Special feature for the Olympics: effects of exercise on the immune system: neuropeptides and their interaction with exercise and immune function

It is known today that the immune system is influenced by various types of psychological and physiological stressors, including physical activity. It is well known that physical activity can influence neuropeptide levels both in the central nervous system as well as in peripheral blood. The reported changes of immune function in response to exercise have been suggested to be partly regulated by the activation of different neuropeptides and the identification of receptors for neuropeptides and steroid hormones on cells of the immune system has created a new dimension in this endocrine-immune interaction. It has also been shown that immune cells are capable of producing neuropeptides, creating a bidirectional link between the nervous and immune systems. The most common neuropeptides mentioned in this context are the endogenous opioids. The activation of endogenous opioid peptides in response to physical exercise is well known in the literature, as well as the immunomodulation mediated by opioid peptides. The role of endogenous opioids in the exercise-induced modulation of immune function is less clear. The present paper will also discuss the role of other neuroendocrine factors, such as substance P, neuropeptide Y and vasoactive intestinal peptide, and pituitary hormones, including growth hormone, prolactin and adrenocorticotrophin, in exercise and their possible effects on immune function.     

Immune mechanisms in pain control

Pain inhibition can be induced by immune‐derived opioids interacting with opioid receptors on peripheral sensory nerves. These receptors are up‐regulated in inflammation (1). Opioid peptides are synthesised in circulating immune cells which migrate to injured tissue. This is orchestrated by selectins and other adhesion molecules located on immunocytes and on vascular endothelium (2). In response to releasing stimuli the opioids are secreted, activate peripheral opioid receptors and produce analgesia by inhibiting the excitability of sensory nerves and/or the release of excitatory neuropeptides. These effects occur in the periphery and are devoid of central side‐effects such as respiratory depression, sedation, dysphoria or dependence. Targeting of immune cells containing opioids to injured tissues is a novel concept of pain control and opens potential new therapeutic approaches.     

Opioids in neuropathic pain

Pain inhibition can be induced by immune-derived opioids interacting with opioid receptors on peripheral sensory nerves. These receptors are up-regulated in inflammation (1). Opioid peptides are synthesised in circulating immune cells which migrate to injured tissue. This is orchestrated by selectins and other adhesion molecules located on immunocytes and on vascular endothelium (2). In response to releasing stimuli the opioids are secreted, activate peripheral opioid receptors and produce analgesia by inhibiting the excitability of sensory nerves and/or the release of excitatory neuropeptides. These effects occur in the periphery and are devoid of central side-effects such as respiratory depression, sedation, dysphoria or dependence. Targeting of immune cells containing opioids to injured tissues is a novel concept of pain control and opens potential new therapeutic approaches.     

Role of receptor transactivation in the cardioprotective effects of preconditioning and postconditioning

Analysis of published data indicates that the activity of receptors for adenosine, opioids, bradykinin, calcitonin-gene related peptides (CGRP) and epidermal growth factor (EGF) play important role in triggering the cardioprotective effects of ischemic preconditioning. Cannabinoids mimic the infarct-sparing effects of preconditioning. Endogenous adenosine, opioids, bradykinin and CGRP have also been implicated in infarct-reduction with ischemic postconditioning. Again, cannabinoids also mimic the protective effect of postconditioning. Recent works support heterodimerization of G-protein coupled receptors (GPCRs), and GPCR transactivation of EGF receptors. It was found that cross-talk between delta(j)-opioid receptors and adenosine A(1)-receptors is essential to cardiac protection. Furthermore, evidence implicates EGF receptor transactivation in cardioprotective effect of multiple GPCrs including adenosine, acetylcholine, bradykinin, and opioid receptors. Such findings support a convergent pathway in which multiple GPCRs may interact (or function independently) to transactivate EGF receptor-dependent kinase signaling and cytoprotection.     

Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing

Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2.     

