like enkephalin (Schulz et al., 1977) and endorphin from the myenteric plexus-longitudinal muscle preparations

Transcription

1 Br. J. Pharmaol. (1988), 95, Effets of naloxone and opioid agonists on gastri exitatory responses to stimulation of the vagus nerve in ats Toshihiro Okamoto, Kazuyoshi Kurahashi & Motohatsu Fujiwara Department of Pharmaology, Faulty of Mediine, Kyoto University, Kyoto 66, Japan 1 An investigation was made into the ontriution of endogenous opioids to the initial and delayed exitatory response of the stomah indued y stimulation of the vagal trunk in ats. 2 Naloxone (1 to looougkg-1) had no effet on the initial exitatory response to stimulation of the vagal efferent fires. However, the same treatment dose-dependently enhaned the delayed exitatory response to stimulation of the vagal afferent fires. 3 n omparison with the p-opioid-reeptor agonist, [D-Ala2, MePhe4, Gly-o15]enkephalin and the K-opioid-reeptor agonist dynorphin A (1-13), lower doses of methionine enkephalin ([Met]enkephalin) markedly inhiited the exitation aused y stimulation of the vagal efferent and afferent fires. The inhiitory effet of [Met]enkephalin was antagonized y naloxone. 4 The 5-opioid-reeptor seletive agonist [D-Pen2, DPen5]enkephalin mimiked the inhiitory effets of [Met]enkephalin and inhiition y [D-Pen2, DPen5]enkephalin was antagonized y the -opioid-reeptor antagonist, C 174, t is onluded that the inhiitory effets of exogenous opioids on the exitatory response of the stomah to stimulation of the vagal efferent and afferent fires are mediated, at least in part, y -opioid-reeptors. Naturally ourring opioids may partiipate in the inhiition of the delayed gastri exitation to stimulation of the vagal afferent fires. ntrodution Reently, it was reported that eletrial stimulation of the vagal trunk produed a two-phase exitatory response of the stomah in ats: an initial exitatory response during stimulation and a delayed exitatory response after the stimulation period. These responses seemed to e mediated through the efferent and afferent fires, respetively (Kurahashi et al., 1983; Okamoto et al., 1986). Naroti analgesi drugs, suh as morphine, have a marked effet on gastrointestinal funtion. This effet has long een exploited oth therapeutially and experimentally. Morphine was shown to ause relaxation of the guinea-pig intestine (Weinstok, 1971) and to inhiit the peristalti reflex of the guinea-pig isolated ileum y reduing the output of the aetylholine from the nerves in the myenteri plexuses whih innervate the musle layers (see, Cheruini et al., 1985). Opioid-like materials, ehaving like enkephalin (Shulz et al., 1977) and endorphin (Puig et al., 1976), were found to e released from the myenteri plexus-longitudinal musle preparations of guinea-pigs. These fats suggest the possiility that in these animals, naturally ourring opioids ontriute to the regulation of intestinal motility. Daniel (1966) showed that morphine inhiited gastri emptying in the dog and he further suggested that inhiition of gastri peristalsis either in duodenal spasm or in antral spasm ould aount for delayed gastri emptying. We found that morphine had no effet on the initial exitatory response of the stomah, ut this agent dose-dependently inhiited the delayed exitatory response (Okamoto et al., 1986). V The Mamillan Press Ltd 1988

