The immunomodulation mediated by a delta-opioid receptor for [Met(5)]-enkephalin in oyster Crassostrea gigas.

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met(5)]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met(5)]-enkephalin, the concentration of second messengers Ca(2+) and cAMP in the HEK293T cells decreased significantly (p <0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p <0.05) at 6 h, and reached the highest level (5.00-fold, p <0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met(5)]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met(5)]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca(2+) and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster.

[Anti-inflammatory and synovial-opioid system effects of electroacupuncture intervention on chronic pain in arthritic rats].

OBJECTIVE: To observe the analgesic effect of electroacupuncture (EA) on collagen-induced arthritis (CIA) rats and its regulating effect on inflammation reaction and the endogenous opioid system of synovial tissues. Methods A total of 30 healthy male Wistar rats were randomly divided into a control group, a model group and an EA group, 10 rats in each one. The chronic pain model of CIA rats was made by cattle type-II collagen in the model group and EA group. Rats in the EA group were treated with EA at "Zusanli" (ST 36) and "Kunlun" (BL 60) for 30 min from 16th day after model establishment, once a day for consecutive 10 days. Rats in the control group did not receive any treatment. Rats in the model group were treated with fixation as the EA group. Threshold of pain, arthritis index, paw swelling were measured before model establishment and 16 d, 20 d, 23 d and 25 d after model establishment. The levels of beta-endorphin (ß-END), met-enkephalin (met-ENK), dynorphin A (Dyn A) were measured by radioimmunoassay; the mRNA expressions of mu opioid receptor (MOR), kappa opioid receptor (KOR) and delta opioid receptor (DOR) in synovial tissues of CIA rats were detected by I quantitative polymerase chain reaction (qPCR). RESULTS: Compared with the control group, threshold of pain was reduced (all P<0. 01), arthritis index was increased (all P<0. 01) and paw swelling was increased (all P<0. 01) in the model group on the 16th day, 20th day, 23rd day, 25th day after model establishment. Compared with the model group, the threshold of pain was increased in the EA group (all P<0. 01), arthritis index and paw swelling were reduced (all P<0. 01) on the 23rd day and 25th day after model establishment. Compared with the control group, the level of Dyn A in synovial tissues of CIA rats was increased in the model group (P<0. 01); the mRNA expressions of MOR, KOR and DOR were down-regulated lower than 0. 5 fold of normal level. Compared with the model group, the level of ß-END in synovial tissues of the knee joint was increased in the EA group (P<0. 05), and the mRNA expressions of MOR, KOR and DOR in synovial tissues of CIA rats were up-regulated more than 2 folds of normal level. CONCLUSION: The intervention of EA on chronic pain of CIA rats is superior, which is likely to be related with effects of EA on anti-inflammation and up-regulation of synovial tissue ß-END and MOR, KOR, DOR.

Immune modulation in response to stress and relaxation.

Traditional medical science has kept the mind separate from the body. Recently people realize the effect of mind on health and psychoneuroimmunology is the new evolved science that describes the interactions between psyche and soma. In this review through a typical psycho-neuro-endocrino-immune network the effects of psychological stress (acute, brief naturalistic and chronic) and relaxation on immune modulation has been shown. From this network Corticotrophin Releasing Factor (CRF), Adrenocorticotrophic Hormone (ACTH), Glucocorticoids (GC), alpha-endorphin and Met-enkephalin are found as important endocrine components and T cells, B cells, monocytes/macrophages, Natural Killer (NK) cells and their cytokines that is Tumor Necrosis Factor-alpha (TNF-alpha), Interferon Gamma (IFN-alpha) and interleukins such as IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 etc. are found as important immune components. Finally, it has been shown that, acute, brief naturalistic and chronic stress have different immune modulatory activities which are harmful to one's homeostasis and relaxation can help to maintain that homeostasis.

Lymphocytic ppENKmRNA, MEK-IR, and Dyn-IR in electroacupuncture.

We studied the effect of acupuncture analgesia on the expression of ppENKmRNA, MEK-IR, and Dyn-IR in circulating mouse lymphocytes. Electroacupuncture stimulated cell immunity. The release of irDyn during electrostimulation at 5 Hz frequency was less active than irMEK release.

Modulation of delayed-type hypersensitivity responses in hairless guinea pigs by peptides derived from enkephalin.