The opioid agonist ethylketocyclazocine reverts the rapid, non-genomic effects of membrane testosterone receptors in the human prostate LNCaP cell line

Neuropeptides influence cancer cell replication and growth. Opioid peptides, and opiergic neurons are found in the prostate gland, and they are proposed to exert a role in tumor regulation, influencing cancer cell growth, as opioid agonists inhibit cell growth in several systems, including the human prostate cancer cell line LNCaP. In the same cell line, the existence of membrane testosterone receptors was recently reported, which increase, in a non-genomic manner, the secretion of PSA, and modify actin cytoskeleton dynamics, through the signaling cascade FAK-->PI-3 kinase-->Cdc42/Rac1. In the present work, we present data supporting that the general opioid agonist Ethylketocyclazocine (EKC) decreases testosterone-BSA (a non-internalizable testosterone analog) induced PSA secretion. Furthermore, we report that this opioid affects this non-genomic testosterone action, by modifying the distribution of the actin cytoskeleton in the cells, disrupting the above signaling cascade. In addition, after long (>24 h) incubation, opioids decrease the number of membrane testosterone receptors, and reverse their effect on the signaling molecules. In conclusion, our results provide some new insights of a possible action of opioids in prostate cancer control by interfering with the action and the expression of membrane testosterone receptors and signaling.     

Effects of opioids on the immune system

Both therapeutic and chronic uses of opioids compromise the optimal functioning of the immune system. Overwhelming evidence suggests that opioid use affects both innate immunity and adaptive immunity. Chronic administration of opioids decreases the proliferative capacity of macrophage progenitor cells and lymphocytes. Additionally, the differentiated function of immune cells is significantly affected by opioids. These effects are mediated by either a direct action of opioids on the target cells or by indirect centrally mediated pathways. Molecular biological and biochemical characterization suggest that immune cells differentially express classical opioid receptors. Interestingly, these studies also reveal the presence of a novel class of opioid receptors in immune cells. We believe that this low affinity morphine binding site mediates the antiproliferative effects of morphine. Special issue dedicated to Dr. Eric J. Simon.     

Endogenous opioids and ventilatory responses to hypercapnia in normal humans

Though administration of opioid peptides depresses ventilation and ventilatory responsiveness, the role of endogenous opioid peptides in modulating ventilatory responsiveness is not clear. We studied the interaction of endogenous opioids and ventilatory responses in 12 adult male volunteers by relating hypercapnic responsiveness to plasma levels of immunoactive beta-endorphin and by administering the opiate antagonist naloxone. Ventilatory responsiveness to hypercapnia was not altered by pretreatment with naloxone, and this by itself suggests that endogenous opioids have no role in modulating this response. However, there was an inverse relationship between basal levels of immunoactive beta-endorphin in plasma and ventilatory responsiveness to CO2. Furthermore, plasma beta-endorphin levels rose after short-term hypercapnia but only when subjects had been pretreated with naloxone. We conclude that measurement of plasma endorphin levels suggests relationships between endogenous opioid peptides and ventilatory responses to CO2 that are not apparent in studies limited to assessing the effect of naloxone.     

Signaling diversity of mu- and delta- opioid receptor ligands: Re-evaluating the benefits of β-arrestin/G protein signaling bias

Opioid analgesics are elective for treating moderate to severe pain but their use is restricted by severe side effects. Signaling bias has been proposed as a viable means for improving this situation. To exploit this opportunity, continuous efforts are devoted to understand how ligand-specific modulations of receptor functions could mediate the different in vivo effects of opioids. Advances in the field have led to the development of biased agonists based on hypotheses that allocated desired and undesired effects to specific signaling pathways. However, the prevalent hypothesis associating β-arrestin to opioid side effects was recently challenged and multiple of the newly developed biased drugs may not display the superior side effects profile that was sought. Moreover, biased agonism at opioid receptors is now known to be time- and cell-dependent, which adds a new layer of complexity for bias estimation. Here, we first review the signaling mechanisms underlying desired and undesired effects of opioids. We then describe biased agonism at opioid receptors and discuss the different perspectives that support the desired and undesired effects of opioids in view of exploiting biased signaling for therapeutic purposes. Finally, we explore how signaling kinetics and cellular background can influence the magnitude and directionality of bias at those receptors.     

