Delta opioid peptide [D-ala2, D-leu5]-Enkephalin's ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells.

BACKGROUND: Local brain tissue can suffer from ischaemia/reperfusion (I/R) injury, which lead to vascular endothelial damage. The peptide δ opioid receptor (δOR) agonist [D-ala2, D-leu5]-Enkephalin (DADLE) can reduce apoptosis caused by acute I/R injury in brain microvascular endothelial cells (BMECs). OBJECTIVE: This study aims to explore the mechanism by which DADLE enhances the level of mitophagy in BMECs by upregulating the expression of transient receptor potential vanilloid subtype 4 (TRPV4). METHODS: BMECs were extracted and made to undergo oxygen-glucose deprivation/reoxygenation (OGD/R) accompanied by DADLE. RNA-seq analysis revealed that DADLE induced increased TRPV4 expression. The CCK-8 method was used to assess the cellular viability; quantitative PCR (qPCR) was used to determine the mRNA expression of Drp1; western blot was used to determine the expression of TRPV4 and autophagy-related proteins; and calcium imaging was used to detect the calcium influx. Autophagosomes in in the cells' mitochondria were observed by using transmission electron microscopy. ELISA was used to measure ATP content, and a JC-1 fluorescent probe was used to detect mitochondrial membrane potential. RESULTS: When compared with the OGD/R group, OGD/R+DADLE group showed significantly enhanced cellular viability; increased expression of TRPV4, Beclin-1, LC3-II/I, PINK1 and Parkin; decreased p62 expression; a marked rise in calcium influx; further increases in mitophagy, an increase in ATP synthesis and an elevation of mitochondrial membrane potential. These protective effects of DADLE can be blocked by a TRPV4 inhibitor HC067047 or RNAi of TRPV4. CONCLUSION: DADLE can promote mitophagy in BMECs through TRPV4, improving mitochondrial function and relieving I/R injury.

MicroRNA-221-3p inhibits the inflammatory response of keratinocytes by regulating the DYRK1A/STAT3 signaling pathway to promote wound healing in diabetes.

Diabetic foot ulcer (DFU), a serious complication of diabetes, remains a clinical challenge. MicroRNAs affect inflammation and may have therapeutic value in DFU. Here, we find that an miR-221-3p mimic reduces the inflammatory response and increases skin wound healing rates in a mouse model of diabetes, whereas miR-221-3p knockout produced the opposite result. In human keratinocytes cells, miR-221-3p suppresses the inflammatory response induced by high glucose. The gene encoding DYRK1A is a target of miR-221-3p. High glucose increases the expression of DYRK1A, but silencing DYRK1A expression decreases high glucose-induced inflammatory cytokine release via dephosphorylation of STAT3, a substrate of DYRK1A. Application of miR-221-3p mimic to human keratinocytes cells not only decreases DYRK1A expression but also inhibits high glucose-induced production of inflammatory cytokines to promote wound healing. This molecular mechanism whereby miR-221-3p regulates inflammation through the DYRK1A/STAT3 signaling pathway suggests targets and therapeutic approaches for treating DFU.

Tibial cortex transverse transport regulates Orai1/STIM1-mediated NO release and improve the migration and proliferation of vessels via increasing osteopontin expression.