2 33 T. OKAMOTO et al. The purpose of these experiments was to explore the ontriution of endogenous opioid to the exitatory response of the stomah to stimulation of the vagal efferent and afferent fires in ats. Methods Thirty-five ats of either sex, weighing 2.5 to 4.kg were used. The animals were deprived of food ut allowed free aess to water 12h efore the experiments. After initial anaesthesia with ether, sodium pentoaritone (1mgkg-1, i.v.) was administered. A traheal annula was inserted. The right femoral vein was atheterized and gallamine triethiodide (2mgml-1) was ontinuously infused at a onstant rate (1.48mlh 1). Artifiial respiration was maintained y a respiration pump. The respiration rate was 15 per min with the air volume at 7ml per stroke. The left femoral vein was atheterized for drug injetion. f the heart rate inreased y aout 1% or the pupils dilated, additional pentoaritone (1mgkg-1) was injeted into the left femoral vein; additional anaestheti was given at least twie during an experiment. The ervial vagal trunks on oth sides were ut and the ends were ligated. The distal trunk of the left vagus was plaed on a ipolar platinum eletrode and overed with otton wool soaked in liquid paraffin. Propranolol (1 mgkg -) and phentolamine (2mg kg-') pretreatment was used to inhiit a and f-adrenoeptors. Gastri motility was reorded with a alloon introdued via the oesophagus. The system was filled with water and onneted to a pressure transduer, thus reording hanges in intragastri pressure. The level of intragastri pressure was set at 1 to 15 mh2o. Changes in intragastri pressure were then reorded on a polygraph (San-ei nstrument, Tokyo, Japan) through a pressure transduer. A stimulator giving square wave pulses was used (1OHz in frequeny, 3 ms in duration and 1 V in intensity for O s). The following drugs were used: naloxone, gallamine triethiodide (Sigma Chemial Co., St Louis, MO); methionine enkephalin ([Met]enkephalin), dynorphin A (1-13) (Protein Researh Foundation, Osaka, Japan); [D-Ala2, MePhe4, Gly-o15]enkephalin (DAGOL), [D-Pen2, D-Pen5]enkephalin (DPDPE) (Peninsula Laoratories); C 174,864 (allyl 2-Try-Ai-Phe-Leu-OH, Camridge Researh Biohemials) and ether (Nakarai Chemial Co., Kyoto, Japan). The height of the exitatory response of the stomah efore administration of drug was regarded as 1%. Statistial analysis was performed y use of Student's t test for unpaired data. Results Effets of naloxone The effets of naloxone on the initial and the delayed exitatory responses of the stomah to stimulation of the vagal trunk were studied in 7 ats. Naloxone was administered 1 min efore the stimulation. Naloxone (1 to looopgkg-1, i.v.) had no effet on the initial exitatory response. The same treatment augmented the delayed exitatory response dose-dependently (Figures 1 and 2), to aout 18% of the ontrol response. Exitatory responses returned to the ontrol level after 2 to 3min. Basal tone was not affeted y naloxone. Effets of [Met]enkephalin, dynorphin A (1-13) and DAGOL The effets of [Met]enkephalin, dynorphin A (1-13) and DAGOL were examined in 13 ats. Eah drug Naloxone (1 jpg kg-', iv.) J _N~~~~~~~~~~~~~~~~~~~~~ 1 mino Figure 1 Effets of naloxone (1ig kg-1, i.v.) on the initial and the delayed exitatory response of the stomah to eletrial stimulation of the vagal trunk in ats. Vertial sale indiates 1 mh2o; horizontal sale 1 min; (-) the initial exitatory response. a 2 i) n QCL a) 1..! 1T T -- so 2r 1F ai.1~ ol Naloxone (,ug kg-1) Figure 2 Effets of naloxone (1 to looo1gkg-1, i.v.) on the initial (a) and the delayed () exitatory response of the stomah to eletrial stimulation of the vagal trunk in ats. Ordinate sale: % response as ompared with ontrol response efore administration of naloxone. Asissa sale: dose of naloxone. ** Statistially signifiant differene from orresponding ontrol, y Student's t test (P <.5).