Morphine regulates gene expression of alpha- and beta-chemokines and their receptors on astroglial cells via the opioid mu receptor

The brain is a target organ for recreational drugs and HIV-1. Epidemiological data demonstrate that opioid abuse is a risk factor for HIV-1 infection and progression to AIDS. Chemokines and their receptors have been implicated in the neuropathogenesis of HIV-1 infections. However, little is known about the effects of opioids on the expression of chemokines and their receptors (the latter also are HIV-1 coreceptors) by cells of the CNS. Herein we describe the effects of morphine on gene expression of the alpha- and beta-chemokines and their receptors by the astrocytoma cell line U87 and by primary normal human astrocyte (NHA) cultures. U87 cells treated with morphine showed significant down-regulation of IL-8 gene expression, whereas expression of the IL-8 receptor CXCR2 was reciprocally up-regulated as detected by RT-PCR. Treatment of NHAs with morphine suppressed IL-8 and macrophage-inflammatory protein-1beta gene expression, whereas expression of their receptor genes, CCR3 and CCR5, was simultaneously enhanced. These morphine-induced effects on U87 and NHA cells were reversed by the opioid mu receptor antagonist beta-funaltrexamine. Morphine also enhanced the constitutive expression of the opioid mu receptor on astroglial cells. Our results support the hypothesis that opioids play a significant role in the susceptibility of the CNS to HIV-1 infection and subsequent encephalopathy by inhibiting local production of HIV-1-protective chemokines (IL-8 and macrophage-inflammatory protein-1beta) and enhancing expression of HIV-1 entry coreceptor genes (CCR3, CCR5, and CXCR2) within the CNS. These effects of opioids appear to be mediated through the opioid mu receptor that we demonstrated on astroglial cells.     

PD98059 Influences Immune Factors and Enhances Opioid Analgesia in Model of Neuropathy

Neuropathic pain treatment remains challenging due to ineffective therapy and resistance to opioid analgesia. Mitogen-activated protein kinase kinase (MAPKK) have been identified as the crucial regulators of pro- and antinociceptive factors. We used PD98059, an inhibitor of the MAPKK family members MEK1/2. The aim of study was to examine the influence of single and/or repeated PD98059 on nociception and opioid effectiveness in neuropathy. Moreover, we examined how PD98059 influences selected members of cellular pathways and cytokines. The PD98059 (2.5 mcg) was intrathecally preemptively administered before chronic constriction injury (CCI), and then once daily for 7 days. Additionally, at day 7 after CCI the PD98059-treated rats received a single injection of opioids. Using Western blot and qRT-PCR techniques in PD98059-treated rats we analyzed the mRNA and/or protein level of p38, ERK1/2, JNK, NF-kappaB, IL-1beta, IL-6, iNOS and IL-10 in the lumbar spinal cord. Our results indicate that PD98059 has an analgesic effects and potentiates morphine and/or buprenorphine analgesia. Parallel we observed that PD98059 inhibit upregulation of the CCI-elevated p38, ERK1/2, JNK and NF-kappaB protein levels. Moreover, PD98059 also prevented increase of pro- (IL-1beta, IL-6, and iNOS) but enhances anti-nociceptive (IL-10) factors. Summing up, PD98059 diminished pain and increased the effectiveness of opioids in neuropathy. The inhibition of MEKs might inactivate a variety of cell signaling pathways that are implicated in nociception.     

Synergistic interactions between cannabinoid and opioid analgesics

Cannabinoids and opioids both produce analgesia through a G-protein-coupled mechanism that blocks the release of pain-propagating neurotransmitters in the brain and spinal cord. However, high doses of these drugs, which may be required to treat chronic, severe pain, are accompanied by undesirable side effects. Thus, a search for a better analgesic strategy led to the discovery that delta 9-tetrahydrocannabinol (THC), the major psychoactive constituent of marijuana, enhances the potency of opioids such as morphine in animal models. In addition, studies have determined that the analgesic effect of THC is, at least in part, mediated through delta and kappa opioid receptors, indicating an intimate connection between cannabinoid and opioid signaling pathways in the modulation of pain perception. A host of behavioral and molecular experiments have been performed to elucidate the role of opioid receptors in cannabinoid-induced analgesia, and some of these findings are presented below. The aim of such studies is to develop a novel analgesic regimen using low dose combinations of cannabinoids and opioids to effectively treat acute and chronic pain, especially pain that may be resistant to opioids alone.     