Background: Diabetic foot is a major complication of diabetes. The bone transverse transport method could be applied in clinics for treatment, which could improve the metabolism of the tissues via lasting distraction forces. However, the process' specific regulating mechanism is still unknown. Methods: Based on the notion that the healing of bones involves the recruitment of calcium ions, in this study, we established the model of tibial cortex transverse transport (TTT) on rats and then used tissue immunologic detection, such as the double fluorescent staining to explore the expression of the calcium channels' calcium release-activated calcium modulator 1 (Orai1)/stromal interaction molecule 1 (STIM1), which belong to the store-operated calcium entry (SOCE) signaling pathways on the tissues around the bone transport area. By using the laser capture microdissection (LCM) tool, we acquired samples of tissues around the bone and endeavored to identify pivotal protein molecules. Subsequently, we validated the functions of key protein molecules through in vitro and in vivo experiments. Results: After protein profile analysis, we found the differentially expressed key protein osteopontin (OPN). The in vitro experiments verified that, being stimulated by OPN, the migration, proliferation, and angiogenesis of human umbilical vein endothelial cells (HUVEC) were observed to be enhanced. The activation of Orai1/STIM1 might increase the activity of endothelial nitric oxide synthase (eNOS) and its effect on releasing nitric oxide (NO). Subsequently, the migration and proliferation of the HUVECs are improved, which ultimately accelerates wound healing. These signaling pathway was also observed in the OPN-stimulated healing process of the skin wound surface of diabetic mice. Conclusion: This study identifies the molecular biological mechanism of OPN-benefited the migration and proliferation of the HUVECs and provides ideas for searching for new therapeutic targets for drugs that repair diabetes-induced wounds to replace invasive treatment methods. The translational potential of this article: The OPN is highly expressed in the tissues surrounding the TTT bone transfer area, which may possibly stimulate the activation of eNOS to increase NO release through the SOCE pathway mediated by Orai1/STIM1. This mechanism may play a significant role in the angiogenesis of diabetic foot's wounds promoted by TTT, providing new therapeutic strategies for the non-surgical treatment for this disease.

Role of thrombospondin-1 in high-salt-induced mesenteric artery endothelial impairment in rats.

The matrix glycoprotein thrombospondin-1 (THBS1) modulates nitric oxide (NO) signaling in endothelial cells. A high-salt diet induces deficiencies of NO production and bioavailability, thereby leading to endothelial dysfunction. In this study we investigated the changes of THBS1 expression and its pathological role in the dysfunction of mesenteric artery endothelial cells (MAECs) induced by a high-salt diet. Wild-type rats, and wild-type and Thbs1-/- mice were fed chow containing 8% w/w NaCl for 4 weeks. We showed that a high salt diet significantly increased THBS1 expression and secretion in plasma and MAECs, and damaged endothelium-dependent vasodilation of mesenteric resistance arteries in wild-type animals, but not in Thbs1-/- mice. In rat MAECs, we demonstrated that a high salt environment (10-40 mM) dose-dependently increased THBS1 expression accompanied by suppressed endothelial nitric oxide synthase (eNOS) and phospho-eNOS S1177 production as well as NO release. Blockade of transforming growth factor-ß1 (TGF-ß1) activity by a TGF-ß1 inhibitor SB 431542 reversed THBS1 up-regulation, rescued the eNOS decrease, enhanced phospho-eNOS S1177 expression, and inhibited Smad4 translocation to the nucleus. By conducting dual-luciferase reporter experiments in HEK293T cells, we demonstrated that Smad4, a transcription promoter, upregulated Thbs1 transcription. We conclude that THBS1 contributes to endothelial dysfunction in a high-salt environment and may be a potential target for treatment of high-salt-induced endothelium dysfunction.

Up-regulation of BDNF/TrkB signaling by δ opioid receptor agonist SNC80 modulates depressive-like behaviors in chronic restraint-stressed mice.

