[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.

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.

Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK).

The aim of this study was to investigate the immunological mechanisms by which synthetic methionine enkephalin (MENK) exerts therapeutic effects on tumor growth. Our findings in vivo or in vitro show that MENK treatment either in vivo or in vitro could up-regulate the percentages of CD8+T cells, induce markers of activated T cells, increased cytotoxic activity against mouse S180 tumor cells and increase secretion of IFNγ. In addition, the adoptively transferred CD8+T cells, after either in vitro or in vivo treatment with MENK, result in significantly increased survival of S180 tumor-bearing mice and significant shrinkage in tumor growth. Opioid receptors are detected on normal CD8+T cells and exposure to MENK leads to increased expression of opioid receptors. Interaction between MENK and the opioid receptors on CD8+T cells appears to be essential for the activation of CTL, since the addition of naltrexone (NTX), an opioid receptor antagonist, significantly inhibits all of the effects of MENK. The evidence obtained indicates that the MENK-induced T cell signaling is associated with a significant up-regulation of Ca2+ influx into the cytoplasm and the translocation of NFAT2 into nucleus, and these signaling effects are also inhibited by naltrexone.

Pain inhibition by blocking leukocytic and neuronal opioid peptidases in peripheral inflamed tissue.

Inflammatory pain can be controlled by endogenous opioid peptides. Here we blocked the degradation of opioids in peripheral injured tissue to locally augment this physiological system. In rats with hindpaw inflammation, inhibitors of aminopeptidase N (APN; bestatin) or neutral endopeptidase (NEP; thiorphan), and a dual inhibitor, NH(2)-CH-Ph-P(O)(OH)CH(2)-CH-CH(2)Ph(p-Ph)-CONH-CH-CH(3)-COOH (P8B), were applied to injured paws. Combined bestatin (1.25-5 mg)/thiorphan (0.2-0.8 mg) or P8B (0.0625-1 mg) alone elevated mechanical nociceptive thresholds to 307 and 227% of vehicle-treated controls, respectively. This analgesia was abolished by antibodies to methionine-enkephalin, leucine-enkephalin, and dynorphin A 1-17, by peripherally restricted and by selective µ-, δ-, and κ-opioid receptor antagonists. Flow cytometry and photospectrometry revealed expression and metabolic activity of APN and NEP on macrophages, granulocytes, and sciatic nerves from inflamed tissue. Radioimmunoassays showed that inhibition of leukocytic APN and NEP by bestatin (5-500 µM)/thiorphan (1-100 µM) combinations or by P8B (1-100 µM) prevented the degradation of enkephalins. Blockade of neuronal peptidases by bestatin (0.5-10 mM)/thiorphan (0.1-5 mM) or by P8B (0.1-10 mM) additionally hindered dynorphin A 1-17 catabolism. Thus, leukocytes and peripheral nerves are important sources of APN and NEP in inflamed tissue, and their blockade promotes peripheral opioid analgesia.

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.

B lymphocyte proliferation is suppressed by the opioid growth factor-opioid growth factor receptor axis: Implication for the treatment of autoimmune diseases.

Endogenous opioids are known to repress the incidence and progression of autoimmune diseases. One native opioid peptide, [Met5]-enkephalin, termed the opioid gowth factor (OGF), interacts with the OGF receptor (OGFr) to suppress the expression of experimental autoimmune encephalomyelitis. The present study examined the role of the OGF-OGFr axis in the regulation of B lymphocyte proliferation. Murine B lymphocytes were stimulated with lipopolysaccharide. Both OGF and OGFr were present in all B lymphocytes. OGF had a dose-dependent effect on growth, with cell number inhibited by up to 43% at 72 h; no other synthetic or native opioid altered cell proliferation. Exogenous OGF depressed cell number in cultures treated with siRNAs for the classical opioid receptors, MOR (µ), DOR (δ), and KOR (κ), however this peptide had no effect in preparations exposed to siRNA for OGFr. The decrease in cell number by exogenous OGF was dependent on p16 or p21 cyclin-dependent inhibitory kinase pathways. Exposure to the opioid antagonist, naltrexone, did not change cell number from control levels. These results suggest that the OGF-OGFr axis is present and functional in B lymphocytes, but this system is not an autocrine regulator of cell proliferation. Thus, at least exogenous OGF and perhaps endogenous OGF by paracrine/endocrine sources, can be an immunosuppressant. Modulation of the OGF-OGFr axis may be a novel paradigm for the treatment of autoimmune diseases.

