Involvement of delta-and mu-opioid receptors in the delayed cerebral ischemic tolerance induced by repeated electroacupuncture preconditioning in rats.

BACKGROUND: Preconditioning with repeated electroacupuncture (EA) could mimic ischemic preconditioning to induce cerebral ischemic tolerance in rats. The present study was designed to investigate whether mu (micro)-, delta (delta)- or kappa (kappa)-opioid receptors are involved in the neuroprotection induced by repeated EA preconditioning. METHODS: The rats were pretreated with naltrindole (NTI), nor-binaltorphimine (nor-BNI) or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), which is a highly selective delta-, kappa- or micro-opioid receptor antagonist respectively, before each EA preconditioning (30 minutes per day, 5 days). Twenty-four hours after the last EA treatment, the middle cerebral artery occlusion (MCAO) was induced for 120 minutes. The brain infarct volume was determined with 2, 3, 5-triphenyltetrazolium chloride staining at 24 hours after MCAO and compared with that in rats which only received EA preconditioning. In another experiment, the met-enkephalin-like immunoreactivity in rat brain was investigated by immunohistochemistry in both EA preconditioning and control rats. RESULTS: The EA preconditioning reduced brain infarct volume compared with the control rats (P = 0.000). Administration of both NTI and CTOP attenuated the brain infarct volume reduction induced by EA preconditioning, presenting with larger infarct volume than that in the EA preconditioning rats (P < 0.001). But nor-BNI administration did not block the infarct volume reduction induced by EA preconditioning, presenting with smaller infarct volume than the control group rats (P = 0.000). The number of met-enkephalin-like immunoreactivity positive neurons in the EA preconditioning rats was more than that of the control rats (P = 0.000). CONCLUSION: Repeated EA preconditioning stimulates the release of enkephalins, which may bind delta- and micro-opioid receptors to induce the tolerance against focal cerebral ischemia.

Beta-endorphin, Met-enkephalin and corresponding opioid receptors within synovium of patients with joint trauma, osteoarthritis and rheumatoid arthritis.

OBJECTIVE: Intra-articularly applied opioid agonists or antagonists modulate pain after knee surgery and in chronic arthritis. Therefore, the expression of beta-endorphin (END), Met-enkephalin (ENK), and mu and delta opioid receptors (ORs) within synovium of patients with joint trauma (JT), osteoarthritis (OA) and rheumatoid arthritis (RA) were examined. METHODS: Synovial samples were subjected to double immunohistochemical analysis of opioid peptides with immune cell markers, and of ORs with the neuronal markers calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH). RESULTS: END and ENK were expressed by macrophage-like (CD68(+)) and fibroblast-like (CD68(-)) cells within synovial lining layers of all disorders. In the sublining layers, END and ENK were mostly expressed by granulocytes in patients with JT, and by macrophages/monocytes, lymphocytes and plasma cells in those with OA and RA. Overall, END- and ENK-immunoreactive (IR) cells were more abundant in patients with RA than in those with OA and JT. ORs were found on nerve fibres and immune cells in all patients. OR-IR nerve fibres were significantly more abundant in patients with RA than in those with OA and JT. muORs and deltaORs were coexpressed with CGRP but not with TH. CONCLUSIONS: Parallel to the severity of inflammation, END and ENK in immune cells and their receptors on sensory nerve terminals are more abundant in patients with RA than in those with JT and OA. These findings are consistent with the notion that, with prolonged and enhanced inflammation, the immune and peripheral nervous systems upregulate sensory nerves expressing ORs and their ligands to counterbalance pain and inflammation.

Production and characterization of antibodies for the specific determination of the opioid peptide Met5-Enkephalin-Arg6-Phe7.

Endogenous opioids serve as modulators of neuroendocrine and immune system processes, the investigation of which calls for high-specificity radioimmunoassays (RIAs). This study focuses on the development and use of a specific radioimmunoassay for the opioid peptide Met5-Enkephalin-Arg6-Phe7 (MEAP), the C-terminus part of proenkephalin A. Antibodies were raised in four rabbits and investigated in terms of titre, avidity and specificity, followed by finding ideal conditions for these antibodies in RIA. MEAP concentrations were determined in crude extracts of rat hypothalamus, dorsal root ganglia, adrenals and ankle using this standardized assay after an oxidizing process. At reverse-phase high-pressure liquid chromatography (HPLC), the position of immunoreactive material from rat hypothalamus eluted as two peaks out of which one was compatible with that of synthetic MEAP. All rabbits exhibited individual differences in relative immune response and time of its onset. The avidity constant was 10 times higher on a molar basis for ab 4108 compared with ab 4182. There was no cross-reactivity for ab 4182 to related peptides, such as enkephalins and dynorphin B, and negligible background values for ab 4108. The relative levels ofimmunoreactive MEAP from the CNS versus peripheral tissues contrasted in accordance with current knowledge. It is suggested that reports with RIA results should include characterization of antibodies, extraction procedures, standard curves and compositions of buffers. Furthermore, the results should preferably be expressed in relation to total protein content.

