Myenteric Plexus-longitudinal Muscle Strips Research Articles

Alkylation with beta-funaltrexamine suggests differences between mu-opioid receptor systems in guinea-pig brain and myenteric-plexus.

1. The effects of pre-incubation with beta-funaltrexamine (beta-FNA) on the binding of [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]-DAMGO) to homogenates of guinea-pig brain and myenteric-plexus longitudinal muscle have been studied. 2. beta-FNA pretreatment of brain homogenates in Tris-HCl buffer reduced the amount of [3H]-DAMGO binding. This was principally due to a reduction in the maximal number of binding sites measurable. However, approximately 30% of sites labelled by 1 nM [3H]-DAMGO were insensitive to 1 microM beta-FNA. Similar findings were obtained when the alkylation was performed in brain homogenates prepared in Krebs solution buffered with HEPES. 3. beta-FNA pretreatment of whole myenteric-plexus longitudinal muscle strips caused an increase in the IC50 values of mu-agonists, but not of kappa-agonists. However, the binding of [3H]-DAMGO to homogenates of myenteric-plexus longitudinal muscle was not altered by pre-incubation with beta-FNA in Tris-HCl buffer. On the other hand when the pretreatment was carried out in whole tissue in Krebs solution, or in homogenates in the presence of NaCl and Gpp(NH)p, a marked reduction in [3H]-DAMGO binding was observed. 4. These results suggest that a low affinity form of the mu-opioid receptor is the physiologically relevant site for beta-FNA alkylation in the myenteric-plexus and that differences exist between mu-receptor systems in guinea-pig myenteric plexus and brain.

Effect of cinitapride in isolated ileum obtained from guinea-pigs treated with morphine

1. 1. Cinitapride enhanced the contractile response induced by electrical stimulation in the guinea-pig myenteric plexus-longitudinal muscle strip preparations. 2. 2. The contractile force was significantly increased in strips pretreated with morphine “ in vitro” and in tolerant strips. 3. 3. However when tissues were obtained from tolerant guinea-pigs and morphine was not added to the organ bath (morphine-abstinence), the cinitapride effect was significantly decreased. 4. 4. Although further work is required to explain the changes in the effect of cinitapride after acute morphine treatment and in morphine tolerant tissues, the changes observed suggest that some of the cinitapride effects could be linked with the perypheral opioid system.

An examination of the 5‐HT3 receptor mediating contraction and evoked [3H]‐acetylcholine release in the guinea‐pig ileum

1. The relative contributions of two classes of 5-hydroxytryptamine (5-HT) receptor (5-HT2 and 5-HT3) to the contractile action of 5-HT, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT) and alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) were studied in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. Contractility studies were combined with an analysis of the effects of the three agonists on [3H]-acetylcholine ([3H]-ACh) release from preparations preincubated with [3H]-choline. 2. In contracting the LMMP, 5-HT was approximately one order of magnitude more active than 2-methyl-5-HT and alpha-methyl-5-HT, with relative activities for 5-HT: 2-methyl-5-HT: alpha-methyl-5-HT of 1.00: 0.13: 0.10. 3. Ketanserin (1 microM) was without effect on the concentration-response curves for concentration to 5-HT. 2-methyl-5-HT or alpha-methyl-5-HT, whilst ondansetron (GR38032F: 1 microM) produced a parallel rightward displacement of the upper part of the concentration-response curves to 5-HT and alpha-methyl-5-HT and of the entire curve to 2-methyl-5-HT. 4. In increasing the spontaneous release of [3H]-ACh from the LMMP, 5-HT was again approximately one order of magnitude more active than 2-methyl-5-HT and alpha-methyl-5-HT with relative activities for 5-HT: 2-methyl-5-HT: alpha-methyl-5-HT of 1.00: 0.19: 0.11. 5. Ondansetron (1 microM) greatly attenuated the increase in spontaneous [3H]-ACh release evoked by all three agonists. pKB estimates of 7.62 + 0.12 and 7.64 + 0.09 were obtained for ondansetron antagonism of 5-HT and 2-methyl-5-HT-evoked increases respectively. 6. These data suggest that the contractile action of 5-HT, 2-methyl-5-HT and a-methyl-5-HT in the guinea-pig ileum can, under these conditions, be accounted for largely in terms of 5-HT3 receptor activation. Estimates for pKB obtained with ondansetron are in accordance with those previously obtained from contractility studies in this preparation and these findings are discussed in terms of the postulated existence of subtypes of 5-HT3 receptors.

