Enkephalin-containing neurons in the inferior mesenteric ganglion projecting to the distal colon of cat: evidence from combined retrograde tracing by fluorescent microspheres and immunohistochemistry

Retrograde tracing with rhodamine fluorescent microspheres combined with fluorescein immunolabelling of methionine-enkephalin showed the presence of enkephalin-like material in neurons of the inferior mesenteric ganglion (sympathetic prevertebral ganglion) projecting to the distal colon in cat. Two weeks after injecting the microspheres into the wall of the distal colon, the inferior mesenteric ganglion was dissected out and incubated for 24 hours in a colchicine-containing culture medium in order to facilitate the detection of enkephalins in the soma of ganglion neurons. It was observed that retrogradely labelled ganglion cells contained enkephalin-like immunoreactive material. These ganglion cells corresponded to enkephalin-like postganglionic neurons, the terminals of which were located inside the wall of the distal colon. These enkephalin-like neurons were numerous and scattered throughout the ganglion. Sometimes enkephalin-like immunoreactive fibers, probably originating from spinal preganglionic neurons, ran close to immunoreactive and non-immunoreactive retrogradely labelled ganglion cells. This suggests that enkephalin-like immunoreactive fibers may make synaptic connections with enkephalin-like and non-enkephalin-like postganglionic neurons projecting to the distal colon. The present study establishes for the first time the existence of an enkephalin-like postganglionic pathway to the digestive tract originating from a sympathetic prevertebral ganglion. This finding indicates that the enkephalinergic innervation of the cat digestive tract may have at least two possible sources: (i) the sympathetic prevertebral ganglia; and (ii) the enteric nervous ganglia.     

Nicotine-induced alterations in peripheral tissue concentrations of native and cryptic Met- and Leu-enkephalin

This study examined the peripheral tissue distribution of native and cryptic Met- and Leu-enkephalin, and regulation of tissue enkephalins by nicotine. Met- and Leu-enkephalin concentrations showed widespread variation in tissue concentration and degree of processing. HPLC characterization of homogenate of spleen revealed that both native and cryptic immunoreactive Met-enkephalin are comprised of two peaks, one representing authentic Met-enkephalin pentapeptide and the other its sulfoxide. Subacute repeated administration of nicotine 0.1 mg/kg ip, six times at 30 min intervals, increased native Met- and Leu-enkephalin in adrenal medulla without affecting cryptic Met- and Leu-enkephalin concentrations, consistent with increased processing of larger peptides to Met- and Leu-enkephalin. Subacute nicotine decreased splenic concentrations of native and cryptic Met-enkephalin and native Leu-enkephalin, consistent with increased release of Met- and Leu-enkephalin from spleen and decreased synthesis of proenkephalin A or inadequate processing of larger peptides to enkephalin pentapeptides in spleen to compensate for the increased release during this period. HPLC characterization revealed that nicotine-induced decrease in native Met-enkephalin in spleen resulted from reductions in both pentapeptide and its sulfoxide. Nicotine also increased native Met-enkephalin in jejunum, decreased cryptic Met-enkephalin in heart atrium, increased native Leu-enkephalin in anterior pituitary and decreased cryptic Leu-enkephalin in jejunum. Nicotine may produce some of its effects through alterations in release of enkephalins from peripheral tissues.     

Actions of morphine and enkephalins on the internal anal sphincter of the cat: relevance for the physiological role of opiates