Altered opioid-mediated control of the spinal release of dynorphin and met-enkephalin in polyarthritic rats.

Previous studies showed that spinal opioidergic neurotransmission is markedly altered in the polyarthritic rat, a model of chronic inflammatory pain. Present investigations aimed at assessing possible changes in opioid-mediated control of the spinal outflow of met-enkephalin (ME) and dynorphin (DYN) in these animals. Intrathecal (i.t.) perfusion under halothane anesthesia showed that polyarthritis was associated with both a 40% decrease in the spinal outflow of ME-like material (MELM) and a 90% increase in that of DYNLM. Local treatment with the mu-opioid agonist DAGO (10 microM i.t.) inhibited equally (-30%) the MELM outflow in polyarthritic and control rats, whereas the delta agonist DTLET (10 microM i.t.) also reduced the peptide outflow in controls (-27%) but enhanced it in polyarthritic animals (+56%). On the other hand, both DAGO (10 microM i.t.) and DTLET (10 microM i.t.) decreased (-40 and -49%) DYNLM outflow in polyarthritic rats, but were inactive in controls. Finally, neither MELM outflow nor that of DYNLM were affected by the kappa-agonist U50488H (10 microM i.t.) in both groups of rats. In all cases, the changes due to active agonists could be prevented by specific antagonists which were inactive on their own except the kappa antagonist nor-binaltorphimine (10 microM i.t.) that decreased (-38%) DYNLM outflow in polyarthritic rats. These data indicate that functional changes in spinal opioid receptors may promote enkephalinergic neurotransmission and reduce dynorphinergic neurotransmission in polyarthritic rats, thereby contributing to the analgesic efficacy of opioids in inflammatory pain.

Regional distribution of gonadotropin-releasing hormone-like, beta-endorphin-like, and methionine-enkephalin-like immunoreactivities in the central nervous system of the goat.

Regional distribution of gonadotropin-releasing hormone (GnRH)-like-, beta-endorphin (beta-end)-like-, and methionine-enkephalin (met-enk)-like-immunoreactivity was quantified across various regions of the central nervous system (CNS) of male and female goats by using highly specific radioimmunoassays. All the animals were sacrificed during the months of March through June (non-breeding season). Although the distribution of these three neuropeptides was similar to other mammalian species, species-specific gender differences in the levels of neuropeptides were noticed in the goat CNS. Highest levels of GnRH-like immunoreactivities were found in the hypothalamus. The hypothalamus of male goats exhibited significantly higher levels of GnRH-like immunoreactivities compared to female goats. Other regions exhibiting GnRH-like immunoreactivities included olfactory bulbs, preoptic and supraoptic regions, and mamillary bodies. Both beta-end- and met-enk immunoreactivities were detected in all selected regions of goat CNS, but highest levels of these opioid peptide-like immunoreactivities were limited to the forebrain regions of the goat. The supraoptic area of the female goats contained significantly higher levels of beta-end-like immunoreactivities than that of the male goats. Met-enk-peptide-like immunoreactivity also exhibited gender-specific differences in its content in some regions of the CNS. The male goats exhibited significantly higher levels of met-enk-like immunoreactivity in both the striatal and hypothalamic regions of the brain.

Immunocytochemical characterization of quisqualic acid- and N-methyl-D-aspartate-induced excitotoxicity in the retina of chicks.