Evaluation by reverse phase HPLC of [3H]acetylcholine release evoked from the myenteric plexus of the rat

Myenteric plexus-longitudinal muscle strips isolated from the small intestine of rats were incubated with [3H]choline to measure the synthesis and the release of [3H]acetylcholine. To separate different radioactive compounds (acetylcholine, choline, phosphorylcholine) from both the tissue and the overflow a new method, the reverse phase HPLC, was used. The radiochromatogram following the injection of a [3H]choline-standard and a [14C]acetylcholine-standard onto the HPLC showed a clear separation of both isotopes with a recovery rate of roughly 100%. Incubation of the muscle strips with [3H]choline caused the synthesis of [3H]acetylcholine (30,000 dpm/preparation) that increased 2-fold, when the electrical field stimulation during labelling was increased from 0.2 Hz to 1 Hz. Electrical field stimulation (3 Hz, 2 min) caused an increase in tritium efflux that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. Analysis by reverse phase HPLC of the overflow showed that the stimulated increase in tritium overflow was balanced by the enhanced release of [3H]acetylcholine. whereas the overflow of [3H]choline was not affected by the electrical field stimulation. Oxotremorine (1 mumol/l) suppressed the release of [3H]acetylcholine by 60%. Scopolamine (0.1 mumol/l) prevented this inhibition and, given alone, enhanced the release of [3H]acetylcholine by 43%. The release of [3H]acetylcholine evoked at 0.2, 2 or 20 Hz did not consistently decline at increasing frequencies. The present experiments show the synthesis and the calcium-dependent release of [3H]acetylcholine from the myenteric plexus-longitudinal muscle preparation of rats correspondingly to the same in-vitro preparation isolated from guinea-pigs. Muscarinic autoinhibition operates also in the small intestine of rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of morphine and acetylcholine on contractile activity and cyclic AMP in guinea-pig ileum

Neither acute nor prolonged exposure to morphine altered cAMP content or spontaneous movements of longitudinal muscle-myenteric plexus strips of the guinea-pig ileum. By contrast, exogenous acetylcholine or electrical stimulation of the strips elicited both a decrease of cAMP concentration and a twitch response. Atropine blocked the effects of stimulation on these parameters. Addition of morphine to electrically stimulated strips inhibited the twitch response but did not affect cAMP levels. Incubation with morphine led to the development of tolerance to the inhibitory effect on twitch activity and prevented the fall in cAMP normally elicited by electrical stimulation. These results suggest that muscarinic activation is associated with a reduction of cAMP content, an effect which would be impaired in opiate-tolerant tissues.

The effect of substance P on nerve action potential propagation and cholinergic transmission in the myenteric plexus of the guinea-pig ileum

The effect of substance P on nerve terminals in myenteric plexus of the guinea-pig ileum was investigated. Neurogenic twitches of the myenteric plexus longitudinal muscle strip were recorded. Twitches of the strip portion where excitation involved the most distal parts of cholinergic nerve terminals were more increased by local application of substance P (0.1 and 0.4 nmol/l) than twitches of the portion where excitation involved both distal and proximal parts of nerve terminals. Substance P addition to a portion of the strip conducting nerve action potentials to invade the neighbouring strip portion also augmented twitches of the latter portion so that the interference with the propagation process was considered. The effect of substance P was poorly antagonized by the addition of a substance P antagonist, (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-substance P. Compound nerve action potentials were evoked in strands of fibres of the myenteric plexus by low-frequency train stimulation (1 Hz). The addition of substance P prevented a decrease of the amplitude of responses observed under control conditions. Using high-frequency train stimulation (30 Hz) the amplitude of responses to impulses 2-7 was augmented over that to the first impulse; substance P further increased such facilitation regularly. It seems that substance P might promote nerve action potential invasion of the distal parts of nerve terminals.