Although opioid peptides such as methionine (met)-enkephalin have been previously shown to enhance or suppress immune responses, few studies in animal models have addressed the immunomodulatory activity of their metabolic derivatives. Hairless (IAF/HA-HO) guinea pigs immunized with Freund's complete adjuvant containing Mycobacterium tuberculosis and repeatedly skin tested with purified protein derivative of tuberculin (PPD) display high levels of stable delayed-type hypersensitivity (DTH) to PPD. Met-enkephalin (YGGFM) and two of its metabolites (YGG, YG) enhanced and accelerated PPD-elicited DTH inflammatory reactions when injected together with elicitor in these animals. At 24 h, 5 x 10(-3) pmol met-enkephalin significantly enhanced DTH responses by 30% over PPD alone, while 5 x 10(-5) pmol of YGG and 5 x 10(-9) pmol of YG significantly enhanced these responses by 62 and 32%, respectively. At much higher doses (5 x 10(3) pmol), met-enkephalin and its metabolites significantly suppressed DTH reactions by 25-32%. Tyrosine and glycine had no effect on PPD-elicited DTH. All DTH reactions (control, enhanced, suppressed) displayed typical perivascular mononuclear cell infiltrates. We conclude that the immunoactivity of met-enkephalin resides in its first two amino acids and suggest that cleavage of enkephalin molecules to YG occurs in serum and/or on the cell surface.

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.

[Effect of anti-opioid peptide sera on the enhancement of electroacupuncture analgesia induced by neurotensin in PAG of rats].

With the use of potassium iontophoresis induced tail-flick for measuring the pain threshold, the effects of injecting neurotensin (NT), naloxone (NX), anti-metenkephalin serum (AMEKS), anti-beta-endorphin serum (AEPS) and anti-dynorphin A1-13 serum (ADYNS) into periaqueductal gray (PAG) on electroacupuncture (EA) analgesia in rats were investigated. NT administration enhanced EA analgesia remarkably. Pre-injection of NX, AMEKS and AEPS into PAG could significantly attenuate the enhancement of EA analgesia induced by NT, but not by administration of ADYNS. The results indicate that NT in PAG is responsible for the enhancement of EA analgesia. The effect of NT may be partly mediated by met-enkephalin and beta-endorphin.

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.

Effects of hypnosis on plasma proenkephalin peptide F and perceptual and cardiovascular responses during submaximal exercise.

Little information is available concerning the influence of subconscious mechanisms on neuroendocrine function, more specifically, proenkephalin peptide F release. Ten men [5 middle distance runners (21.6 (SD 0.54 years) and 5 untrained men (24.0 (SD 4.3 years)] consented to be volunteers in this investigation. Submaximal exercise intensities of 25% and 50% of peak oxygen consumption (VO2) (8 min stages) were used for both the control and hypnosis treatments. A traditional hypnotic induction was used, with the suggestion of two higher intensities of exercise stress (50% and 75% peak VO2) previously experienced in familiarization and testing by each subject. Each minute oxygen consumption was measured using open circuit spirometry, heart rate via an ECG, and ratings of perceived exertion (RPE) using the Borg scale. Plasma peptide F immunoreactivity (ir) [preproenkephalin-(107-140)] in blood sampled from an indwelling cannula was measured by radioimmunoassay at 7-8 min of each stage of the exercise test. Expected significant increases were observed for all cardiorespiratory and perceptual variables over the increasing exercise intensities and there were no significant differences between trained and untrained groups for peptide F if response patterns. Hypnosis did not significantly affect peptide F ir concentrations (P > 0.05) and did not significantly alter exercise heart rate, RPE or minute ventilation (P > 0.05). However, hypnosis did significantly increase oxygen consumption during exercise (P = 0.0095) but not of the magnitude needed for the metabolic demands of the higher exercise intensities. Thus, traditional hypnosis was unable to make functionally significant changes in the cardiorespiratory variables.(ABSTRACT TRUNCATED AT 250 WORDS)

SCPB-and FMRFamide-like immunoreactivities in lobster neurons: colocalization of distinct peptides or colabeling of the same peptide(s)?