3 a A 1 EFFECTS OF NALOXONE j.~~~~ 2E t [Metlenkephalin (1 jig kg-', i.v.) 1 min q Dynorphin A (1-13) E (1 jig kg-', i.v.) min 3 " t DAGOL (1 jig kg-,, i.v.) 3 min - Figure 3 Effets of [Met]enkephalin, dynorphin A (1-13) and [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGOL) on the initial and the delayed exitatory response of the stomah to eletrial stimulation of the vagal trunk in ats. (a) Effets of [Met]enkephalin (1 ygkg- 1, i.v.). Vertial sale indiates 2mH2, horizontal sale min; (-) initial exitatory response. () Effets of dynorphin A (1-13) (1pgkg- 1, i.v.). Vertial sale indiates 15mH2, horizontal sale 3min; () initial exitatory response. () The same as in () exept DAGOL (loopgkg-', i.v.) was used instead of dynorphin A (1-13). was administered 1 min efore stimulation. The results were as follows; in 7 ats, the initial and the delayed exitatory responses were inhiited dosedependently within 3 min after injetion of [Met]enkephalin (.1 to lpgkg-1) (Figures 3a and 4A). Basal tone was not affeted y [Met]enkephalin. Suh inhiition did not ontinue for more than 7min. Judging from the D5o for inhiition of the initial and the delayed exitatory responses, [Met]enkephalin was aout 6 times more effetive on the delayed exitation than on the initial exitation (Tale 1). There was no signifiant differene etween the effets of [Met]enkephalin and [Leu]enkephalin (not shown). n 3 ats, dynorphin A (1-13) (1 to 1/tgkg-1) did not affet asal tone and aused only weak inhiition on oth the initial and the delayed exitatory responses (Figures 3 and 4B). n these ases, the inhiitory effet of dynorphin A (1-13) (1 to loopgkg-1) was less powerful than that of [Met]enkephalin (Tale 1). Finally, in 3 ats, DAGOL (1 to loojgkg-1) did not affet asal 'A )~~~~ L_ T~ a * - -,Os..--, o, [Metlenkephalin (jig kg-') B 1 1$**T ) Ch a ) U) C 14 ) CL U) -) 51. ~~**L a 1 51 o Dynorphin A (1-13) (jig kg-') O lj DAGOL(jg kg-') Figure 4 Effets of [Met]enkephalin, dynorphin A (1-13) and [D-Ala2, MePhe', Gly-ol5]enkephalin (DAGOL) on the initial (a) and the delayed () exitatory response of the stomah to eletrial stimulation of the vagal trunk in ats. (A) Effets of [Met]enkephalin (.1 to 1pgkg-1, i.v.). Ordinate sale: % response as ompared with ontrol response efore administration of [Met]enkephalin. Asissa sale: dose of [Met]enkephalin. () Control, () pretreatment with naloxone (1,ugkg-1, i.v.) and (V) pretreatment with naloxone (1pgkg-1, i.v.). ** Statistially signifiant differene from orresponding ontrol (P <.5). * Statistially signifiant differene from orresponding [Met]enkephalin-indued inhiition, y Student's t test (P <.5). (B) Effets of dynorphin A (1-13) (1 to 1lgkg-1, i.v.). Ordinate sale: % response as ompared with ontrol response efore administration of dynorphin A (1-13). () Control, () pretreatment with naloxone (5pgkg-1, i.v.). ** Statistially signifiant differene from orresponding ontrol, y Student's t test (P <.5). (C) The same as in (B) exept that DAGOL was used instead of dynorphin A (1-13).