Opioid receptor agonists and Ca2+ modulation in human B cell lines.

Opiates and opioid peptides have been shown to modulate lymphocyte functions; however, little attention has been given to the type of receptors or receptor signaling mechanisms that are involved. Receptor-mediated signaling via ionized free Ca2+ is an event thought to be important in the triggering of lymphocyte activities. We report use of the calcium indicator dye, indo-1, and flow cytometry to identify B lymphocyte calcium responses to physiologic concentrations of opioid peptides. The human B cell lines Nalm 6 and JY responded to the naturally occurring opioid pentapeptide methionine-enkephalin or other opiate receptor agonists with a rapid, dose-dependent rise in free cytoplasmic Ca2+. This opioid peptide effect on Ca2+ modulation was inhibited by the opiate receptor antagonist naloxone. The synthetic enkephalin analogue DAMGO with specificity for mu-type opiate receptors and the synthetic opiate receptor agonists U50,488H and U69,593 with selectivity for kappa-type sites also stimulated calcium responses when applied to the B cell lines. These studies provide evidence that human B cell lines express functional opiate receptors of the mu- and kappa-types and suggest that such receptors, coupled with Ca2+ modulation, are instrumental in the B cell response to opiates and endogenous opioid neuropeptides.     

Opioids trigger alpha 5 beta 1 integrin-mediated monocyte adhesion

Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.     

Opioid mechanisms in insects,with special attention toLeucophaea maderae

1. This review article provides information on the evolutionary history of neuroendocrine and related regulatory mechanisms. It focuses on the presence, diverse roles, and modes of operation of one class of neuropeptides, the endogenous opioids, in insects. 2. Opioid peptides, closely resembling those of vertebrates, have been identified in the brain and related neuroendocrine structures by means of immunocytochemistry and high-pressure liquid chromatography. 3. The demonstration of naloxone-sensitive, high-affinity binding sites for Met-enkephalin-like neuropeptides in the brain and digestive tract of Leucophaea deserves special attention because it provides new insights into the functional significance of opiate receptors paralleling those known in vertebrates. 4. Possible roles of receptor-mediated opioid systems in the insects discussed are regulation of the cyclicity of the female reproductive system, maintenance of normal midgut function mediated by the recurrent nerve, and locomotor activity.     

Nonopiate active proenkephalin-derived peptides are secreted by T helper cells

Recent investigations have shown that the neuroendocrine and immune systems profoundly affect each other. In part, these interactions occur via common chemical messengers and receptors. One possible shared chemical messenger is the opioid precursor preproenkephalin, for which high concentrations of messenger RNA are present in brain, adrenal, and activated T helper cells. Because the biologic action of most peptide messengers depends on the posttranslational processing of the precursor, we have examined T helper cell lines for the production of proenkephalin-derived peptides. These peptides were characterized by multiple radioimmunoassays, gel filtration chromatography, and opiate radioreceptor assays. We found that activated T helper cells secrete significant concentrations of high-molecular-weight, opiate-inactive peptides, which are distinct from the proenkephalin-derived peptides of the neuroendocrine system. These studies clearly indicate cell-specific processing of proenkephalin, and suggest that the T helper cell-secreted products may have nonopiate receptor-mediated actions.     

Molecular and cellular mechanisms of the age-dependency of opioid analgesia and tolerance

ABSTRACT: The age-dependency of opioid analgesia and tolerance has been noticed in both clinical observation and laboratory studies. Evidence shows that many molecular and cellular events that play essential roles in opioid analgesia and tolerance are actually age-dependent. For example, the expression and functions of endogenous opioid peptides, multiple types of opioid receptors, G protein subunits that couple to opioid receptors, and regulators of G protein signaling (RGS proteins) change with development and age. Other signaling systems that are critical to opioid tolerance development, such as N-methyl-D-aspartic acid (NMDA) receptors, also undergo age-related changes. It is plausible that the age-dependent expression and functions of molecules within and related to the opioid signaling pathways, as well as age-dependent cellular activity such as agonist-induced opioid receptor internalization and desensitization, eventually lead to significant age-dependent changes in opioid analgesia and tolerance development.