The effects of Leu-enkephalin, Met-enkephalin and morphine on the electrical activity of the internal anal sphincter were studied in anesthetized spinalized cats and in vitro on sphincteric muscle strips. All the effects of enkephalins and morphine were antagonized by naloxone (2 mg/kg, i.v. in vivo and 10(-6)M in vitro). In vivo, the enkephalins (0.01 mg/kg i.v.) and morphine (2 mg/kg, i.v.) decreased the amplitude of the excitatory responses evoked in the sphincter by stimulation of the hypogastric nerves. Opiates presumably act on the sympathetic nerve endings by reducing the release of noradrenaline. In vitro, the enkephalins (10(-6)M) and morphine (10(-6)M) had a similar inhibitory effect, indicating that opiates act, at least partly, at intramural level. In vivo, the enkephalins and morphine produced an inhibition of the spontaneous electrical activity of the internal anal sphincter. This inhibition occurs also in vitro; it is thus due to a peripheral effect of opiates acting either directly on the sphincteric smooth muscle cells, or through the nervous structures controlling sphincteric motility. In addition, the distribution of nerves containing enkephalin-like immunoreactivity, using whole mount preparations of cat internal anal sphincter, indicates that this area is supplied with a dense Leu- and Met-enkephalinergic innervation. Met- and Leu-enkephalin-like immunoreactive axons were detected within the circular and longitudinal muscles.     

The proenkephalin-A-derivative Met-enkephalin-Arg-Gly-Leu is not present in the feline species

No opioid octapeptide Met-enkephalin-Arg-Gly-Leu was detected either in the brain or in the adrenal gland of the cat using a specific radioimmunoassay. Whereas it was possible to determine the Met-enkephalin and Leu-enkephalin contents. The Met-enkephalin versus Leu-enkephalin concentration ratio was around five in each area of the brain assayed. The presence of authentic Met-enkephalin and Leu-enkephalin was confirmed by high performance liquid chromatography analysis. All in all, these data seem to indicate that the cat proenkephalin is partly different from that previously analysed in mammalian species including humans, rats and cows.     

Several distinct receptor binding enkephalins in olivocochlear fibers and terminals in the organ of corti

Biochemical studies centering on the use of reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassays (RIA) demonstrate the presence in the guinea pig organ of Corti of at least 3 enkephalin-related peptides, two of which are identified as Met- and Leu-enkephalin, respectively. Enkephalins were identified and quantitated by HPLC-RIA in the isolated second turn of the organ of Corti, but were not found in stria vascularis or auditory nerve dissected from the cochlea. Three enkephalin-immunoreactive HPLC fractions inhibited the binding of labeled naloxone to rat brain membranes. All enkephalins identified by the combined HPLC-RIA procedure had an apparent molecular weight similar to that of Met- and Leu-enkephalin peptide standards. Immunocytochemistry, performed with the best-characterized Met-enkephalin antiserum used in the RIAs, localized the enkephalin-like immunoreactivity to lateral efferent fibers and terminals under inner hair cells of the organ of Corti. Other antisera raised against Met-enkephalin, not used for RIA, visualized enkephalin-like immunoreactivity in medial efferent fibers under outer hair cells as well. This enkephalin-like immunoreactivity may reflect the presence in the medial efferent system of other structurally similar peptides in addition to those detected biochemisally. Efferent fiber lesion, by evulsion of the vestibular nerve close to the vestibulocochlear anastomosis in which the olivocochlear fibers run, eliminated enkephalin-like immunoreactivity and the enkephalin-related peptides identified by HPLC-RIA.     

Immunocytochemical evidence for the presence of met-enkephalin and leu-enkephalin distinct neurons in the brain of the elasmobranch fish Scyliorhinus canicula

Immunohistochemical methods have been used to investigate the distribution of various opioid peptides derived from mammalian proenkephalin in the central nervous system of Scyliorhinus canicula. The results indicate that both Leu- and Met-enkephalin-immunoreactive peptides are present in the dogfish brain. In contrast, enkephalin forms similar to Met-enkephalin-Arg-Phe or Met-enkephalin-Arg-Gly-Leu, and mammalian α-neo-endorphin, dynorphin A (1–8), dynorphin A (1–13), and dynorphin A (1–17) were not detected. Met- and Leu-enkephalin immunoreactivities were found in distinct neurons of the telencephalon and hypothalamus. In particular, cell bodies reacting only with the Met-enkephalin antiserum were localized in the preoptic nucleus and in the suprachiasmatic region of the hypothalamus. Conversely, cell bodies reacting only with the Leu-enkephalin antiserum were localized in the pallium and the nucleus lobi lateralis hypothalami. Several areas of the telencephalon and diencephalon exhibited both Met- and Leu-enkephalin-like immunoreactivity, but the two immunoreactive peptides were clearly contained in distinct perikarya. The overall distribution of Met-enkephalin-immunoreactive elements in the dogfish brain exhibited similarities to the distribution of proenkephalin-derived peptides previously reported for the brain of tetrapods. The fact that Met- and Leu-enkephalin-like peptides were detected in distinct neurons, together with the absence of dynorphin-related peptides, suggests the existence of a novel Leu-enkephalin-containing precursor in the dogfish brain. © 1994 Wiley-Liss, Inc.     