Depressive disorder is a psychiatric disease characterized by its main symptoms of low mood and anhedonia. Due to its complex etiology, current clinical treatments for depressive disorder are limited. In this study, we assessed the role of the δ opioid receptor (δOR) system in the development of chronic-restraint-stressed (CRS)-induced depressive behaviors. We employed a 21-day CRS model and detected the c-fos activation and protein levels' changes in enkephalin (ENK)/δOR. It was found that the hippocampus and amygdala were involved in CRS-induced depression. The expression of pro-enkephalin (PENK), the precursors of the endogenous ligand for δOR, was significantly decreased in the hippocampus and amygdala following CRS. We then treated the mice with SNC80, a specific δOR agonist, to examine its anti-depressant effects in the tail suspension test (TST), forced swimming test (FST), and sucrose preference test (SPT). SNC80 administration significantly reversed depressive-like behaviors, and this antidepressant effect could be blocked by a TrkB inhibitor: ANA-12. Although ANA-12 treatment had no significant effect on the expression of ENK/δOR, it blocked the promoting effects of brain-derived neurotrophic factor (BDNF)/tyrosine kinase B(TrkB) signaling by SNC80 in the hippocampus and amygdala. Therefore, the present study demonstrates that SNC80 exerts anti-depressant effects by up-regulating the BDNF/TrkB signaling pathway in the hippocampus and amygdala in CRS-induced depression and provides evidence that δOR's agonists may be potential anti-depressant therapeutic agents.

A Diagnostic Model for Alzheimer's Disease Based on Blood Levels of Autophagy-Related Genes.

Alzheimer's disease (AD) is a common neurodegenerative disease. The major problems that exist in the diagnosis of AD include the costly examinations and the high-invasive sampling tissue. Therefore, it would be advantageous to develop blood biomarkers. Because AD's pathological process is considered tightly related to autophagy; thus, a diagnostic model for AD based on ATGs may have more predictive accuracy than other models. We obtained GSE63060 dataset from the GEO database, ATGs from the HADb and screened 64 differentially expressed autophagy-related genes (DE-ATGs). We then applied them to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses as well as DisGeNET and PaGenBase enrichment analyses. By using the univariate analysis, least absolute shrinkage and selection operator (LASSO) regression method and the multivariable logistic regression, nine DE-ATGs were identified as biomarkers, which are ATG16L2, BAK1, CAPN10, CASP1, RAB24, RGS19, RPS6KB1, ULK2, and WDFY3. We combined them with sex and age to establish a nomogram model. To evaluate the model's distinguishability, consistency, and clinical applicability, we applied the receiver operating characteristic (ROC) curve, C-index, calibration curve, and on the validation datasets GSE63061, GSE54536, GSE22255, and GSE151371 from GEO database. The results show that our model demonstrates good prediction performance. This AD diagnosis model may benefit both clinical work and mechanistic research.

Parathyroid Hormone Promotes Human Umbilical Vein Endothelial Cell Migration and Proliferation Through Orai1-Mediated Calcium Signaling.

Parathyroid hormone is the main endocrine regulator of extracellular calcium and phosphorus levels. Secondary hyperparathyroidism-induced endothelial dysfunction may be related to calcium homeostasis disorders. Here, we investigated the effects of parathyroid hormone on human umbilical vein endothelial cells (HUVECs) and characterized the involvement of store-operated Ca2+ entry (SOCE) and the nuclear factor of activated T cells (NFAT) signaling pathway. We used immunoblot experiments to find that parathyroid hormone significantly enhanced the expression of the Orai1 channel, a type of channel mediating SOCE, SOCE activity, and Orai1-mediated proliferation of HUVECs but did not increase Orai2 and Orai3. RNA-seq was utilized to identify 1,655 differentially expressed genes (823 upregulated and 832 downregulated) in parathyroid hormone-treated HUVECs as well as enhanced focal adhesion signaling and expression levels of two key genes, namely, COL1A1 and NFATC1. Increased protein and mRNA expression levels of COL1A1 and NFATC1 were confirmed by immunoblotting and quantitative RT-PCR, respectively. Cytosol and nuclei fractionation experiments and immunofluorescence methods were used to show that parathyroid hormone treatment increased NFATC1 nuclear translocation, which was inhibited by a calcineurin inhibitor (CsA), a selective calmodulin antagonist (W7), an Orai channel inhibitor (BTP2), or Orai1 small interfering RNA (siRNA) transfection. Parathyroid hormone also increased COL1A1 expression, cell migration, and proliferation of HUVECs. The PTH-induced increase in HUVEC migration and proliferation were inhibited by CsA, W7, BTP2, or COL1A1 siRNA transfection. These findings indicated that PTH increased Orai1 expression and Orai1-mediated SOCE, causing the nuclear translocation of NFATC1 to increase COL1A1 expression and COL1A1-mediated HUVEC migration and proliferation. These results suggest potential key therapeutic targets of Orai1 and the downstream calmodulin/calcineurin/NFATC1/COL1A1 signaling pathway in parathyroid hormone-induced endothelial dysfunction and shed light on underlying mechanisms that may be altered to prevent or treat secondary hyperparathyroidism-associated cardiovascular disease.