[Distribution of met-enkephalin expression in the visual pathway. Experimental study by inmunocytochemistry]. / Distribución de la metencefalina en la vía óptica. Estudio experimental inmunocitoquímico.

PURPOSE: The localization and distribution of neuropeptide expression in the cat visual pathway can provide information about the function of that pathway. METHOD: Study of optic pathway in eight cats. Following extraction of the brain, slices were prepared using a microkeratome. The slices were examined by indirect immunocytochemistry using anti-metenkephalin as antibody to determine the presence or absence of this pentapeptide in the visual pathway. RESULTS: Met-enkephalin receptors in both cortical and subcortical regions of the brain were detected. This suggests that met-enkephalin could be involved in the visual mechanism. CONCLUSIONS: The presence of met-enkephalin receptors in both cortical and subcortical regions of the brain suggests that this pentapeptide could be involved in the visual mechanism.

Methionine-enkephalin secreted by human colorectal cancer cells suppresses T lymphocytes.

The role of methionine-enkephalin (MENK) as an immunomodulator in colorectal carcinomas (CRC) was examined. MENK was produced in CT26, IEC6A, Colo320, and HT29 CRC cell lines but not in IEC6 intestinal cells. MENK secretion was associated with tumorigenicity and metastasis of CRC cells in syngeneic rodent models. The MENK concentration in subcutaneous tumors of CT26 and IEC6A CRC cells exhibited an inverse correlation with the number of tumor-infiltrating T lymphocytes. MENK inhibited the growth of MOLT-4 T-lymphoblastic cells in a dose-dependent manner. Furthermore, it increased the phosphorylation level of c-Jun N-terminal kinase and induced apoptosis in MOLT-4 cells. MENK-induced apoptosis was abrogated by a c-Jun N-terminal kinase inhibitor. Immunohistochemical analysis revealed moderate to strong expression of MENK in 33 (54%) of 61 CRC. MENK expression was associated with Dukes' staging, nodal metastasis, and liver metastasis. The MENK concentration in tumor tissues was higher in Dukes' C cases than in Dukes' B cases. MENK expression was associated with tumor-infiltrating T lymphocytes, especially those belonging to the CD4(+) subset. These findings suggest that MENK secreted by CRC cells caused escape of the host from the effects of immunity.

An in vitro comparison of microdialysis relative recovery of Met- and Leu-enkephalin using cyclodextrins and antibodies as affinity agents.

Cyclodextrins and antibodies have been used as affinity agents to improve relative recovery during microdialysis sampling. Two neuropeptides, methionine-enkephalin (ME) and leucine-enkephalin (LE), were chosen to compare the use of cyclodextrins and antibodies as possible affinity agents for improving their relative recovery across polycarbonate and polyethersulfone membranes during in vitro sampling. Cyclodextrins (CD) including beta-CD, 2-hydroxypropyl-beta-cyclodextrin (2HPbeta-CD), and gamma-CD gave improvements of relative recovery for both peptides of less than 2-fold as compared to controls. Comparisons of relative recovery between tyrosine-glycine-glycine, tyrosine, and phenylalanine using different cyclodextrins in the perfusion fluid were also obtained. Inclusion of an antibody against met-enkephalin in the microdialysis perfusion fluid resulted in relative recovery increases of up to 2.5-fold. These results show that using antibodies as affinity agents during microdialysis sampling may be more effective agents to improve the relative recovery of these opioid neuropeptides.

The involvement of spinal bovine adrenal medulla 22-like peptide, the proenkephalin derivative, in modulation of nociceptive processing.

Bovine adrenal medulla 22 (BAM22), one of the cleavage products of proenkephalin A, possesses high affinity for opioid receptors and sensory neuron-specific receptor (SNSR). The present study was designed to examine the expression of BAM22 in the spinal cord and dorsal root ganglion (DRG) of naive rats as well as in a model of inflammation. BAM22-like immunoreactivity (BAM22-IR) was expressed in fibers in the spinal cord, with high density seen in lamina I in naïve rats. The expression of BAM22-IR in the superficial laminae was greatly reduced following dorsal rhizotomy. BAM22-IR was also located in 19% of DRG cells, mainly in the small- and medium-sized subpopulations. Following injection of complete Freund's adjuvant (CFA) in the hindpaw, the expression of BAM22-IR in the superficial laminae of the spinal cord and small-sized DRG neurons on the ipsilateral side was markedly increased. Double labeling showed that the Fos-positive nucleus was surrounded by BAM22-IR cytoplasm in the spinal dorsal horn neurons or closely associated with BAM22-IR fibers in the superficial laminae. Furthermore, CFA-induced mechanical allodynia in the inflamed paw was potentiated by intrathecal administration of anti-BAM22 antibody. Together, these results demonstrate for the first time that BAM22-like peptide is mainly located in the superficial laminae of the spinal cord and mostly originates from nociceptive DRG neurons. BAM22 could thus act as a ligand for presynaptic opioid receptors and SNSR. Our study also provides evidence suggesting that BAM22 plays a role in the modulation of nociceptive processing at the spinal level under normal and inflammatory conditions.