A single, large dose of N-methyl-D-aspartate (NMDA) or quisqualic acid (QA) injected into the chick eye has been shown previously to destroy many retinal amacrine cells and to induce excessive ocular growth accompanied by myopia. The purpose of this study was to identify distinct populations of retinal cells, particularly those believed to be involved in regulating ocular growth, that are sensitive to NMDA or QA. Two pmol of NMDA or 0.2 micromol of QA were injected unilaterally into eyes of 7-day-old chicks, and retinas were prepared for observation 1, 3, or 7 days later. Retinal neurons were identified by using immunocytochemistry, and cells containing fragmented DNA were identified by 3'-nick-end labelling in frozen sections. NMDA and QA destroyed many amacrine cells, including those immunoreactive for vasoactive intestinal polypeptide, Met-enkephalin, and choline acetyltransferase, but they had little effect upon tyrosine hydroxylase-immunoreactive cells. Other cells affected by both QA and NMDA included those immunoreactive for glutamic acid decarboxylase, gamma-aminobutyric acid, parvalbumin, serotonin, and aminohydroxy methylisoxazole propionic acid (AMPA) receptor subunits GluR1 and GluR2/3. Cells largely unaffected by QA or NMDA included bipolar cells immunoreactive for protein kinase C (alpha and beta isoforms) and amacrine cells immunoreactive for glucagon. DNA fragmentation was detected maximally in many amacrine cells and in some bipolar cells 1 day after exposure to QA or NMDA. We propose that excitotoxicity caused by QA and NMDA induces apoptosis in specific populations of amacrine cells and that these actions are responsible for the ocular growth-specific effects of QA and NMDA reported elsewhere.

Determination of peptides by high-performance liquid chromatography with laser-induced fluorescence detection.

Peptides were determined by high-performance liquid chromatography (HPLC) with laser-induced fluorescence detection. Detection was based on pre-column fluorescence derivatization of peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in acetonitrile (MeCN)-0.1 M borate buffer (pH 8.0) at 40 degrees C for 10 min. The peptide derivatives were separated on a reversed-phase column with trifluoroacetic acid-MeCN and determined fluorometrically at 530 nm with excitation at 470 nm. The method was applied to the determination of enkephalins in rat brain and to a degradation study of bradykinin in human plasma. Optimization of the reaction conditions and the use of a semi-micro-column (100 x 2 mm I.D., 2 microns) made the detection limit of the peptides as low as 5-10 fmol. The detection limits of enkephalin and bradykinin were 20 and 5 fmol, respectively, using HPLC with laser-induced fluorescence detection. The method was sensitive enough to permit the quantitative determination of opioid peptides and bradykinin in tissue and plasma samples.

Estradiol alters ethanol-induced effects on beta-endorphin and met-enkephalin levels in specific brain regions of ovariectomized rats.

The present investigation was conducted to evaluate the effects of estradiol on ethanol-induced alterations of beta-endorphin (beta-EN) and met-enkephalin (ME) levels in specific brain regions of ovariectomized rats. Female Sprague-Dawley rats (100-124 g) adapted to a 12-hour light, 12-hour dark illumination cycle were used in these studies. Animals were ovariectomized under pentobarbital anesthesia. After a recovery period of 14 days, ethanol (3 g/kg as 22.5% solution in saline, i.p.), estradiol (50 micrograms/kg in 0.2 ml of olive oil, s.c. in the dorsal neck region), or a combination of ethanol and estradiol were administered to rats at 11:00 h. Control animals were injected intraperitoneally with 2 ml saline and subcutaneously with 0.2 ml olive oil. Animals were sacrificed by decapitation 2 h later. The brains were immediately removed; the cortex, hippocampus, hypothalamus, and midbrain were dissected and their beta-EN and ME levels were measured by radioimmunoassay. Ethanol administration significantly decreased both beta-EN and ME levels in the hypothalamus. Administration of estradiol alone also resulted in a significant decrease in beta-EN and ME levels in the hypothalamus. Additionally, concurrent administration of ethanol and estradiol showed a decrease in the levels of beta-EN and ME in the hypothalamus. Co-administration of ethanol with estradiol also caused a significant decrease in the levels of beta-EN in midbrain. However, ME levels were increased in the midbrain after concurrent administration of estradiol and ethanol. ME levels also increased in the hippocampus and cortex after co-administration of estrogen and ethanol. These results clearly indicate that estradiol significantly alters ethanol-induced effects on beta-EN and ME levels in specific brain regions of ovariectomized rats. The present findings may in part explain sex differences in alcohol effects.

The distribution of Met-enkephalin like immunoreactivity in the brain of Apteronotus leptorhynchus, with emphasis on the electrosensory system.