Expression of SH3 and Multiple Ankyrin Repeat Domains Protein 3 in Mouse Retina.

Synapse-associated gene mutations of SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) may lead to autism spectrum disorder (ASD). In some ASD cases, patients may also have vision disorders. However, the effects of SHANK3 in the retina are barely mentioned in the literature. In this study, we used wild-type mice to systematically map the distribution of SHANK3 expression in entire mouse retinas. Using Western blot analysis and immunofluorescence double labeling, we identified a large number of prominent cells expressing high levels of SHANK3 in the inner retina, in particular, the ganglion cell layer (GCL) and inner nucleus layer. The inner plexiform layer and outer nucleus layer were also exhibited positive SHANK3 signals. In the inner layer, GABAergic amacrine cells (ACs) labeled by glutamate decarboxylase were colocalized with SHANK3-positive cells. Dopaminergic ACs (labeled by tyrosine hydroxylase) and cholinergic ACs (labeled by choline acetyltransferase) were also found to contain SHANK3-positive signals. Additionally, most GCs (labeled by Brn3a) were also found to be SHANK3 positive. In the outer retina, bipolar cells (labeled by homeobox protein ChX10) and horizontal cells (labeled by calbindin) were SHANK3 positive. In the outer nucleus layers, the somata of blue cones (labeled by S-opsin) were weekly co-labeled with SHANK3. The inner segments of blue cones and the outer segments of red/green cones (labeled by L/M-opsin) were partially colocalized with SHANK3 and the outer segments of rods (labeled by Rho4D2) were partially SHANK3 positive too. Moreover, SHANK3-positive labeling was also observed in Müller cells (labeled by cellular retinaldehyde-binding protein). These wide expression patterns indicate that SHANK3 may play an important role in the visual signaling pathway.

Oxidative Stress-Induced TRPV2 Expression Increase Is Involved in Diabetic Cataracts and Apoptosis of Lens Epithelial Cells in a High-Glucose Environment.

Cataracts are a serious complication of diabetes. In long-term hyperglycemia, intracellular Ca2+ concentration ([Ca2+]i) and reactive oxygen species (ROS) are increased. The apoptosis of lens epithelial cells plays a key role in the development of cataract. We investigated a potential role for transient receptor potential vanilloid 2 (TRPV2) in the development of diabetic cataracts. Immunohistochemical and Western blotting analyses showed that TRPV2 expression levels were significantly increased in the lens epithelial cells of patients with diabetic cataracts as compared with senile cataract, as well as in both a human lens epithelial cell line (HLEpiC) and primary rat lens epithelial cells (RLEpiCs) cultured under high-glucose conditions. The [Ca2+]i increase evoked by a TRPV2 channel agonist was significantly enhanced in both HLEpiCs and RLEpiCs cultured in high-glucose media. This enhancement was blocked by the TRPV2 nonspecific inhibitor ruthenium red and by TRPV2-specific small interfering (si)RNA transfection. Culturing HLEpiCs or RLEpiCs for seven days in high glucose significantly increased apoptosis, which was inhibited by TRPV2-specific siRNA transfection. In addition, ROS inhibitor significantly suppressed the ROS-induced increase of TRPV2-mediated Ca2+ signal and apoptosis under high-glucose conditions. These findings suggest a mechanism underlying high-glucose-induced apoptosis of lens epithelial cells, and offer a potential target for developing new therapeutic options for diabetes-related cataracts.