Overexpression of the opioid growth factor receptor downregulates cell proliferation of human squamous carcinoma cells of the head and neck.

The opioid growth factor (OGF) is a constitutively expressed negative growth regulator whose action is mediated by the OGF receptor (OGFr). The OGF-OGFr axis tonically regulates the growth of human squamous cell carcinoma of the head and neck (SCCHN). To examine the repercussions of amplifying OGFr in SCCHN, constructs were prepared to overexpress OGFr in SCC-1 cells; six clonal lines were examined. OGFr binding assays of clonal cells revealed a 2.4- to 8.4-fold increase in binding capacity compared to wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by quantitative immunohistochemistry and Western blotting, was increased in clonal cell lines compared to controls. Under standard growth conditions the cell number of the OGFr clonal lines was reduced by 11 to 68% from the WT group, and doubling times were 7 to 67% longer. Addition of exogenous OGF further reduced (8 to 37%) cell growth of the clonal lines. Depletion of endogenous OGF with antibodies to this peptide increased growth 2-fold in cells amplifying OGFr relative to increases of 32 and 34% for the WT and EV groups, respectively. DNA synthesis of cells overexpressing OGFr was reduced from the WT group by 46 to 75%. These data indicate that the OGF receptor is integral to cell replication of SCCHN, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.

Influence of pain treatment by epidural fentanyl and bupivacaine on homing of opioid-containing leukocytes to surgical wounds.

Endogenous opioids released from leukocytes extravasating into injured tissue can interact with peripheral opioid receptors to inhibit nociception. Animal studies have shown that the homing of opioid-producing leukocytes to the injured site is modulated by spinal blockade of noxious input. This study investigated whether epidural analgesia (EDA) influences the migration of beta-endorphin (END) and/or met-enkephalin (ENK)-containing leukocytes into the subcutaneous wound tissue of patients undergoing abdominal surgery. In part I patients received general anesthesia combined either with intra- and postoperative EDA (with bupivacaine and fentanyl) or with postoperative patient controlled intravenous analgesia (PCIA; with the opioid piritramide). In part II patients received general anesthesia combined with either epidural fentanyl or bupivacaine which was continued postoperatively. Samples of cutanous and subcutanous tissue were taken from the wound site at the beginning, at the end and at various times after surgery, and were examined by immunohistochemistry for the presence of END and ENK. We found that (i) epidural bupivacaine, fentanyl and PCIA provided similar and clinically acceptable postoperative pain relief; (ii) compared to PCIA, epidural bupivacaine or fentanyl did not change the gross inflammatory reaction within the surgical wound; (iii) opioid-containing leukocytes were almost absent in normal subcutaneous tissue but migrated to the inflamed wound tissue in ascending numbers within a few hours, reaching a peak at about 24 h after surgery; (iv) compared to PCIA, EDA resulted in significantly decreased homing of END-containing leukocytes to the injured site at 24 h after surgery; and (v) the magnitude of this decrease was similar regardless of the epidural medication. These findings suggest that nociceptive but not sympathetic neurons are primarily involved in the attraction of opioid-containing leukocytes during early stages of inflammation.

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.

In vitro modulation of cytokine expression by enkephalin-derived peptides.

