excitability by blocking sodium channels [13], and the administered dose is known to block the effects of electrical stimulation of the splanchnic nerves in the feline biliary tract [14]. The increase in SO activity produced by administration of TTX confirms previous results [15] and may be explained by increased spontaneous activity in the SO when denervated. Naloxone, which blocks the opiate receptors, by itself neither affected the basal F R nor FC in the sphincters, suggesting that there is no basal enkephalinergic tone under the conditions studied. This disagrees with earlier findings by Behar & Biancani [l]. The discrepancy may A. Thune e t al. I72 2mm SM .2mm O.bmm H .. Fig. 3. Autoradiographic localization of enkephalinase immunoreactivity i n t h e cat SO. ( a ) Haemalinierythrosinstained section from which the autoradiogram ( b ) was generated. ( b ) Labelling obtained with 'lsl-labelled mAb 135, which recognizes cat enkephalinase. The immunoreactivity is found in the epithelium of the small intestinal mucosa (SIM) and also in the smooth muscle (SM) surrounding the distal common bile duct (CBD) and main pancreatic duct (PD). (c) Non-specific labelling determined on an adjacent section incubated with 'lsl-labelled mAb 85A2, which does not recognize cat enkephalinase. be explained by the repeated doses of ketamine used for anaesthesia in the quoted study. Ketamine was shown to exhibit opiate-like properties in this study with naloxone-sensitive increases in FR and FC in the SO of the same magnitude as those produced by morphine. These effects exclude ketamine as an appropriate anaesthetic agent when studying opiates in vivo. Dose-response analysis for [Leu'lenkephalin demonstrated that the feline SO is highly sensitive to this drug and that the effect is mediated by opiate receptors as the response to a maximal dose was significantly reduced by naloxone. Pretreatment with TTX also significantly decreased the change in FR in the SO by a maximal dose of [Leu']enkephalin, indicating that the response of the SO to [Leus]enkephalin was influenced by nervous activity. An increase in FC in the SO was observed after injection of [Le~~lenkaphalin, ketamine and morphine in this study. These results agree with those of Helm et al. [lG], who found an increased FC in the human SO after injec- tion of morphine. In the present study, FC was also increased in response to TTX. This may be explained by blocking of activity in inhibitory nerves to the SO. Further, the increase in FC in the SO was accompanied by an increase in FR. It thus seems that, in the cat, an increased FC is associated with reduced flow through the sphincter rather than an increased flow due to propulsion of bile and pancreatic juice, as has been postulated for other species. The present findings correspond to those obtained earlier in the guinea pig [ 171and in the cat [ 181. This study demonstrates the presence of enkephalinase in the smooth muscle of the SO. This finding suggests that enkephalinergic neurons may be active in the regulation of the smooth muscle activity of the SO. To further investigate the activity of endogenous enkephalins the effect on the SO by enkephalinase inhibitors were studied. These drugs reduce the inactivation of endogenous enkephalins. In previous studies the prodrug acetorphan was shown to have actions on the central nervous system as well as on the peripheral tissues [4].Thiorphan, on the [Leus]enkephalin and t h e sphincter other hand, is only effective in peripheral tissues owing to poor passage across the blood-brain barrier [4]. The contraction observed in response to acetorphan demonstrates that endogenous enkephalins have a stimulating effect on the SO. This effect is probably of central nervous system origin, as no response by the SO was seen with the administration of thiorphan under basal conditions. However, these results do not exclude the possibility that endogenous enkephalins in peripheral tissue may be active under conditions other than basal. This study also shows that administration of the opiate agonist [Leu5]enkephalin contracts the feline SO. As enkephalins d o not pass the blood-brain barrier [ 191, the site of action of [Leus]enkephalin is suggested to be in the intrinsic neural network of the SO. This assumption is supported by the observation of enkephalinase activity in the SO. The results suggest that both central and peripheral enkephalinergic mechanisms may be implicated in a possible excitatory regulation of the activity of the feline SO. The lack of effect of naloxone per se indicates that there is no basal tone in the enkephalinergic system under basal conditions. ACKNOWLEDGMENTS This study was supported by grants from the Goteborg Medical Society, the Swedish Medical Research Council (17X-04984)and the University of Goteborg. REFERENCES I. Behar, J. & Biancani, P. Neural control o f the sphincter o f Oddi. Gastroenterology 1984; 86, 134-41. 2. Wolter, H.J. Localization o f Met-enkephalin-Arg 6-Gly 7-Leu 8, immunoreactivity in rat duodenum. Neuropeptides 1986; 7, 20 1-6. 3. Llorens-Cortes, C. & Schwartz, J.C. 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