Virtually all of the SCPB-like immunoreactive neurons (ca. 60 cells) in the lobster Homarus americanus also contain FMRFamide-like immunoreactivity. Control experiments reveal that SCPB-and FMRFamide-like immunoreactivities are successfully preadsorbed with their specific antigens, while the normal staining pattern is retained following preadsorption of each antibody with the alternate peptide. These experiments potentially lead to the conclusion that the anti-SCPB and anti-FMRFamide antibodies are labeling distinct compounds that are colocalized in lobster neurons. The lobster nervous system does not, however, contain authentic FMRFamide, but rather several FMRFamide-like compounds (Trimmer et al., J. Comp. Neurol. 266:16-26, 1987). The most abundant of these is the octapeptide TNRNFLRFamide. Experiments demonstrate that SCPB-like immunoreactivity is completely preadsorbed with synthetic TNRNFLRFamide, while there is a significant or complete loss of staining after preadsorption of the FMRFamide antibody with this molecule. Met-enkephalin-Arg-Phe-amide (YGGFMRFamide), an extended opioid peptide containing the FMRFamide sequence, also preadsorbs SCPB- and FMRFamide-like immunoreactivities, while Met-enkephalin-Arg-Phe (YGGFMRF) has no effect on the staining properties of these antibodies. These results suggest that the SCPB antibody can bind to extended forms of FMRFamide-like molecules, and that anti-SCPB and anti-FMRFamide antibodies may be colabeling one or more FMRFamide-like molecules in lobster neurons.

GABA axon terminals in synaptic contact with enkephalin neurons in the hypothalamus of the guinea pig. Demonstration by double immunocytochemistry.

By using a method combining pre-embedding immunoperoxidase staining for enkephalin and postembedding immunocolloidal gold labeling for gamma-aminobutyric acid (GABA) it has been demonstrate that many GABAergic boutons made synapses on enkephalin-reacting soma in the magnocellular dorsal nucleus of the guinea pig hypothalamus. The gold particles revealing the presence of GABA were essentially located over the small clear vesicles and mitochondria present in these GABAergic nerve endings. All the synapses observed were symmetrical. Taking into account the great number of these nerve endings, we conclude for a strong regulatory role of GABA on enkephalin-containing cells of the magnocellular dorsal nucleus.

Immunohistochemical distribution of cholecystokinin, dynorphin A and Met-enkephalin neurons in sheep hypothalamus.

The distribution of cholecystokinin (CCK)-, Met-enkephalin (M-ENK)- and dynorphin (DYN)-like immunoreactive perikarya were examined in the sheep hypothalamus using the peroxidase-anti-peroxidase technique. CCK- and DYN-containing neurons were found primarily in the suprachiasmatic nucleus (SCH) and supraoptic nucleus (SO). No CCK- or DYN-containing neurons were found in the paraventricular nucleus (PVN). M-ENK-containing neurons were found mainly in the PVN of the hypothalamus. In addition, M-ENK neurons were found in the dorsomedial (DMH), lateral (LH), anterior (AH) and periventricular hypothalamic areas. The distribution of these neuropeptides may provide a basis for understanding differences in responsiveness to centrally administered peptides.

Electroacupuncture and morphine analgesia potentiated by bestatin and thiorphan administered to the nucleus accumbens of the rabbit.

The endogenous opioid peptide enkephalin (EK) is known to be degraded mainly by two enzymes, the dipeptidyl carboxypeptidase 'enkephalinase' and aminopeptidase. Microinjection of the enkephalinase inhibitor thiorphan or the aminopeptidase inhibitor bestatin into the nucleus accumbens of the rabbit produced a dose-dependent analgesic effect. This analgesic effect was totally reversed by the narcotic antagonist naloxone or by antibodies against [Met5]enkephalin (MEK) administered to the same site. Antibodies against [Leu5]enkephalin were not effective. Moreover, microinjection of thiorphan or bestatin into the nucleus accumbens resulted in a marked potentiation of the aftereffect of electroacupuncture (EA) produced analgesia, as well as the analgesia induced by a small dose of morphine. It is concluded that the analgesic effect elicited by EA and morphine is mediated, at least in part, by MEK-like immunoreactive substance(s) in the nucleus accumbens.

Acupuncture mechanisms in rabbits studied with microinjection of antibodies against beta-endorphin, enkephalin and substance P.

Injection of protein-A purified antibodies against Met-enkephalin and beta-endorphin into the periaqueductal gray matter (PAG) was shown to decrease the analgesic effect of electroacupuncture (EA) in rabbits. Met-enkephalin antibodies were more potent than the beta-endorphin antibodies in causing a statistically-significant effect on electroacupuncture analgesia. Antibodies to Met-enkephalin were also active at the spinal level, whereas antibodies against beta-endorphin were without effect: this is in agreement with a rich enkephalinergic innervation and absence of beta-endorphin-containing fibres in the spinal cord. Substance P, the other neuropeptide of this study, also seems to be important in mediating effects of electroacupuncture. Injection of antibodies into the periaqueductal gray caused decrease of the effect of electroacupuncture whereas intrathecal administration of Fab-fragment substance P antibodies caused a marked potentiation. The demonstration of site specificity of the neuropeptides in mediating analgesia induced by electroacupuncture supports the validity of this experimental approach.