Delta Opioid Peptide Targets Brain Microvascular Endothelial Cells Reducing Apoptosis to Relieve Hypoxia-Ischemic/Reperfusion Injury.

Stroke is one of the leading causes of death. (D-ala2, D-leu5) enkephalin (DADLE) is a synthetic peptide and highly selective delta opioid receptor (δOR) agonist that has exhibited protective properties in ischemia. However, the specific target and mechanism are still unclear. The present study explores the expression of δOR on brain microvascular endothelial cells (BMECs) and whether DADLE could relieve I/R-induced injury by reducing apoptosis. A lateral ventricular injection of DADLE for pretreatment, the neurofunctional behavior score, and TTC staining, were used to evaluate the protective effect of DADLE. Immunofluorescence technology was used to label different types of cells with apoptosis-positive signals to test co-localization status. Primary cultured BMECs were separated and treated with DADLE, accompanied by OGD/R. The CCK-8 test was conducted to evaluate cell viability and TdT-mediated dUTP Nick-end Labelling (TUNEL) staining to test apoptosis levels. The levels of apoptosis-related proteins were analyzed by Western blotting. The co-localization results showed that BMECs, but not astrocytes, microglia, or neurons, presented mostly TUNEL-positive signals, especially in the Dentate gyrus (DG) area of the hippocampus. Either activation of δORs on rats' brains or primary BMECs mainly reduce cellular apoptosis and relieve the injury. Interference with the expression δOR could block this effect. DADLE also significantly increased levels of Bcl-2 and reduced levels of Bax. δOR's expressions can be detected on the BMECs, but not on the HEK293 cells, by Western blotting and IFC. Therefore, DADLE exerts a cytoprotective effect, primarily under hypoxia-ischemic injury/reperfusion conditions, by targeting BMECs to inhibit apoptosis.

X-Ray Causes mRNA Transcripts Change to Enhance Orai2-Mediated Ca2+ Influx in Rat Brain Microvascular Endothelial Cells.

Background: Radiation-induced brain injury is a serious and treatment-limiting complication of brain radiation therapy. Although endothelial cell dysfunction plays a critical role in the development of this pathogenesis, the underlying molecular mechanisms remain elusive. Methods: Primary cultured rat brain microvascular endothelial cells (BMECs) were divided into five groups without or with exposure of x-rays delivered at 5 Gy or 20 Gy. For the irradiated groups, cells were continued to cultivate for 12 or 24 h after being irradiated. Then the mRNA libraries of each group were established and applied for next-generation sequencing. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to analyze the sequencing results. Quantitative polymerase chain reaction, western blotting, cck8 assay and intracellular calcium concentration assays were conducted to analyze the role of Orai2-associated SOCE in x-ray induced cellular injury. Results: In total, 3,005 transcripts in all the four x-ray-exposed groups of BMECs showed expression level changes compared with controls. With the dose of x-ray augment and the following cultured time extension, the numbers of differentially expressed genes (DEGs) increased significantly in BMECs. Venn diagrams identified 40 DEGs common to all four exposure groups. Functional pathway enrichment analyses indicated that those 40 DEGs were enriched in the calcium signaling pathway. Among those 40 DEGs, mRNA and protein expression levels of Orai2 were significantly upregulated for 24 h. Similarly, calcium influx via store-operated calcium entry, which is modulated by Orai2, was also significantly increased for 24 h in x-ray-exposed BMECs. Moreover, the change in SOCE was suppressed by btp-2, which is a non-selective inhibitor of Orai. Additionally, x-ray exposure induced a significant decrease of proliferation in BMECs in the dose- and time-dependent manner. Conclusion: These findings provide evidence for molecular mechanisms underlying BMECs dysfunction in development of radiation-induced brain injury and suggest new approaches for therapeutic targets.