The distribution of Met-enkephalin-like immunoreactivity in the brain of the electric fish, Apteronotus leptorhynchus was analysed by immunohistochemistry. The majority of Met-enkephalin immunoreactive neurons were found in the hypothalamus. Dense Met-enkephalin immunoreactive fiber plexuses were seen in the hypothalamus and ventral forebrain. In the dorsal telencephalon an olfacto-recipient region (ventral subdivision of the dorsolateral forebrain) was specifically and densely innervated. Regions of the brain known to be involved in electrocommunication also received a substantial innervation by Met-enkephalin-like immunoreactive fibers. This distribution of immunoreactive fibers in the brain of this gymnotiform fish indicates that Met-enkephalin may be generally involved in the regulation of sensory, neuroendocrine and reproductive functions and specifically in the regulation of electrocommunication.

Immunocytochemical evidence for the presence of Met-enkephalin and Leu-enkephalin in 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 alpha-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 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.

Direct and indirect enkephalinergic synaptic inputs to the rat arcuate nucleus studied by combination of retrograde tracing and immunocytochemistry.

The origin of both direct and indirect enkephalinergic innervation potentially able to influence neurons of the rat arcuate nucleus has been investigated by combining enkephalin immunocytochemistry and retrograde axonal transport of a wheatgerm agglutinin-Apo horseradish peroxidase-gold complex. Twenty four hours after tissue injections of small volumes (20 nl) of the tracer into the arcuate nucleus, rats were treated with colchicine and killed. In order to localize the enkephalinergic cells which directly innervate the arcuate nucleus, Vibratome sections were first silver-stained for detection of the wheatgerm agglutinin-Apohorseradish peroxidase-gold complex and then processed for enkephalin immunohistochemistry. To study the indirect enkephalinergic input to the arcuate nucleus, an electron microscope detection of immunoreactive synapses was carried out in areas rich in retrogradely labeled perikarya. Perikarya both immunoreactive and retrogradely labeled were observed ipsilaterally to the injection site in telencephalic structures such as the bed nucleus of the stria terminalis, medial preoptic and adjacent periventricular areas. Hypothalamic ipsilateral doubly labeled cells were localized principally in the dorsomedial nucleus and rostral arcuate nucleus. The major direct inputs arising from brainstem structures concerns the dorsal and ventral parabrachial nuclei. Moreover, at the ultrastructural level, numerous enkephalinergic terminals were demonstrated to synapse with retrogradely labeled perikarya and dendrites localized in the medial preoptic area, the hypothalamic paraventricular nucleus and the parabrachial nuclei providing evidence for an important enkephalinergic input on neurons projecting to the arcuate nucleus. Taken together, our light and electron microscope studies strongly suggest that the arcuate nucleus is the target of an enkephalinergic control originating from several regions and acting either directly or indirectly on neurons projecting to the arcuate nucleus.

Combination of immunocytochemistry and in situ hybridization in the same semi-thin sections: detection of Met-enkephalin and pro-enkephalin mRNA in the hypothalamic magnocellular dorsal nucleus of the guinea pig.

We describe a procedure for combining pre-embedding peroxidase immunocytochemistry with pre-embedding autoradiographic in situ hybridization in the same vibratome sections of paraformaldehyde-fixed brain tissue. The simultaneous detection of Met-enkephalin (Met-enk)-immunoreactive product and pro-enkephalin (PE) mRNA in neurons of the magnocellular dorsal nucleus (MDN) in the guinea pig hypothalamus was carried out as a model for this procedure. Vibratome slices were processed for Met-enk immunodetection followed by the incubation with a 45-base synthetic oligonucleotide complementary to PE mRNA labeled with 35S. Tissues were embedded in araldite, cut into semi-thin sections, and processed for autoradiography. Many neurons double labeled for Met-enk and PE mRNA were viewed in the MDN. The histological quality and the spatial resolution of both signals were optimized, since precise intracellular localization of hybridization sites was possible. This method allows simultaneous study of peptide immunoreactivity and mRNA expression levels in neurons within the same semi-thin sections. It may be useful for a variety of quantitative analyses, and might also be extended to ultrastructural analysis.

The regional distribution of thyrotropin releasing hormone, leu-enkephalin, met-enkephalin, substance P, somatostatin and cholecystokinin in the rat brain and pituitary.