OBJECTIVE: We have previously reported that low doses of [Met(5)]-enkephalin (YGGFM, met-enkephalin) and two of its derivatives (YGG and YG) enhanced and accelerated delayed-type hypersensitivity responses while much higher doses of these compounds suppressed these reactions. Since the underlying mechanisms by which this and other immunomodulatory effects occur have not been established, this report explores the in vitro modulation of Th1 and Th2 cytokine expression by these peptides. METHODS: Murine splenocytes were stimulated with suboptimal concentrations of concanavalin A (ConA) in serum-free medium in the absence or presence of met-enkephalin, YGG, YG, [des-Tyr(1)]-met-enkephalin (GGFM), [D-Ala(2)], [D-Met(5)]-enkephalin or tyrosine (Y). Cell-conditioned supernatants were assayed for interferon-gamma (IFN-gamma), interleukin (IL)-2 and IL-4. Relative IFN-gamma and IL-2 mRNA levels were assessed by reverse transcription-polymerase chain reaction. The enhancing and suppressive effects of met-enkephalin and YG on IFN-gamma production were also tested in the presence of naloxone (Nx). RESULTS: Met-enkephalin, YGG and YG modulated the in vitro production of IFN-gamma in a biphasic manner: stimulation at low doses and inhibition at high doses. At higher concentrations, met-enkephalin and YG also suppressed the production of IL-2 (type 1) and IL-4, a type 2 cytokine. Nx reversed the enhancing effect of met-enkephalin on IFN-gamma production without affecting its suppressive action or any of the immunomodulating effects of YG. The degradation-resistant analog [D-Ala(2)], [D-Met(5)]-enkephalin enhanced IFN-gamma production but did not suppress it. CONCLUSIONS: YG, the minimal molecular requirement for enhancement and suppression of immune responses by these metabolites, appears to mediate exclusively an across-the-board suppression via low-affinity, nonclassical, nonopioid receptors.

Methionine-enkephalin stimulates hydrogen peroxide and nitric oxide production in rat peritoneal macrophages: interaction of mu, delta and kappa opioid receptors.

OBJECTIVE: Methionine-enkephalin (MET) modulates various functions of macrophages related to both immune and inflammatory reactions in a naloxone reversible manner, suggesting that opioid receptors are involved in the regulation of macrophage activity. Since an endogenous opioid ligand might interact with more than one type of opioid receptor, the receptor interaction determines its effect on a particular function. METHODS: In the present study we have investigated the involvement of different opioid receptor types/subtypes in MET-induced modulation of H(2)O(2) and NO production in macrophages. Thioglycollate-elicited or resident rat peritoneal macrophages were treated in vitro with MET and/or specific antagonists of delta(1,2), delta(1), delta(2), mu and kappa opioid receptors. RESULTS: MET increased H(2)O(2)production in phorbol myristate acetate-stimulated rat peritoneal macrophages mainly through delta(1) opioid receptor. MET also enhanced NO production in rat peritoneal macrophages stimulated with lipopolysaccharide through delta(1) and mu opioid receptors. The blockade of mu and kappa receptor facilitated a potentiating effect of MET on H(2)O(2) release, and blockade of kappa receptor further raised the MET-induced increase of NO production in macrophages. CONCLUSION: It is concluded that both negative and positive functional interaction between delta, mu and kappa opioid receptors regulate the influence of MET on H(2)O(2) and NO production in rat peritoneal macrophages.

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.

Effects of endogenous and synthetic opioid peptides on neutrophil function in vitro.

BACKGROUND: Opioid peptides released from immunocytes during inflammation and stress in critically ill patients are associated with an altered immune response. Moreover, concentrations of opioid peptides are increased in peripheral blood and at the sites of inflammatory reactions. METHODS: Using flow cytometric assay of whole human blood, we investigated direct effects of endogenous and synthetic opioid peptides on surface expression of complement receptors CD35 and CD11b/CD18 and Fcã receptor III CD16, and superoxide anion generation of neutrophils. RESULTS: The endogenous opioid peptides beta-endorphin(1-31) and met-enkephalin, representing the N-terminal fragment of beta-endorphin(1-31), and the synthetic delta opioid receptor agonists D-Ala(2)-D-Leu(5)-enkephalin and D-Pen(2)-enkephalin produced concentration-dependent stimulation of neutrophil activity. Incubation with met-enkephalin 10(-7) M or beta-endorphin(1-31) 10(-7) M led to an increase in receptor expression of up to 10% (met-enkephalin) and 15% (beta-endorphin(1-31)). After incubation with D-Ala(2)-D-Leu(5)-enkephalin or D-Pen(2/5)-enkephalin, receptor expression was increased by up to 30%. This correlated with concentration-dependent stimulation of the production of reactive oxygen intermediates, as shown by an increase of up to 40% in oxidative burst activity. All effects were abolished after preincubation with naloxone or with the selective delta opioid antagonist naltrindole, whereas the selective micro receptor antagonist d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) showed only partial inhibitory effects. CONCLUSIONS: Our data suggest a delta opioid receptor-mediated stimulatory effect on neutrophil function. beta-Endorphin(27-31), the C-terminal fragment of beta-endorphin(1-31), did not alter neutrophil function, indicating that beta-endorphin(1-31) mediates its effect on neutrophils via the N-terminal fragment. This study may contribute to a better understanding of neuroimmune interaction.