Regional and subcellular distribution of Met- and Leu-enkephalin-degrading activity in rat brain

Met- and Leu-enkephalin were degraded rapidly by brain aminopeptidases with the K_m's 9.1 mM and 5.7 mM respectively; the V_max, 100 μmol/mg protein per min for Met-enkephalin and 50 μmol/mg protein per min for Leu-enkephalin. The major product for Met-enkephalin was des-Tyr-Met-enkephalin. The enkephalin-degrading activity (EDA) in the brain was 16-fold higher than in plasma and was 15% of that in the kidney. The hydrolytic activity was heterogeneous in rat brain regions. For Met-enkephalin, the activity in decreasing order was striatum, hypothalamus, hippocampus, cerebellum, cortex, mid-brain, and medulla oblongata; for Leu-enkephalin the order was hippocampus, striatum, mid-brain, cortex, hypothalamus, cerebellum, and medulla oblongata. The subcellular distribution of the EDA in the whole brain, the hippocampus, and the striatum was similar, with the soluble fraction having the highest, the synaptosomal fraction the lowest, activity. The distribution of EDA was different from the arylamidase activity with Tyr-β-naphthylamide and Leu-β-naphthylamide as substrates. Our results indicate that a group of aminopeptidases is responsible for the degradation of both enkephalins.     

Effects of chemical and surgical lesions on levels of chromatographically identified enkephalin-like peptides in rat hippocampus

The present study quantitates the content of Met- and Leu-enkephalin in the rat hippocampus, and provides information on the localization of the enkephalins within the hippocampal neuronal circuitry. Several enkephalins were identified in rat hippocampus, two of which are shown to be Met- and Leu-enkephalin. The levels of these enkephalins, and of other unidentified enkephalin-related peptides, were not depleted by intrahippocampal colchicine, which destroyed the great majority of the hippocampal granule cells and the associated mossy fiber pathway. Entorhinal cortical lesions ablating the perforant pathway input to the hippocampus also did not significantly lower enkephalin levels in the hippocampus. Unilateral fimbrial transection caused a significant bilateral increase in both Met- and Leu-enkephalin levels. This may result from loss of a stimulatory input to putative enkephalin containing interneurons within the hippocampus. The extents of all lesions were verified histologically in hippocampi used for biochemical analysis. No evidence was seen for the presence of enkephalins in the perforant pathway, nor in nerve fibers in the fimbria/fornix, which provide the other main source of hippocampal efferents. The enkephalins are likely to be intrinsic to the hippocampus, in which neuronal cell bodies containing enkephalin-like immunoreactivity have been extensively reported.     

Role of vesical afferent nerve fibres involved in the control of internal anal sphincter motility

The neural pathways involved in the interactions between urinary bladder and internal anal sphincter (IAS) were studied in anaesthetized spinal cats. Activation of vesical afferents produced in the IAS a reflex increase in the electrical activity and a reflex inhibition of the excitatory responses evoked by stimulation of one hypogastric nerve. Both reflexes are achieved partly in the lumbar spinal cord and partly within the inferior mesenteric ganglion.     

Release of immunoreactive Met-enkephalin from the spinal cord by intraventricular beta-endorphin but not morphine in anesthetized rats

Effect of beta-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin1-13 from the spinal cord was studied in anesthetized rats. Intraventricular beta-endorphin, 16 micrograms, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 micrograms, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin1-13 was not released by either beta-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by beta-endorphin had a retention time identical to [3H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for beta-endorphin and morphine.     