There was no apparent difference in the regional distribution of neuropeptides in the brain of male and female rats. The highest levels of immunoreactive leu-enkephalin, TRH, substance P and somatostatin were found in the hypothalamus, while the striatum and the cerebral cortex had the highest concentrations of met-enkephalin and cholecystokinin respectively. The lowest concentrations of these were found in the cerebellum. Enkephalins (cerebral cortex), substance P (cerebral cortex and brain stem), and somatostatin (brain stem and striatum) showed higher level in the female while enkephalin and substance P contents in the anterior pituitary were higher in the male.

Effect of dietary energy, body condition and calf removal on pituitary gonadotropins, gonadotropin-releasing hormone (GnRH) and hypothalamic opioids in beef cows.

Beef cows (n = 64) were slaughtered to evaluate effects of dietary energy and calf removal (CR) on hypothalamic and adenohypophysial endocrine characteristics. From d 190 of gestation until parturition, cows received maintenance (ME; n = 32) or low (LE; n = 32) energy diets (ME = 100%, LE = 70% NRC recommendations). After parturition, half (n = 16) of each prepartum diet group received low (LE; n = 32) or high (HE = 130% NRC; n = 32) energy diets. At 30 d postpartum, cows were slaughtered 0 or 48 hr after CR. Hypothalami [preoptic area (POA), hypothalamus (HYP), stalk-median eminence (SME)] and pituitaries were collected. Basal and K(+)-induced release of GnRH from SME, and pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) did not differ among groups (P greater than .05). Hypophyseal LH was correlated (P less than .01) with body condition score (BCS) at parturition and slaughter (r = .36 and .47, respectively). Prepartum LE diet increased (P less than .05) met-enkephalin in POA compared to prepartum ME (.59 +/- .05 vs. .44 +/- .04 pmol/mg) regardless of postpartum diet or suckling status. Concentrations of beta-endorphin in combined HYP + POA were decreased (P less than .05) by 48 hr CR (15.1 +/- 1.1 vs. 18.1 +/- 0.7 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

The immunostaining for the hypothalamic vasoactive intestinal peptide, but not for beta-endorphin, dynorphin-A or methionine-enkephalin, is affected by the glucocorticoid milieu in the rat: correlation with the prolactin secretion.

Effects of the glucocorticoid milieu on the basal and ether stress-induced prolactin (PRL) release and on the immunostaining for hypothalamic vasoactive intestinal peptide (VIP), beta-endorphin (beta-EP), dynorphin-A (DYN-A) and methionine-enkephalin (Met-ENK), were examined in separate groups of male rats. After colchicine treatment in intact rats, VIP-containing cell bodies were observed only in the suprachiasmatic nucleus (SCN). Adrenalectomy (ADX), performed 7 days previously, resulted in the additional appearance of VIP-immunoreactive neurons in the parvocellular subdivision of the paraventricular nucleus (PVN), as well as in significantly higher basal and stressed PRL levels than intact values. Treatment of intact rats with a high dose (500 micrograms/kg body weight (s.c.) daily for 7 days) of dexamethasone (DEX), but not with a low dose (50 micrograms/kg) of DEX, significantly reduced both the basal and stressed PRL release. Administration of either the low or high dose of DEX to ADX rats prevented the appearance of the PVN-VIP neurons. In addition, the ADX-induced high basal and stressed PRL levels were restored to intact values by the low dose of DEX, and completely suppressed by the high dose of DEX. The staining of SCN-VIP-, beta-EP-, DYN-A or Met-ENK neurons was not affected by any treatment employed in this study. These results suggest that the appearance of PVN-VIP immunostaining in ADX rats may, at least in part, be responsible for the enhanced PRL secretion observed in this group. However, SCN-VIP-, beta-EP-, DYN-A- or Met-ENK neurons do not seem to play a pivotal role in the glucocorticoid regulation of PRL secretion.

Antineoplastic properties of Maharishi-4 against DMBA-induced mammary tumors in rats.

Maharishi-4 (M-4), an ayurvedic food supplement, was tested for anticarcinogenic and anticancer properties against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. The 6% M-4-supplemented diet protected DMBA-induced carcinogenesis by reducing both tumor incidence and multiplicity during initiation and promotion phases. The control animals who developed tumors when supplemented with M-4 diet for four weeks showed tumor regression in 60% of cases. There was no significant difference in the food intake or weight gain in rats who were on M-4-supplemented diet compared to control group. Possible mechanisms of action of M-4 are discussed.

Measurement of methionine enkephalin and leucine enkephalin in rat brain regions by high-performance liquid chromatography with coulometric electrochemical detection.