Endogenous opioid peptides contribute to antinociceptive potency of intrathecal [Dmt1]DALDA.

[Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) is a dermorphin analog that shows high affinity and selectivity for the mu opioid receptor. The intrathecal potency of [Dmt(1)]DALDA far exceeded its affinity at mu receptors and suggests that other mechanisms must be involved in its action in the spinal cord. The affinity and selectivity of [Dmt(1)]DALDA was determined using cell membranes expressing cloned human mu, delta, and kappa opioid receptors. Competitive displacement binding with [(3)H][Dmt(1)]DALDA, [(3)H]DPDPE (H-Tyr-d-Pen-Gly-Phe-d-Pen), and [(3)H]U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)-benzeneacetamide] revealed K(i) of 156 +/- 26 pM for mu opioid receptor (MOR), 1.67 +/- 0.04 microM for delta opioid receptor (DOR), and K(i) of 4.4 +/- 1.7 nM for kappa opioid receptor (KOR), respectively. [Dmt(1)]DALDA increased guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding in MOR, DOR, and KOR membranes, with EC(50) being 17 (8.8-33) nM, 2 (1.2-3.2) microM, and 124 (15-1000) nM, respectively. Intrathecal [Dmt(1)]DALDA inhibited the tail-flick response in mice with ED(50) = 1.22 (0.59-2.34) pmol. Intrathecal administration of an antiserum against dynorphin A(1-17) or [Met(5)]enkephalin significantly attenuated the response to i.t. [Dmt(1)]DALDA, resulting in ED(50) of 6.2 (3.6-12.6) pmol and 6.6 (3.5-19.6) pmol, respectively. Neither antisera had any effect on the response to i.t. morphine. Intracerebroventricular (i.c.v.) [Dmt(1)]DALDA was not affected by previous i.c.v. administration of anti-Dyn or anti-ME. Pretreatment with norbinaltorphimine or naltriben also attenuated the antinociceptive response to i.t., but not i.c.v., [Dmt(1)]DALDA. These data suggest that i.t. [Dmt(1)]DALDA causes the release of dynorphin and [Met(5)]enkephalin-like substances that act at kappa and delta receptors, respectively, to contribute to the extraordinary potency of [Dmt(1)]DALDA.

Met-enkephalin immunoreactive neurons recruited by acute stress are innervated by axon terminals immunopositive for tyrosine hydroxylase and dopamine-alpha-hydroxylase in the anterolateral division of bed nuclei of the stria terminalis in the rat.

The bed nuclei of the stria terminalis (BST) are highly heterogeneous forebrain structures, which play a central role in the regulation/modulation of stress responses. Studies using the inducible immediate early gene c-fos as a marker of activated neurons have demonstrated significant stress-induced neuronal activation in this limbic region. The BST also exhibit a dense network of dopamine and noradrenaline immunoreactive (ir) axon terminals. These catecholaminergic projections from various brainstem sources to the BST play an important role in a neurochemically mediated coordination of stress responses. In the anterolateral division of bed nuclei of the stria terminalis, the distribution of several Met-enkephalin immunopositive perikarya overlaps with that of catecholaminergic axon terminals. Both monoaminergic and enkephalinergic structures have been postulated to play a role in the regulation/modulation of the central regulatory pathways of endocrine, behavioural and physiological responses during stress. Therefore the aims of this study were: (i). to study the possible involvement of dopaminergic fibre terminals in stress-induced activation of BST perikarya; (ii). to investigate whether Met-enkephalin-immunoreactive neurons are recruited by acute volumen/osmotic challenge; and (iii). to demonstrate synaptic interactions between Met-enkephalin-ir neurons and fibre terminals immunopositive for dopamine or noradrenaline in the anterolateral division of the BST. From the results of this study we can conclude that depletion of dopamine in fibre terminals completely abolished stress-induced activation of perikarya in the anterolateral division of BST. Furthermore, the innervation of stress-induced Met-enkephalin-ir perikarya by dopaminergic fibre terminals in the oval nucleus of BST was demonstrated, whereas noradrenergic axons contacted enkephalinergic structures in the fusiform and subcomissural nuclei of BST. These interactions can be central in the modulatory control of the major stress regulatory pathway, the limbic hypothalamo-pituitary-adrenal axis.