The topographical basis of cholinergic transmission in guinea-pig ileum myenteric plexus

Myenteric plexus-longitudinal muscle strips were used to study nerve action potential propagation and transmission and their differences between the proximal and the distal regions of cholinergic terminals. Neurogenic twitches of a portion of the strip were evoked by focal electrical stimulation. Twitches mediated by the distal regions of cholinergic nerve terminals were more influenced by drugs affecting Ca 2+ “utilization” (Bay K 8644, kappa opiate ligand ethylketocyclazocine, changes in extracellular Ca 2+ or Co 2+ concentration) in contrast to twitches mediated by proximal regions of these terminals which were more influenced by drugs affecting sodium-potassium spike (tetrodotoxin, dendrotoxin, 4-aminopyridine, tetraethylammonium). Post-tetanic potentiation of twitches was prominent with that portion of the strip where the distal regions of nerve terminals were involved. Drugs interfering with Na +/K + spikes indiscriminately influenced both the twitch height and post-tetanic potentiation whereas changes in extracellular Ca 2+ concentration affected selectively only post-tetanic potentiation. Release of [ 3H]acetylcholine from pre-labelled strips evoked by 1 Hz continuous stimulation or by train stimulation at 30 Hz was measured selectively from portions containing either proximal and distal or only distal regions of nerve terminals. The release from portions containing the distal regions was relatively higher when evoked by 30 Hz than by 1 Hz. The distal regions of nerve terminals might be thus recruited to participate in transmission by a frequency-dependent process. Nerve impulses were recorded from strands of nerve fibres in the myenteric plexus. At 1 and 5 mm distance from the stimulation focus nerve impulses were completely suppressed by tetrodotoxin. At 5 mm, in some strands the amplitude of nerve impulses was also subject to the effect of drugs affecting Ca 2+ “utilization”; facilitation of nerve impulse amplitude during 30 Hz train stimulation was always influenced by drugs affecting Ca 2+ “utilization”. Propagation of nerve impulses in the distal region of cholinergic nerve terminals was found to be Ca-sensitive and frequency-dependent; this might form the basis for facilitation and post-tetanic potentiation of muscarinic transmission.

The topography of cholinergic transmission in the mechanism of drug action at muscarinic synapses of the guinea-pig ileum

The pharmacology of cholinergic neurogenic responses evoked by the participation of only the endings of axon terminals was compared to that of responses evoked by participation of the more proximal parts of the terminals also. Myenteric plexus-longitudinal muscle strips of the guinea-pig ileum were drawn through narrow orifices in 2 rubber membranes dividing a bath into 3 separate compartments. Oral segments were stimulated electrically by single impulses or by trains and local neurogenic contractions were evoked. The contractions of the aboral segment due to nerve impulses transmitted from the oral segment via the middle segment were also recorded. The opioid ligands ketocyclazocine and [D-Ala 2,MePhe 4,Met(O) 5-ol]enkephalin and noradrenaline inhibited the twitches of the aboral segment evoked by oral segment stimulation more than the local twitches of the oral segment when these agents were applied directly to the respective compartments. The twitches of the aboral segment were also inhibited by the application of these drugs into the middle compartment adjusted to 10 mm width. Verapamil and the alkaline earth metal ions cobalt and lanthanum had similar effects. 4-Aminopyridine increased twitch amplitude more in the aboral segment than in the oral segment when applied directly; similar effects in the aboral segment were seen when the agents were applied to the middle compartment. The action of atropine, papaverine, d-tubocurarine and prostigmine did not discriminate between twitches in the oral and aboral segment when applied directly and all drugs except prostigmine were without effect when applied to the middle compartment. It is concluded that drugs which are known to affect transmitter release from cholinergic nerve terminals show distinct topographical differences in pharmacological effects. Distal parts rather than proximal parts of the terminals were likely to be more affected. Propagation of action potentials within the terminals might be primarily subject to the action of these drugs.

Yohimbine reduces morphine tolerance in guinea-pig ileum

Opiates are known to inhibit electrically-evoked twitches of longitudinal muscle-myenteric plexus strips from guinea-pig ileum. When this preparation was incubated with morphine for 1 h tolerance developed to the inhibitory effect, since dose-response curves were shifted to the right. In the present study, the effects of alpha-2 adrenergic agents on the tolerance induced by morphine in this preparation was investigated. Addition of yohimbine 10 μM (but not 0.1 or 1 μM) to the incubating medium reduced the magnitude of opiate tolerance. This effect did not appear in the presence of the alpha-2 agonists clonidine or guanfacine (10 μM). Our results provide evidence of the longitudinal muscle-myenteric plexus as a useful model for the study of the relationship between morphine tolerance and alpha-2 adrenergic mechanisms.