Modulation of superoxide anion release from human polymorphonuclear cells by Met- and Leu-enkephalin

The present work describes the ability of Met- and Leu-enkephalin to modulate the superoxide anion (O2-) release from unstimulated human polymorphonuclear cells (PMN) and from PMN stimulated with phorbol myristate acetate (PMA). The direction (stimulation or suppression) and the magnitude of change were dependent upon the baseline reactivity of the donor's PMN. Both opioid peptides stimulated O2- release by PMN from donors with low baseline reactivity in a concentration-dependent manner. PMNs collected from donors with medium baseline reactivity incubated with Leu-enkephalin regardless of concentration released less O2- than control, nontreated PMNs. Met-enkephalin stimulated O2- release but only at 2 X 10(-15) M concentration. Superoxide anion release from PMNs of individuals with high baseline reactivity was concentration dependent and suppressed by Met- and Leu-enkephalin. Leu-enkephalin induced baseline reactivity was dependent upon progressive increase in the magnitude of change on O2- release (i.e., the higher the baseline the higher the magnitude of change in O2- generation). Met-enkephalin data show this also, but to a lesser extent. In cells stimulated with PMA, Met-enkephalin caused additional O2- release, while Leu-enkephalin was ineffective in triggering already stimulated cells. The modulating effect of both opioid peptides on superoxide anion release by human PMN is a short phenomenon that lasts up to 10 min after the addition of the peptide.     

Regional differences in the sympathetic innervation of the guinea pig large intestine by neuropeptide Y- and tyrosine hydroxylase-immunoreactive nerves of divergent extrinsic origin.

Region-specific patterns of nerves with immunoreactivity to neuropeptide Y (NPY) have been described previously in the submucous plexus of guinea pig large intestine. Because these may have functional significance, the possibility of similar, characteristic variations of NPY-like immunoreactivity (NPY-ir) in the myenteric plexus was explored. Regional differences were found in the occurrence and pattern of distribution of NPY-ir in the myenteric plexus of the guinea pig large intestine. NPY-ir was present rarely within neuron somata in any region of the large intestine, and NPY-ir nerve fibers were present only within the distal large intestine, increasing progressively in density from the distal spiral to the rectum. Lesion of the colonic nerves, but not the hypogastric, intermesenteric, or lumbar splanchnic nerves, resulted in a loss of NPY-ir in the distal spiral and transverse colon but not in the descending colon or rectum. Ring myotomies in the descending colon resulted in a loss of NPY-ir proximal to the lesion. Dual-labeling immunohistochemical studies revealed that the NPY-ir nerve fibers rarely contained immunoreactivity for tyrosine hydroxylase (TH). Extrinsic nerve lesions resulted in an unequivocal reduction in NPY-ir in intraganglionic fibers of the submucosal plexuses of the transverse colon and a partial loss in the distal spiral and descending colon: the rectum was unaffected; in only a minority of guinea pigs, however, was any decrease in the NPY-ir innervation of submucosal blood vessels detected. The principal projections of NPY-ir nerves were from and through the inferior mesenteric ganglion; however, NPY-ir was not colocalized with TH-ir. It is proposed that nonnoradrenergic, NPY-containing neurons located in the inferior mesenteric ganglion project through the colonic nerves and that these proximally directed fibers innervate the transverse colon and the distal spiral. Nonnoradrenergic, NPY-ir neurons lying in the pelvic ganglia or sacral sympathetic chain may make an important contribution to the innervation of the myenteric plexus of the rectum and the descending colon.     

Neuronal projections to the guinea pig stellate ganglion investigated by retrograde tracing