Alteration of twinfilin1 expression underlies opioid withdrawal-induced remodeling of actin cytoskeleton at synapses and formation of aversive memory.

Exposure to drugs of abuse induces alterations of dendritic spine morphology and density that has been proposed to be a cellular basis of long-lasting addictive memory and heavily depend on remodeling of its underlying actin cytoskeleton by the actin cytoskeleton regulators. However, the actin cytoskeleton regulators involved and the specific mechanisms whereby drugs of abuse alter their expression or function are largely unknown. Twinfilin (Twf1) is a highly conserved actin-depolymerizing factor that regulates actin dynamics in organisms from yeast to mammals. Despite abundant expression of Twf1 in mammalian brain, little is known about its importance for brain functions such as experience-dependent synaptic and behavioral plasticity. Here we show that conditioned morphine withdrawal (CMW)-induced synaptic structure and behavior plasticity depends on downregulation of Twf1 in the amygdala of rats. Genetically manipulating Twf1 expression in the amygdala bidirectionally regulates CMW-induced changes in actin polymerization, spine density and behavior. We further demonstrate that downregulation of Twf1 is due to upregulation of miR101a expression via a previously unrecognized mechanism involving CMW-induced increases in miR101a nuclear processing via phosphorylation of MeCP2 at Ser421. Our findings establish the importance of Twf1 in regulating opioid-induced synaptic and behavioral plasticity and demonstrate its value as a potential therapeutic target for the treatment of opioid addiction.

Activation of the δ opioid receptor relieves cerebral ischemic injury in rats via EGFR transactivation.

Delta opioids are thought to relieve ischemic injury and have tissue-protective properties. However, the detailed mechanisms of delta opioids have not been well identified. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), have been shown to mediate downstream signals of δ opioid receptor (δOR) activation through the metalloproteinase (MMP)-dependent EGF-like growth factor (HB-EGF) excretion pathway, which is called transactivation. In this study, to investigate the role of EGFR in δOR-induced anti-ischemic effects in the brain, we applied the middle cerebral artery occlusion (MCAO) model followed by reperfusion to mimic ischemic stroke injury in rats. Pre-treatment with the δOR agonist [D-ala2, D-leu5] enkephalin (DADLE) improved the neurologic deficits and the decreased infarct volume caused by cerebral ischemia/reperfusion injury, which were blocked by the EGFR inhibitor AG1478 and the MMP inhibitor GM6001, respectively. Further results indicated that DADLE activated EGFR, Akt and ERK1/2 and upregulated EGFR expression in the hippocampus in a time-dependent manner, which were inhibited by AG1478 and GM6001. The enzyme-linked immunosorbent assay (ELISA) results showed that δOR activation led to an increase in HB-EGF release, but HB-EGF in tissue was downregulated at the mRNA and protein levels. Moreover, this protective action caused by δOR agonists may involve attenuated hippocampal cellular apoptosis. Overall, these results demonstrate that MMP-mediated transactivation of EGFR is essential for δOR agonist-induced MCAO/reperfusion injury relief. These findings provide a potential molecular mechanism for the neuroprotective property of δOR and may add new insight into mitigating or preventing injury.

Blue light insertion at night is involved in sleep and arousal-promoting response delays and depressive-like emotion in mice.