A method is described for the determination of two pentapeptides, methionine enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) (ME) and leucine enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) (LE) in discrete rat brain regions. Separation and quantitation were performed by reversed-phase high-performance liquid chromatography with coulometric detection. Perchloric acid extracts of the tissue after enzyme inactivation by heat treatment were passed through a normal-phase solid-phase extraction diol (COHCOH) column, and endogenous ME and LE were subsequently eluted with methanol. The mobile phase was 1-propanol-phosphate buffer (pH 5.5) (9:91). Eluted samples were detected electrochemically using dual coulometric electrodes operated in screen mode. Each of these enkephalins gave a linear response over the range 40-160 ng/ml cerebellar homogenate (0.8-3.2 ng absolute amount on column). Analytical recoveries of synthetic ME and LE, added to the homogenates, were 70 +/- 3 and 70 +/- 10%, respectively, when compared with enkephalins dissolved in water. The mean between-assay coefficients of variation for synthetic ME and LE were lower than 10.7 and 7.4%, respectively, over the concentration range studied. The within-assay coefficients of variation for synthetic ME and LE were 11.4 and 9.5%, respectively, at the lowest concentration. The present method has been applied to a study determining the levels of endogenous ME and LE in discrete rat brain regions.

Changes in met-enkephalin and beta-endorphin contents in the hypothalamus and the pituitary in diabetic rats: effects of insulin therapy.

1. Immunoreactive (IR)-met-enkephalin and beta-endorphin contents in the hypothalamus and the pituitary were measured in alloxan-diabetic rats with or without insulin treatment. 2. Both IR-met-enkephalin and IR-beta-endorphin in the pituitary were substantially reduced in alloxan-diabetic rats 1 month after treatment. 3. Hypothalamic IR-beta-endorphin content was also significantly lower. 4. Gel-filtration chromatography showed that the peaks co-eluting with met-enkephalin precursor, met-enkephalin and beta-endorphin were lower in the pituitaries from the diabetic rats, whereas the peaks co-eluting with beta-endorphin precursor and beta-lipotropin were not. 5. In another experiment, the IR-beta-endorphin contents of the neuro-intermediate lobe and hypothalamus, but not the anterior lobe were significantly lowered in diabetic rats, whereas IR-met-enkephalin contents were significantly reduced in both the anterior and neuro-intermediate lobe. 6. All these changes were reversed by insulin treatment. 7. As a decrease in general protein synthesis could not explain the recorded changes, these results suggest a possible direct role of insulin in regulating the opioid peptide content of the hypothalamus and pituitary.

Characterization of metorphamide-like immunoreactivity in the zona incerta and lateral hypothalamus: co-localization with alpha-melanocyte-stimulating hormone-like immunoreactivity.

Double-staining in either vibratome or paraffin sections using contrasting chromogens revealed an alpha-melanocyte-stimulating hormone (alpha-MSH)-containing cell group in the arcuate nucleus, a metorphamide-containing cell group in the paraventricular hypothalamus, and an extensive group of magnocellular perikarya in the zona incerta (ZI) and the lateral hypothalamus (LH) that appeared to contain both antigens. Staining of adjacent paraffin sections also suggested that most (and perhaps all) of the magnocellular perikarya in the ZI and LH that contained metorphamide-like immunoreactivity also contained alpha-MSH-like immunoreactivity. Metorphamide-like immunoreactivity in the ZI and the LH was abolished by absorption of the antiserum with metorphamide but was unaffected by absorption with alpha-MSH. alpha-MSH-like immunoreactivity in the ZI and LH was abolished by absorption of the antiserum with alpha-MSH but was unaffected by absorption with metorphamide. Antisera directed against [Met5]-enkephalin (Met-ENK), [Met5]-enkephalin-Arg6,Gly7,Leu8 (ENK-8), [Met5]-enkephalin-Arg6,Phe7 (ENK-7), neuropeptide Y, and FMRF-amide did not stain magnocellular perikarya in the ZI and LH. Pretreatment of paraffin sections with trypsin resulted in the appearance of [Met5]-enkephalin-Arg6-like immunoreactivity in the ZI and LH. Pretreatment of paraffin sections with trypsin did not reveal any occult Met-ENK-, ENK-7- or ENK-8-like immunoreactivity in either the ZI or the LH. These observations indicate that magnocellular neurons in the ZI and LH contain both a metorphamide-like and an alpha-MSH-like peptide but do not express either the preproenkephalin or the prepro-opiomelanocortin48 gene.