Previous electrophysiological studies have revealed a peripheral sensory input to the stellate ganglion which does not originate from the dorsal root ganglia. The present retrograde tracing study aimed at evaluating whether the parent cell bodies are located in the periphery, i.e. in mediastinal ganglia. Following injection of Fast blue or wheat germ agglutinin-horseradish peroxidase into the right stellate ganglion of the guinea pig, retrogradely labelled cell bodies were observed in the intermediolateral and intercalated nuclei of the spinal cord as well as in dorsal root ganglia at segmental levels C8 to T6. In another case, the stellate ganglion was resected and replaced by a sponge soaked with 10 μl of Fast blue. Labelling of preganglionic and sensory neurons parallelled that obtained by tracer injections. In neither case, however, were retrogradely labelled neurons found within or around the thoracic viscera (thymus, trachea, bronchi, esophagus, heart, great vessels of upper mediastinum) when these were cut serially en bloc. Controls performed by injection of Fast blue into the inferior mesenteric ganglion and investigation of the distal colon showed that our experimental protocol was able to visualize a peripheral projection towards a sympathetic ganglion — in this case from myenteric ganglia to the inferior mesenteric ganglion. We conclude that, in contrast to the circuitry connecting prevertebral sympathetic ganglia with the gut, the neuronal cell bodies providing peripheral sensory input from thoracic viscera to the right stellate ganglion most likely are not located within the mediastinal ganglia. Instead, they may reside within the stellate ganglion itself.     

Inflammation-induced changes in the chemical coding pattern of colon-projecting neurons in the inferior mesenteric ganglia of the pig

The present study examines the chemical coding of the inferior mesenteric ganglia after chemically induced colitis in the pig animal model. In all animals (n = 6), a median laparotomy was performed under anesthesia, and the Fast Blue retrograde tracer was injected into the descending colon wall. In experimental animals (n = 3), the thick descending colon were injected with formalin solution to induce inflammation. The animals were euthanized and the inferior mesenteric ganglion was harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P (SP), vesicular acetylcholine transporter (VAChT), or galanin (GAL). Immunohistochemistry revealed significant changes in the chemical coding pattern of inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, LENK, CB, and NOS. In the experimental group, TH, NPY, SOM, VIP, and LENK expressing neurons were reduced, whereas the number of neurons immunoreactive to CB, NOS, and GAL were increased. The increase of so-called neuroprotective neuropeptides suggests that the changes in the chemical coding of inferior mesenteric ganglion neurons reflect adaption under pathological conditions to promote their own survival.     

Spreading depression induced by microinjection of enkephalins into the hippocampus and neocortex

The opioid peptides Leu-enkephalin, Met-enkephalin and d-Ala²-Met-enkephalinamide were injected at various concentrations into the neocortex and hippocampus of rats to examine their effects on EEG activity and DC potentials. All three compounds were found to elicit spreading depression (SD) in both structures. Higher doses of Met-enkephalin were required to elicit SD as well as seizure activity. In the hippocampus the wave of SD was frequently preceded by seizure activity which was antagonized by naloxone pretreatment (40 mg/kg i.p.). Naloxone also prevented Leu-enkephalin-induced SD in the neocortex (but not in the hippocampus) and Met-enkephalin-induced SD in the hippocampus (but not in the neocortex). It failed to block SD elicited by d-Ala²-Met-enkephalinamide in both structures. Some of the various reported behavioral effects of intracranial injections of enkephalins could be artefacts of hippocampal and/or cortical spreading depression.     

Morphological and chemical identification of neurons that project from the colon to the inferior mesenteric ganglia in the guinea-pig

Labelled nerve cells were located in the distal colon of the guinea-pig 4-5 days after the retrograde tracing agent, Fast blue, was injected into the inferior mesenteric ganglia. Labelled neurons were only found in the myenteric plexus. Their frequency increased from oral to anal and was greater towards the mesenteric border, compared with the anti-mesenteric aspect, of the colon. Many retrogradely labelled neurons were immunoreactive for vasoactive intestinal peptide or calbindin. In the inferior mesenteric ganglia, vasoactive intestinal peptide and calbindin immunoreactive nerve fibres surrounded the same clumps of nerve cell bodies. Almost all calbindin and vasoactive intestinal peptide immunoreactive terminals degenerated after the nerves running from the large intestine to the inferior mesenteric ganglia were cut. It is concluded that the great majority of calbindin and vasoactive intestinal peptide immunoreactive terminals in the inferior mesenteric ganglia arise from nerve cell bodies in the myenteric plexus of the large intestine.     