Evidence for galanin as an inhibitory neuropeptide on myenteric cholinergic neurons in the guinea pig small intestine

The effect of galanin on the [ 3H]ACh release from myenteric plexus-longitudinal muscle strips of the guinea pig small intestine was studied. While galanin did not alter the basal spontaneous efflux of ACh, it significantly depressed the ACh release evoked by electrical stimulation or caused by VIP and substance P. These results suggest an important neuromodulatory role for galanin in the enteric nervous system.

Substance B: an endogenous brain factor that reverses presynaptic inhibition of acetylcholine release.

Evidence is presented of the existence of a substance in brain (substance B) that reverses presynaptic inhibition of evoked acetylcholine (AcCho) release. Addition of guinea pig brain synaptosomal incubation medium or whole brain extracts to assays in which 1,1-dimethyl-4-phenylpiperazinium-evoked release of [3H]AcCho from guinea pig myenteric plexus synaptosomes reversed purinergic, muscarinic, and alpha 2-adrenergic agonist inhibition. However, the extracts did not increase basal or evoked release of AcCho. Results of chromatography on Bio-Gel P-2 of a concentrated whole brain extract suggest this factor has an Mr of 700. Substance B was resistant to boiling under acid or alkaline conditions as well as incubation with phospholipase C and various proteases; ashing, however, completely destroyed activity. This endogenous factor was also found to antagonize agonist-mediated inhibition of electrically evoked contractions of myenteric plexus-longitudinal muscle strips from the guinea pig ileum. The demonstration of substance B with its ability to reverse presynaptic inhibition of AcCho release reveals one mode of presynaptic modulation.

Bradykinin releases acetylcholine from myenteric plexus by a prostaglandin-mediated mechanism

Release of [ 3H]acetylcholine (ACh) under the influence of bradykinin was measured from myenteric plexus-longitudinal muscle strips taken from guinea pig small intestine. Bradykinin stimulated the efflux of [ 3H]ACh in a dose-dependent manner. This stimulation by bradykinin was resistant to the effect of [Des-Arg 9-Leu 8]-bradykinin but not to indomethacin, indicating that the ACh-releasing action of bradykinin was mediated indirectly by a prostaglandin mechanism. Direct evidence for a stimulation of ACh release by prostaglandin E 1 was obtained. Prostaglandin was able to stimulate ACh release in a dose-related fashion. The inhibition of bradykinin-induced ACh release by indomethacin was partly reversed by exogenous prostaglandin E 1. These results suggest a neuromodulatory role for bradykinin in the enteric nervous system.

Interactions between the duration of stimulation and noradrenaline on cholinergic transmission in the myenteric plexus-smooth muscle preparation

Output of acetylcholine (ACh), electromyogram (EMG) recordings and contractions of myenteric plexus-longitudinal muscle strip preparations from the guinea-pig ileum were studied during stimulation by single impulses or by trains (30 Hz; 2 to 128 impulses) under control conditions and in the presence of noradrenaline (NA). During supramaximal stimulation N A (2.5 μM) inhibited both contractions of the smooth muscle and the release of ACh evoked by single impulses more effectively than those evoked by train stimulation so that in a train of 4 impulses the output of ACh per impulse after the 2nd to 4th impulses was 69 to 104% higher than the output after the 1st impulse. During submaximal stimulation, contractions and ACh release evoked by single impulses were almost completely inhibited by NA. The neurogenic EMG, a direct consequence of the localized action of released transmitter (ACh), was recorded in the longitudinal muscle 4 and 10 mm aborally from the focal stimulation site. The incidence of the neurogenic response was much higher at the proximal (4 mm) than at the distal (10 mm) site and was proportional to the number of impulses in a train (100 Hz). NA inhibited propagation of the neurogenic response evoked by single impulses whereas its effect during train stimulation was less. It is concluded that in the course of train stimulation, sites of transmission more distant from the stimulation focus were recruited and consequently the secretion of ACh in succeeding impulses was enhanced. NA could interfere with this process; it might inhibit the invasion by action potentials of cholinergic nerve terminal varicosities, thereby reducing the release of ACh.