Light plays a direct crucial role in the switch between sleep and arousal and the regulation of physiology and behaviour, such as circadian rhythms and emotional change. Artificial lights, which are different from natural light sources with a continuous light spectrum, are composed of three single-colour lights and are increasingly applied in modern society. However, in vivo research on the mechanisms of blue light-regulated sleep and arousal is still insufficient. In this work, we detected the effects of inserting white or blue light for 1 h during the dark period on the wheel-running activity and sucrose preference of C57 mice. The results showed that blue light could induce delays in sleep and arousal-promoting responses. Furthermore, this lighting pattern, including blue light alone, induced depressive-like emotions. The c-fos expression in the blue light group was significantly higher in the arcuate hypothalamic nucleus (Arc) and significantly lower in the cingulate cortex (Cg) and anterior part of the paraventricular thalamic nucleus (PVA) than in the white light group. Compared with the white light group, the phospho-ERK expression in the paraventricular hypothalamic nucleus (PVN) and PVA was lower in the blue light group. These molecular changes indicated that certain brain regions are involved in blue light-induced response processes. This study may provide useful information to explore the specific mechanism of special light-regulated physiological function.

Differentially expressed transcripts and associated protein pathways in basilar artery smooth muscle cells of the high-salt intake-induced hypertensive rat.

The pathology of cerebrovascular disorders, such as hypertension, is associated with genetic changes and dysfunction of basilar artery smooth muscle cells (BASMCs). Long-term high-salt diets have been associated with the development of hypertension. However, the molecular mechanisms underlying salt-sensitive hypertension-induced BASMC modifications have not been well defined, especially at the level of variations in gene transcription. Here, we utilized high-throughput sequencing and subsequent signaling pathway analyses to find a two-fold change or greater upregulated expression of 203 transcripts and downregulated expression of 165 transcripts in BASMCs derived from rats fed a high-salt diet compared with those from control rats. These differentially expressed transcripts were enriched in pathways involved in cellular, morphological, and structural plasticity, autophagy, and endocrine regulation. These transcripts changes in the BASMCs derived from high-salt intake-induced hypertensive rats may provide critical information about multiple cellular processes and biological functions that occur during the development of cerebrovascular disorders and provide potential new targets to help control or block the development of hypertension.

Propofol decreases the excitability of cholinergic neurons in mouse basal forebrain via GABAA receptors.

Propofol is an intravenous anesthetic that can active γ-aminobutyric acid A (GABAA) receptors and generate sedative-hypnotic effects. Propofol has been widely applied clinically to achieve sedation comparable to sleep in humans. The basal forebrain (BF) is a brain region that plays an important role in sleep-wake regulation. Previous studies suggest that propofol affects the sleep-wake circuit via the BF; however, the mechanism remains elusive. In the current study we investigated the effects of propofol on the inherent properties of cholinergic neurons and their ability to convert excitatory inputs into spikes in mouse BF slices using whole-cell patch clamp recordings. Bath application of propofol (10 µM) significantly elevated the threshold potentials (Vts), decreased the number of spikes in response to a depolarizing current injection, and augmented the inter-spike intervals (ISIs), energy barrier (Vts-Vrs), and absolute refractory periods (ARPs). These effects were eliminated by co-application of a GABAA receptor antagonist picrotoxin (50 µM). Altogether, our results reveal that propofol decreases the excitability of cholinergic neurons in mouse BF via GABAA receptors.

Role for engagement of ß-arrestin2 by the transactivated EGFR in agonist-specific regulation of δ receptor activation of ERK1/2.

