ABSTRACT
Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.
Subject(s)
Morphine , Toll-Like Receptor 4 , Mice , Animals , Morphine/pharmacology , Toll-Like Receptor 4/metabolism , Receptors, Opioid, mu/metabolism , Analgesics, Opioid/pharmacology , Receptors, Opioid/metabolism , Hypothalamus/metabolism , Molecular Chaperones/metabolismABSTRACT
Radiation therapy for head and neck cancers causes salivary gland dysfunction leading to permanent xerostomia. Limited progress in the discovery of new therapeutic strategies is attributed to the lack of in vitro models that mimic salivary gland function and allow high-throughput drug screening. We address this limitation by combining engineered extracellular matrices with microbubble (MB) array technology to develop functional tissue mimetics for mouse and human salivary glands. We demonstrate that mouse and human salivary tissues encapsulated within matrix metalloproteinase-degradable poly(ethylene glycol) hydrogels formed in MB arrays are viable, express key salivary gland markers, and exhibit polarized localization of functional proteins. The salivary gland mimetics (SGm) respond to calcium signaling agonists and secrete salivary proteins. SGm were then used to evaluate radiosensitivity and mitigation of radiation damage using a radioprotective compound. Altogether, SGm exhibit phenotypic and functional parameters of salivary glands, and provide an enabling technology for high-content/throughput drug testing.
Subject(s)
Acinar Cells/drug effects , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Radiation Injuries/prevention & control , Salivary Glands/drug effects , Tissue Array Analysis , Xerostomia/prevention & control , Acinar Cells/metabolism , Acinar Cells/radiation effects , Animals , Calcium Signaling/drug effects , Cells, Cultured , Female , Humans , Hydrogels , Male , Mice, Inbred C57BL , Microbubbles , Middle Aged , Parotid Gland/drug effects , Parotid Gland/metabolism , Parotid Gland/radiation effects , Phenotype , Polyethylene Glycols/chemistry , Radiation Injuries/etiology , Radiation Injuries/metabolism , Salivary Glands/metabolism , Salivary Glands/radiation effects , Xerostomia/etiology , Xerostomia/metabolismABSTRACT
Neuropathic pain is often insensitive to morphine. Our previous study has demonstrated that neuron-restrictive silencer factor represses mu opioid receptor (MOP) gene expression in the dorsal root ganglion (DRG) via histone hypoacetylation-mediated mechanisms after peripheral nerve injury, thereby causing loss of peripheral morphine analgesia. Here, we showed that histone deacetylase (HDAC) inhibitors, such as trichostatin A and valproic acid, restored peripheral and systemic morphine analgesia in neuropathic pain. Also, these agents blocked nerve injury-induced MOP down-regulation in the DRG. These results suggest that HDAC inhibitors could serve as adjuvant analgesics to morphine for the management of neuropathic pain.
Subject(s)
Analgesics , Down-Regulation/drug effects , Drug Resistance/drug effects , Gene Expression/drug effects , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Morphine/pharmacology , Morphine/therapeutic use , Neuralgia/drug therapy , Neuralgia/etiology , Peripheral Nerve Injuries/complications , Receptors, Opioid, mu/genetics , Valproic Acid/pharmacology , Valproic Acid/therapeutic use , Acetylation , Analgesia , Animals , Ganglia, Spinal/metabolism , Histone Deacetylases/metabolism , Histone Deacetylases/physiology , Histones/metabolism , Hydroxamic Acids , Male , Mice, Inbred C57BL , Receptors, Opioid, mu/metabolismABSTRACT
Lysophosphatidic acid receptor (LPA(1)) signaling initiates neuropathic pain through demyelination of the dorsal root (DR). Although LPA is found to cause down-regulation of myelin proteins underlying demyelination, the detailed mechanism remains to be determined. In the present study, we found that a single intrathecal injection of LPA evoked a dose- and time-dependent down-regulation of myelin-associated glycoprotein (MAG) in the DR through LPA(1) receptor. A similar event was also observed in ex vivo DR cultures. Interestingly, LPA-induced down-regulation of MAG was significantly inhibited by calpain inhibitors (calpain inhibitor X, E-64 and E-64d) and LPA markedly induced calpain activation in the DR. The pre-treatment with calpain inhibitors attenuated LPA-induced neuropathic pain behaviors such as hyperalgesia and allodynia. Moreover, we found that sciatic nerve injury activates calpain activity in the DR in a LPA(1) receptor-dependent manner. The E-64d treatments significantly blocked nerve injury-induced MAG down-regulation and neuropathic pain. However, there was no significant calpain activation in the DR by complete Freund's adjuvant treatment, and E-64d failed to show anti-hyperalgesic effects in this inflammation model. The present study provides strong evidence that LPA-induced calpain activation plays a crucial role in the manifestation of neuropathic pain through MAG down-regulation in the DR.