Inhibitory peptidergic neurons: functional difference between somatostatin and enkephalin in myenteric plexus

This study reports on a difference in the inhibitory action of the neuropeptides somatostatin and Met-enkephalin on acetylcholine (ACh) release from myenteric plexus-longitudinal muscle strips of guinea pig small intestine. Met-enkephalin (8.7 X 10(-8) M) inhibited ACh release evoked by either substance P (3.7 X 10(-8) M) or neurotensin (7.5 X 10(-11) M), and this inhibition could be reversed by naloxone (5 X 10(-8) to 5 X 10(-5) M). Neurotensin-induced ACh release was also sensitive to the inhibition by somatostatin. However, when tested in a dose range from 6.1 X 10(-8) to 6.1 X 10(-6) M, somatostatin was ineffective in reducing the efflux of ACh evoked by substance P. These observations provided evidence to support the view that inhibitory peptidergic neurons within the myenteric plexus modulate the activity of cholinergic neurons with a high degree of specificity and that both somatostatin and Met-enkephalin have distinct neuromodulatory functions in the gut.

Effects of Peptidase Inhibitors on the (Met5)-Enkephalin Hydrolysis in Ileal and Striatal Preparations of Guinea-Pig: Almost Complete Protection of Degradation by the Combination of Amastatin, Captopril and Thiorphan

AbstractThe contents of [Met5]-enkephalin and its four hydrolysis products. Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe, in the sample obtained after enkephalin was incubated with tissues in either the absence or the presence of the peptidase inhibitor were estimated by high performance liquid chromatography combined with electrochemical detection in order to elucidate the effect of the peptidase inhibitor on enkephalin hydrolysis. Free Tyr was the major degradative product in the absence of the peptidase inhibitor, while the major hydrolysis product was the Tyr-Gly-Gly fragment in the presence of amastatin in both total homogenates and membrane fractions prepared from either the myenteric plexus-longitudinal muscle strip of guinea-pig ileum or the striatum of guinea-pig brain. Additionally. Captopril significantly decreased the generation of Tyr-Gly-Gly in the presence of amastatin in both ileal and striatal membrane fractions. Moreover, thiorphan significantly prevented the formation of Tyr-Gly-Gly in the presence of both amastatin and Captopril in both membrane preparations. Finally, when [Met6]-enkephalin was incubated with either an ileal or a striatal membrane fraction for 60 min at 37°C in the presence of three peptidase inhibitors, amastatin. Captopril, and thiorphan. approximately 98 or 94%, respectively, of enkephalin remained intact. The results indicated that [Met6]-enkephalin was almost exclusively hydrolyzed by three distinct enzymes, amastatin-sensitive ammopeplidase. Captopril-sensitive peptidyl dipeptidase A. and thiorphan-sensitive endopeptidase-24.11 in both ileal and striatal membrane fractions.

How far from the stimulation site in myenteric plexus-longitudinal muscle preparations the neurogenic cholinergic contraction can be evoked?

Neurogenic contractions of a segment of myenteric plexus-longitudinal muscle strip preparations from the guinea-pig ileum were evoked. The site of electric stimulation was separated from the contracting segment by a gap preventing the spread of muscle action potentials set up in other regions. The width of the separating gap (2-20 mm) indicated the length of nerve fibers that could conduct impulses across and trigger cholinergic contractions behind the gap; it was more than 12 and less than 16 mm. Transmission of excitation was more effective in the aboral direction compared to the oral direction and was not apparently affected by noradrenaline nor by substance P.

Adenosine and dipyridamole: actions and interactions on the contractile response of guinea-pig ileum to high frequency electrical field stimulation.