BACKGROUND AND PURPOSE: ß-Arrestins function as signal transducers linking GPCRs to ERK1/2 signalling either by scaffolding members of ERK1/2s cascades or by transactivating receptor tyrosine kinases through Src-mediated release of transactivating factor. Recruitment of ß-arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that δ receptors activate ERK1/2 through a ß-arrestin-dependent mechanism without inducing ß-arrestin binding to the δ receptors. However, the precise mechanisms involved remain to be established. EXPERIMENTAL APPROACH: ERK1/2 activation by δ receptor ligands was assessed using HEK293 cells in vitro and male Sprague Dawley rats in vivo. Immunoprecipitation, immunoblotting, siRNA transfection, intracerebroventricular injection and immunohistochemistry were used to elucidate the underlying mechanism. KEY RESULTS: We identified a new signalling pathway in which recruitment of ß-arrestin2 to the EGFR rather than δ receptor was required for its role in δ receptor-mediated ERK1/2 activation in response to H-Tyr-Tic-Phe-Phe-OH (TIPP) or morphine stimulation. Stimulation of the δ receptor with ligands leads to the phosphorylation of PKCδ, which acts upstream of EGFR transactivation and is needed for the release of the EGFR-activating factor, whereas ß-arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCδ/EGFR/ß-arrestin2 transactivation pathway to δ receptor-mediated ERK1/2 activation was ligand-specific and the Ser(363) of δ receptors was crucial for ligand-specific implementation of this ERK1/2 activation pathway. CONCLUSIONS AND IMPLICATIONS: The δ receptor-mediated activation of ERK1/2 is via ligand-specific transactivation of EGFR. This study adds new insights into the mechanism by which δ receptors activate ERK1/2.

Novel κ-opioid receptor agonist MB-1C-OH produces potent analgesia with less depression and sedation.

AIM: To characterize the pharmacological profiles of a novel κ-opioid receptor agonist MB-1C-OH. METHODS: [(3)H]diprenorphine binding and [(35)S]GTPγS binding assays were performed to determine the agonistic properties of MB-1C-OH. Hot plate, tail flick, acetic acid-induced writhing, and formalin tests were conducted in mice to evaluate the antinociceptive actions. Forced swimming and rotarod tests of mice were used to assess the sedation and depression actions. RESULTS: In [(3)H]diprenorphine binding assay, MB-1C-OH did not bind to µ- and δ-opioid receptors at the concentration of 100 µmol/L, but showed a high affinity for κ-opioid receptor (Ki=35 nmol/L). In [(35)S]GTPγS binding assay, the compound had an Emax of 98% and an EC50 of 16.7 nmol/L for κ-opioid receptor. Subcutaneous injection of MB-1C-OH had no effects in both hot plate and tail flick tests, but produced potent antinociception in the acetic acid-induced writhing test (ED50=0.39 mg/kg), which was antagonized by pretreatment with a selective κ-opioid receptor antagonist Nor-BNI. In the formalin test, subcutaneous injection of MB-1C-OH did not affect the flinching behavior in the first phase, but significantly inhibited that in the second phase (ED50=0.87 mg/kg). In addition, the sedation or depression actions of MB-1C-OH were about 3-fold weaker than those of the classical κ agonist (-)U50,488H. CONCLUSION: MB-1C-OH is a novel κ-opioid receptor agonist that produces potent antinociception causing less sedation and depression.

Serine 363 of the {delta}-opioid receptor is crucial for adopting distinct pathways to activate ERK1/2 in response to stimulation with different ligands.

Distinct opioid receptor agonists have been proved to induce differential patterns of ERK activation, but the underlying mechanisms remain unclear. Here, we report that Ser363 in the δ-opioid receptor (δOR) determines the different abilities of the δOR agonists DPDPE and TIPP to activate ERK by G-protein- or ß-arrestin-dependent pathways. Although both DPDPE and TIPP activated ERK1/2, they showed different temporal, spatial and desensitization patterns of ERK activation. We show that that DPDPE employed G protein as the primary mediator to activate the ERK cascade in an Src-dependent manner, whereas TIPP mainly adopted a ß-arrestin1/2-mediated pathway. Moreover, we found that DPDPE gained the capacity to adopt the ß-arrestin1/2-mediated pathway upon Ser363 mutation, accompanied by the same pattern of ERK activation as that induced by TIPP. Additionally, we found that TIPP- but not DPDPE-activated ERK could phosphorylate G-protein-coupled receptor kinase-2 and ß-arrestin1. However, such functional differences of ERK disappeared with the mutation of Ser363. Therefore, the present study reveals a crucial role for Ser363 in agonist-specific regulation of ERK activation patterns and functions.