4 332 T. OKAMOTO et al. * ft Naloxone (1 plg kg-', i.v.) * f [Metlenkephalin (1,ug kg-, i.v.) N E 1 min ( Figure 5 Effets of naloxone (1lgkg- ',i.v.) on the [Met]enkephalin (1 pgkg- 1, i.v.)-indued inhiition of the initial and the delayed exitatory response of the stomah to eletrial stimulation of the vagal trunk in ats. Vertial sale indiates 2mH2; horizontal sale 1 min; (a) initial exitatory response. a Tale 1 D5 of [Met]enkephalin, dynorphin A (1-13), [D-Ala2, MePhe', Gly-ol5]enkephalin (DAGOL) and [D-Pen2, D-Pen5]enkephalin (DPDPE) on the initial and the delayed exitatory response of the stomah to stimulation of vagal trunk Agonist [Met]enkephalin Dynorphin A (1-13) DAGOL DPDPE 4LX D5 (pgkg-') nitial Delayed exitation exitation 7.6 ± ± 1.1 > 1 > 1 > 5 > , DiPDPE (1,pg kg--,, imv.) f 2mino C 174,864 DPDPE (1tO g kg-ij.v.) (1 pg kg-'i,.) E 2 mi N Flgure 6 Effets of [D-Pen2, D-Pen5]enkephalin (DPDPE) on the initial and the delayed exitatory response of the stomah to eletrial stimulation of the vagal trunk in ats. Vertial sale indiates 2mH2; horizontal sale 2min; () initial exitatory response. (a) DPDPE (lougkg-1, i.v.); () DPDPE (lougkg-1, i-v.) following pretreatment with C 174,864 (lgkg-1, i.v.). tone and aused no signifiant inhiition on either the initial or the delayed exitatory response (Figures 3 and 4C). Effets ofnaloxone on [Met]enkephalin-, dynorphin A (1-13) and DAGOL-indued inhiition [Met]enkephalin has een shown to inhiit the initial and the delayed exitatory response. On the other hand, dynorphin A (1-13) and DAGOL have shown weak inhiition on the exitatory responses. A further set of experiments was onduted on 9 ats to test the effets of naloxone on the inhiitory effets. The results of these experiments were as follows: in 3 ats, the augmentation of gastri response due to naloxone (1 and 1 pgkg-1) was allowed to return to the ontrol level. [Met]enkephalin (.1 to 1pgkg- 1) was then administered intravenously, 1 min efore stimulation. Pretreatment with naloxone attenuated the inhiitory effets of [Met]enkephalin on oth the initial and the delayed exitatory response of the stomah (Figures 4A and 5). n another 3 ats pretreated with naloxone (5pgkg-1), dynorphin A (1-13) (1 to 1pgkg-1) was administered intravenously, 1min efore stimulation. The pretreatment with naloxone did not attenuate the inhiitory effets of dynorphin A (1-13) signifiantly (Figure 4B). n the last 3 ats, naloxone did not affet the inhiitory effets of DAGOL (Figure 4C). Effets ofdpdpe and C 174,864 The effets of DPDPE and C 174,864 on the initial and the delayed exitatory response of the stomah to stimulation of vagal trunk were studied in 6 ats. n 3 ats, DPDPE was administered 1 min efore the stimulation. Basal tone was not affeted y DPDPE. DPDPE (.1 to ygkg- 1) inhiited oth the initial and the delayed exitatory responses dosedependently (Figures 6a and 7). Judging from the

5 EFFECTS OF NALOXONE 333 ) o C (A 4) - o a * response of the stomah. This effet of naloxone may 1 have resulted from the lokade of the ation of ~- * endogenously ourring opioids. t was reported 1 * (Edin, 198; Edin et al., 198) that the gastri ontration indued y stimulation of the vagal efferent fires with pulses of 5 ms duration was onverted to 5 F T** 1\ 5- relaxation y the administration of hexamethonium or atropine and that suh relaxation was redued y naloxone. The administration of enkephalin efore treatment with hexamethonium or atropine, aused relaxation of the stomah and this effet was also 1 '*a redued y naloxone. Thus, endogenous opioids may L, **D have ontriuted to gastri relaxation aused y stimulation of the vagal efferent fires. However, DPDPE (Ag kg-') Delro et al. (1981) and Tsuomura et al. (1987) on- Figure 7 Effets of [D-Pen2, D-Pen' 5]enkephalin sidered that vagal stimulation with pulses of suh (DPDPE,.1 to lopgkg-1, i.v.) on