Evidence for an enkephalinergic system in the nervous system of the pond snail, Lymnaea stagnalis

Evidence for the presence of an enkephalinergic system in the ganglia of the pond snail, Lymnaea stagnalis, has been obtained with 3 experimental approaches. Scatchard analysis with [3H]etorphine reveals a monophasic high-affinity opiate binding site (Kd 2.3 nM) which is naloxone-sensitive. Immunocytochemical localization of Met- and Leu-enkephalin-like substances as well as alpha-MSH- and ACTH-like materials was demonstrated within specific populations of neurons. Substances with Met- and Leu-enkephalin and Met-enkephalin sulfoxide RIA reactivities were detected also in HPLC fractions corresponding to the retention times of authentic enkephalin standards. Together, the results provide strong evidence for the presence of enkephalinergic mechanisms in the nervous system of Lymnaea stagnalis. Additionally, the report provides indirect evidence for the existence of a macromolecular opioid precursor. This enkephalinergic system shows striking similarities to opioid mechanisms found in vertebrates and bespeaks a common evolutionary origin.     

Quantitation and localization of Met-enkephalin-Arg-Gly-Leu in rat brain using highly sensitive antibodies

Met-enkephalin-Arg-Gly-Leu is an endogenous opioid peptide recently identified in bovine adrenal medulla. In the present study, we describe the production of highly sensitive and specific antibodies against this octapeptide. The sensibility of the radioimmunoassay procedure allows us to quantify at the femtomole level, the Met-enkephalin-Arg-Gly-Leu in individual parts of the brain without prior purification or concentration. The antibodies are highly specific for the C terminal part of the molecule, and did not cross-react with the other opioid peptides. Immunochemical techniques were used also to determine the histological location of the immunoreactive substances in individual structures of the brain. In the present paper, the comparative regional distribution of Met-enkephalin-Arg-Gly-Leu and of Met-enkephalin in rat brain are described. Our results are in good agreement with the biosynthetic relationship between Met-enkephalin and Met-enkephalin-Arg-Gly-Leu.     

Internal anal sphincter relaxation associated with bisacodyl‐induced colonic high amplitude propagating contractions in children with constipation: a colo‐anal reflex?

Background Describe the association of internal anal sphincter (IAS) relaxation with colonic high‐ amplitude peristaltic contractions (HAPCs). Methods Retrospective review of colon manometry tracings of children with constipation to determine the IAS relaxation characteristics associated with HAPC’s (HAPC‐IASR) events and compare them to the those seen during the performance of the anorectal manometry (ARMRAIR) events. Key Results A total of 70 HAPC‐ IASRs were observed in 15 patients, 65 after bisacodyl, two during fasting and three after a meal. In 64% of events, the IAS relaxation started when the HAPC reached left colon and in 36% as proximal as the hepatic flexure. High‐ amplitude peristaltic contraction propagation seems to be important in HAPC‐IASR characteristics; those propagating distal to sigmoid colon demonstrated larger and longer IAS relaxation as well as lower residual pressure, but equivalent resting pressure compared with HAPC’s ending proximal to sigmoid colon. Although IAS resting pressure was comparable for ARM‐RAIRs and HAPC‐IASRs, the duration and magnitude of anal relaxation was higher, and the anal residual pressure was lower in HAPC‐IASRs. Conclusions & Inferences We demonstrated that IAS relaxation in constipated children is associated with HAPCs migrating in the proximal and distal colon; in most cases, starting when peristalsis is migrating through left colon and in an important proportion while migrating proximally. We also demonstrated that HAPC‐IASRs are different from ARM‐RAIRs suggesting a neurally mediated reflex. Finally, the IAS relaxation characteristics are highly dependent on the degree of propagation of HAPCs, which could have important implications in the understanding of defecation disorders.     

Release of immunoreactive met-enkephalin from the spinal cord by intraventricular β-endorphin but not morphine in anesthetized rats

Effect of β-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin_1–13 from the spinal cord was studied in anesthetized rats. Intraventricular β-endorphin, 16 μg, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 μg, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin_1–13 was not released by either β-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by β-endorphin had a retention time identical to [³H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for β-endorphin and morphine.