The action of adenosine on the myenteric plexus-longitudinal muscle strips from guinea-pig ileum to high frequency electrical field stimulation (10 Hz) was investigated. Electrically induced contractions were reduced markedly by tetrodotoxin (0.2 microM) and atropine (1 microM), and partially by noradrenaline (3 microM) and morphine (3 microM). Adenosine, adenosine 5'-monophosphate (AMP) and adenosine triphosphate (ATP) produced a concentration-dependent inhibition of the high frequency contractions over the range of 0.1-100 microM, the most potent being adenosine. The concentration-response curve for adenosine was significantly shifted to the left by dipyridamole (10 nM), while dipyridamole at higher concentrations (30 nM-10 microM), depressed the contraction markedly by itself. Dipyridamole decreased [3H]-adenosine uptake into strips of ileum in a concentration-dependent manner. There was a significant correlationship between the reduction of adenosine uptake and the inhibition of the contraction induced by dipyridamole (r = 0.970). In strips desensitized to adenosine or treated with adenosine deaminase, the inhibitory effect of dipyridamole was significantly reduced. The present investigation revealed that adenosine depressed responses of guinea-pig ileum to high frequency electrical stimulation and suggested that the inhibitory effect of dipyridamole may be closely associated with the behaviour of endogenous adenosine or related compounds.

Neurochemical evidence for the release of acetylcholine from the guinea-pig ileum myenteric plexus by capsaicin

Superfision of the sensory stimulant drug capsaicin (3.3 μM) released [ 3H]acetylcholine from isolated myenteric plexus-longitudinal muscle strips of guinea-pig ileum, preincubated with [ 3H]choline. This effect was prevented by tetrodotoxin. There was a fast tachyphylaxis to the effect of capsaicin. It is suggested that capsaicin-sensitive nerve endings capable of activating intrinsic cholinergic neurones are situated in the myenteric plexus of the guinea-pig ileum.

Separation of recycling and reserve synaptic vesicles from cholinergic nerve terminals of the myenteric plexus of guinea pig ileum.

Acetylcholine-rich synaptic vesicles were isolated from myenteric plexus-longitudinal muscle strips derived from the guinea pig ileum by the method of Dowe, Kilbinger, and Whittaker [J. Neurochem. 35, 993-1003 (1980)] using either unstimulated preparations or preparations field-stimulated at 1 Hz for 10 min using pulses of 1 ms duration and 10 V . cm-1 intensity. The organ bath contained either tetradeuterated (d4) choline (50 microM) or [3H]acetate (2 muCi . ml-1); d4 acetylcholine was measured by gas chromatography-mass spectrometry. As with Torpedo electromotor cholinergic vesicle preparations made under similar conditions the distribution of newly synthesized (d4 or [3H]) acetylcholine in the zonal gradient from stimulated preparations was not identical with that of endogenous (d0, [1H]) acetylcholine, but corresponded to a subpopulation of denser vesicles (equivalent to the VP2 fraction from Torpedo) that had preferentially taken up newly synthesized transmitter. The density difference between the reserve (VP1) and recycling (VP2) vesicles was less than that observed in Torpedo but this smaller difference can be accounted for theoretically by the difference in size between the vesicles of the two tissues. At rest, a lesser incorporation of labelled acetylcholine into the vesicle fraction was observed, and the peaks of endogenous and newly synthesized acetylcholine coincided. Stimulation in the absence of label followed by addition of label did not lead to incorporation of labelled acetylcholine, suggesting that the synthesis and storage of acetylcholine in this preparation and its recovery from stimulation is much more rapid than in Torpedo.

Characteristic of antinociceptive effect of a tetrapeptide, Asn-Ala-Gly-Ala (NAGA).

The characteristic of the antinociceptive effect of a tetrapeptide, Asn-Ala-Gly-Ala (NAGA), was examined. Intracisternal (i.cist.) administration of NAGA to mice produced dose-dependent and long-lasting antinociceptive effect as evaluated by the hot plate and tail flick methods. NAGA-induced antinociception was reversed by naloxone (2 mg/kg), reserpine (2 mg/kg X 2 d) and alpha-methyl-p-tyrosine (100 mg/kg). NAGA (10 microM) did not significantly affect the actions of opioid peptides on the electrically evoked twitches of the myenteric plexus-longitudinal muscle strips from the guinea-pig esophagus and strips of the mouse vas deferens. NAGA-induced antinociception was scarcely affected by p-chlorophenylalanine (PCPA, 300 mg/kg X 2 d) and methysergide (2 mg/kg). From the above results, it was suggested that the antinociceptive effect of NAGA may involve the participation of endogenous opioid peptides and endogenous catecholamines.