The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the ß-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the PER2 circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity. To investigate this link, we conducted a 48 h circadian rhythm experiment, during which RNA samples were collected every 5 h. We discovered that the activation of DOPr by its endogenous agonist Met-Enkephalin in N/TERT-1 keratinocytes, synchronized by dexamethasone, resulted in a statistically significant 5.6 h delay in the expression of the core clock gene PER2. Confocal microscopy further confirmed the simultaneous nuclear localization of the DOPr-ß-arrestin-1 complex. Additionally, DOPr activation not only enhanced but also induced a phase shift in the rhythmic binding of ß-arrestin-1 to the PER2 promoter. Furthermore, we observed that ß-arrestin-1 regulates the transcription of its target genes, including PER2, by facilitating histone-4 acetylation. Through the ChIP assay, we determined that Met-Enkephalin enhances ß-arrestin-1 binding to acetylated H4 in the PER2 promoter. In summary, our findings suggest that DOPr activation leads to a phase shift in PER2 expression via ß-arrestin-1-facilitated chromatin remodeling. Consequently, these results indicate that DOPr, much like its role in wound healing, may also play a part in cancer development by influencing PER2.

The bivalent ligand, MMG22, reduces neuropathic pain after nerve injury without the side effects of traditional opioids.

ABSTRACT: Functional interactions between the mu opioid receptor (MOR) and the metabotropic glutamate receptor 5 (mGluR5) in pain and analgesia have been well established. MMG22 is a bivalent ligand containing MOR agonist (oxymorphamine) and mGluR5 antagonist (MPEP) pharmacophores tethered by a 22-atom linker. MMG22 has been shown to produce potent analgesia in several models of chronic inflammatory and neuropathic pain (NP). This study assessed the efficacy of systemic administration of MMG22 at reducing pain behavior in the spared nerve injury (SNI) model of NP in mice, as well as its side-effect profile and abuse potential. MMG22 reduced mechanical hyperalgesia and spontaneous ongoing pain after SNI, with greater potency early (10 days) as compared to late (30 days) after injury. Systemic administration of MMG22 did not induce place preference in naive animals, suggesting absence of abuse liability when compared to traditional opioids. MMG22 also lacked the central locomotor, respiratory, and anxiolytic side effects of its monomeric pharmacophores. Evaluation of mRNA expression showed the transcripts for both receptors were colocalized in cells in the dorsal horn of the lumbar spinal cord and dorsal root ganglia. Thus, MMG22 reduces hyperalgesia after injury in the SNI model of NP without the typical centrally mediated side effects associated with traditional opioids.

Atlas of Immune Cell Populations of the Inflamed Mammalian CNS.

Two processes are known to take place during neuroinflammation: (i) resident immune cells are activated and (ii) inflammatory leukocytes in the periphery begin to infiltrate the central nervous system (CNS).[…].

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.

Emergence of functional delta-opioid receptors induced by long-term treatment with morphine.

Opioid analgesics remain the choice for the treatment of moderate to severe pain. Recent research has established that the mu-opioid receptor is predominantly responsible for mediating many opioid actions, including analgesia and opioid tolerance. However, the function of delta-opioid receptors is rather puzzling at present, with inconsistent reports of system effects by agonists of delta-opioid receptors. The functional interaction between mu-opioid receptors and delta-opioid receptors is also poorly understood. In this study, we demonstrated that in a brainstem site critically involved in opioid analgesia, agonists of delta-opioid receptors, ineffective in opioid naive rats, significantly inhibit presynaptic GABA release in the brainstem neurons from morphine-tolerant rats. In membrane preparation from control brainstem tissues, Western blot detected no proteins of delta-opioid receptors, but consistent delta-opioid receptor proteins were expressed in membrane preparation from morphine-tolerant rats. Immunohistochemical studies revealed that long-term morphine treatment significantly increases the number of delta-opioid receptor-immunoreactive varicosities that appose the postsynaptic membrane of these neurons. The colocalization of delta-opioid receptor-immunoreactive varicosities with the labeling of the GABA-synthesizing enzyme glutamic acid decarboxylase is also significantly increased. From a behavioral perspective, activation of delta-opioid receptors in the brainstem nucleus, lacking an effect in opioid naive rats, became analgesic in morphine-tolerant rats and significantly reduced morphine tolerance. These findings indicate that long-term morphine treatment induces the emergence of functional delta-opioid receptors and delta-opioid receptor-mediated analgesia, probably through receptor translocation to surface membrane in GABAergic terminals. They also suggest that opioid drugs with preference for delta-opioid receptors may have better therapeutic effect in a mu-opioid-tolerant state.