the in3itial (a) and long duration may have ativated not only efferent the delayed () exitatory response of the stomah to ut also afferent fires. Delro et al. (1982) reported eletrial stimulation of the vagal trunk irn ats. Ordi- that the hexamethonium-resistant gastri motor nate sale: % response as ompared awith ontrol response in ats, may e due to antidromi ativaresponse efore administration of DPD?E. Asissa tion of the vagal afferent fires. Unlike our results sale: dose of DPDPE. () Control; () pretreatment however, with C 174,864 (loopgkg-1, i.v.). **Sta this response was not affeted y naloxone. nifiant differene from orrespondi sng ontrol Morphine is known to inhiit gastri emptying in (P <.1). * Statistially signifiant differen from or- the dog and information as to the site of its ation is responding DPDPE-indued inhiition ) y Student's t availale (Daniel, 1966). The latter author argued test (P <.1). that inhiition of gastri peristalsis either in duodenal spasm or in antral spasm ould aount for the delayed gastri emptying. n previous experi- D5 for inhiition of the response, DPDPE has ments (Okamoto et al., 1986), we found that moralmost the same poteny as [Met]enkep: iting the delayed exitatory response Thal in inh- phine had no effet on the initial exitatory response another 3 ats, C 174,864 (looug kg (Tale 1). n ut inhiited the delayed exitatory response of the )was istered aout 15 min efore DPDPE. C] admin- stomah. Although morphine is a prototype p- 174,864 did opioid-reeptor agonist, it has een shown to inter- the delayed at with 6- and K-opioid-reeptors (Paterson et al., not affet the asal tone ut augmentedi exitatory response to aout 35% of the ontrol 1983). Thus, it is not possile to define the reeptor response. C 174,864 antagonized t effets of DPDPE (Figures 6 and 7). ie inhiitory type on whih morphine ats in this ase. n this study, [Met]enkephalin (a 6-opioid-reeptor agonist) in low doses inhiited the initial and the delayed exitatory responses dose-dependently, and these effets were antagonized y naloxone. DAGOL (uopioid-reeptor agonist) did not signifiantly inhiit Disussion either the initial or the delayed exitatory response. We have shown that two types of gastiri exitatory High doses of dynorphin A (1-13) (K-opioid-reeptor responses are produed y stimulation of the vagal agonist) exhiited weak inhiition of oth responses; trunk in ats. The initial exitatory respsonse is inhi- however, this inhiitory effet of dynorphin A (1-13) ited y hexamethonium and atropine; ;the delayed was not antagonized y naloxone. Furthermore, low exitatory response is not inhiite( d y hexa- doses of the -opioid-reeptor seletive agonist, methonium, ut is inhiited y; atropine. n DPDPE (Moserg et al., 1983), mimiked the inhiiats, only a tory effets of [Met]enkephalin and the inhiition y hronially supranodose vagotomized delayed exitatory response was oserved. Thus, it DPDPE was antagonized y the -opioid-reeptor was onsidered that the initial exitatdory response seletive antagonist C 174,864 (Cotton et al., 1984). and the delayed exitatory response vwere indued The results indiate that the inhiitory effets of through stimulation of the efferent and afferent opioids were mediated, at least in part, y 6-opioidfires, respetively (Kurahashi et al., 19M 83; Okamoto reeptors. mmunofluoresene studies have already et al., 1986). demonstrated the presene of enkephalin-like immuenhaned the noreative materials in the vagus nerves (Elde et al., n the present experiments, naloxone hexamethonium-resistant delayed exitatory 1976; Lunderg et al., 1978; Lunderg et al., 1979)

6 334 T. OKAMOTO et al. and stomah (Polak et al., 1977). Our results indiate that [Met]enkephalin is the most proale endogenous opioid to inhiit the delayed exitatory response of the stomah to stimulation of afferent vagal fires. This implies the possiility that an endogenous enkephalin-like opioid may ontriute to the inhiitory modulation of gastri motility. This study was supported in part y a Grant-in-Aid from the Smoking Researh Foundation, Japan. Referenes CHERUBN, E., MORTA, K. & NORTH, R.A. (1985). Opioid inhiition of synapti transmission in the guinea-pig myenteri plexus. Br. J. Pharmaol., 85, COTTON, R., GLES, M.G., MLLER, L., SHAW, J.S. & TMMS, D. (1984). C 174,864: a highly seletive antagonist for the opioid delta reeptor. Eur. J. Pharmaol., 97, DANEL, E.E. (1966). Further studies of the pharmaology of the pylori region. Analysis of the effets of intraarterial histamine, serotonin, phenyliguanide, morphine and other drugs on the antrum and duodenal ul. Can. J. Physiol. Pharmaol., 44, DELBRO, D., FANDRKS, L., LSANDER, B. & ANDERSSON, S.A. (1981). Hexamethonium-resistant, atropine-sensitive vagal exitation of the feline stomah-ativation of an unknown fire system. Ata Physiol. Sand., 112, DELBRO, D., FANDRKS, L., LSANDER, B. & ANDERSSON, S.A. (1982). Gastri atropine-sensitive exitation y peripheral vagal stimulation after hexamethonium. Antidromi ativation of afferents? Ata Physiol. Sand., 114, EDN, R. (198). The vagal ontrol of the pylori motor funtion. Ata Physiol. Sand., Suppl., 485, 1-3. EDN, R., LUNDBERG, J., TERENUS, L., DAHLSTROM, A., HOKFELT, T., KEWENTER, J. & AHLMAN, H. (198). Evidene for vagal enkephalinergi neural ontrol of the feline pylorus and stomah. Gastroenterol., 78, ELDE, R., HOKFELT, T., JOHANSSON,. & TERENUS, L. (1976). mmunohistohemial studies using antiodies to leuine-enkephalin: initial oservations on the nervous system of the rat. Neurosi., 1, KURAHASH, K., FUJWARA, M. & TANGUCH, T. (1983). Cholinergi exitatory response of stomah to stimulation of the vagal trunk in ats with hroni supranodose vagotomy. RCS Medial Si., 11, 62. LUNDBERG, J.M., HOKFELT, T., KEWENTER, J., PETTER- SON, G., AHLMAN, H., EDN, R., DAHLSTROM, A., NLSSON, G., TERENUS, L., WALLENSTEN, K.U. & SAD, S. (1979). Sustane P-, VP-, and enkephalin-like immunoreativity in the human vagus nerve. Gastroenterol., 77, LUNDBERG, J.M., HOKFELT, T., SCHULZBERG, M., NORELL, G., NLSSON, G., WALLENSTEN, K.U., TERE- NUS, L., DAHLSTR(5M, A., REHFELD, J.F., ELDE, R.P. & SAD, S. (1978). Pathways of peripheral peptide neurons with speial referene to the vagus nerve. Neurosi. Lett., Suppl., 1, 224. MOSBERG, H.., HURST, R., HRUBY, V.J., GEE, K., YAMA- MURA, H.., GALLGAN, JJ. & BURKS, T.F. (1983). Bispeniillamine enkephalins possess highly improved speifiity toward delta opioid reeptors. Pro. Nati. Aad. Si. U.S.A., 8, OKAMOTO, T., KURAHASH, K., TSUBOMURA, T. & FUJ- WARA, M. (1986). Effets of morphine on hexamethonium-sensitive and resistant exitatory responses of stomah to stimulation of vagal trunk in ats. Life Si., 39, PATERSON, S.J., ROBSON, L.E. & KOSTERLTZ, H.W. (1983). Classifiation of opioid reeptors. Br. Med. Bull., 39, POLAK, J.M., SULLVAN, S.M., BLOOM, S.R., FACER, P. & PEARSE, A.G.E. (1977). Enkephalin-like immunoreativity in the human gastrointestinal trat. Lanet, ii, PUG, M.M., GASCON, P., CRAVSO, G.L. & MUSACCHO, J.M. (1976). Endogenous opiate reeptor ligand; eletrially indued release in the guinea-pig ileum. Siene, 195, SCHULZ, R., WOSTER, M., SMANTOV, R., SNYDER, S. & HERZ, A. (1977). Eletrially stimulated release of opiate-like material from the myenteri plexus of the guinea-pig ileum. Eur. J. Pharmaol., 41, TSUBOMURA, T., OKAMOTO, T., KURAHASH, K. & FUJ- WARA, M. (1987). Gastri exitation y stimulation of the vagal trunk after hroni supranodose vagotomy in ats. J. Pharmaol. Exp. Ther., 241, WENSTOCK, M. (1971). Site of ation of naroti analgesi drugs in peripheral tissues: naroti drugs. Biohemial Pharmaology. ed. Clouet, D.H., p New York: Plenum Press. (Reeived August 4, 1987 Revised May 4, 1988 Aepted May 17,1988)