Astrocyte D-serine modulates the activation of neuronal NOS leading to the development of mechanical allodynia in peripheral neuropathy.

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the N-Methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here, we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt (LSOS) or the D-serine degrading enzyme, D-amino acid oxidase (DAAO) on post-operative days 0-3 significantly reduced the CCI-induced increase in nitric oxide (NO) levels and nicotinamide adenine dinucleotide phosphate-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and protein kinase C (PKC)-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1). In naïve mice, exogenous D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechanical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively, we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1 and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

Yes-associated protein mediates human embryonic stem cell-derived cardiomyocyte proliferation: Involvement of epidermal growth factor receptor signaling.

Unlike mature cardiomyocytes, human pluripotent stem cell-derived cardiomyocytes exhibit higher proliferative capacity; however, the underlying mechanisms involved are yet to be elucidated. Here, we revealed that the Yes-associated protein (YAP) plays a critical role in regulating cell proliferation in association with epidermal growth factor receptor (EGFR) in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Our results show that low-density culture significantly promotes the proliferation of hESC-CMs via YAP. Interestingly, the low-density culture-induced YAP expression further induced EGFR expression, without any alterations in the activity of EGFR and its two major downstream kinases, ERK, and AKT. However, treatment of a low-density-culture of hESC-CMs with epidermal growth factor (EGF) increased proliferation via phosphorylation of EGFR, ERK, and AKT, and the EGF-induced phosphorylation of EGFR, ERK, and AKT was significantly higher in low-density hESC-CMs than in high-density hESC-CMs. Furthermore, the EGF-induced activation of EGFR, ERK, and AKT increased YAP expression and subsequently proliferation. In conclusion, YAP mediates both low-density culture-induced and EGF-induced proliferation of hESC-CMs in low-density culture conditions.

Neuronal NOS Activates Spinal NADPH Oxidase 2 Contributing to Central Sigma-1 Receptor-Induced Pain Hypersensitivity in Mice.

We recently demonstrated that activation of spinal sigma-1 receptors (Sig-1Rs) induces pain hypersensitivity via the activation of neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2). However, the potential direct interaction between nNOS-derived nitric oxide (NO) and Nox2-derived reactive oxygen species (ROS) is poorly understood, particularly with respect to the potentiation of N-methyl-D-aspartate (NMDA) receptor activity in the spinal cord associated with the development of central sensitization. Thus, the main purpose of this study was to investigate whether Sig-1R-induced and nNOS-derived NO modulates spinal Nox2 activation leading to an increase in ROS production and ultimately to the potentiation of NMDA receptor activity and pain hypersensitivity. Intrathecal pretreatment with the nNOS inhibitor, 7-nitroindazole or with the Nox inhibitor, apocynin significantly inhibited the mechanical and thermal hypersensitivity induced by intrathecal administration of the Sig-1R agonist, 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride (PRE084). Conversely, pretreatment with 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron(III) (FeTPPS; a scavenger of peroxynitrite, a toxic reaction product of NO and superoxide) had no effect on the PRE084-induced pain hypersensitivity. Pretreatment with 7-nitroindazole significantly reduced the PRE084-induced increase in Nox2 activity and concomitant ROS production in the lumbar spinal cord dorsal horn, whereas apocynin did not alter the PRE084-induced changes in nNOS phosphorylation. On the other hand pretreatment with apocynin suppressed the PRE084-induced increase in the protein kinase C (PKC)-dependent phosphorylation of NMDA receptor GluN1 subunit (pGluN1) at Ser896 site in the dorsal horn. These findings demonstrate that spinal Sig-1R-induced pain hypersensitivity is mediated by nNOS activation, which leads to an increase in Nox2 activity ultimately resulting in a ROS-induced increase in PKC-dependent pGluN1 expression.

High glucose-induced O-GlcNAcylated carbohydrate response element-binding protein (ChREBP) mediates mesangial cell lipogenesis and fibrosis: the possible role in the development of diabetic nephropathy.

Carbohydrate response element-binding protein (ChREBP) is a transcription factor responsible for carbohydrate metabolism in the liver. However, the role of ChREBP in diabetic nephropathy has not been elucidated. Thus, we investigated the role of ChREBP in mesangial cells in diabetic nephropathy. Treatment with 25 mM glucose (high glucose; HG) increased cellular O-GlcNAc and O-GlcNAcylated ChREBP in mesangial cells compared with normal 5.5 mM glucose. O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate (PUGNAc), a drug that increases O-GlcNAc, augmented the expression of ChREBP targets, whereas DON, a drug that decreases O-GlcNAc and O-GlcNAcase overexpression, mitigated the increase with HG. O-GlcNAc augmented the protein stability, transcriptional activity, and nuclear translocation of ChREBP. HG treatment also stimulated lipid accumulation and the contents of triglyceride and cholesterol in mesangial cells. In addition, HG triggered expression of hypoxia-inducible factor 1-α, vascular endothelial growth factor, and extracellular matrix components related to nephrosclerosis. The ChREBP mutant, W130A, did not exhibit HG-induced lipid accumulation and fibrotic proteins, suggesting that the Trp-130 residue in the MCR3 domain is important in the development of glomerulosclerosis. O-GlcNAcylated ChREBP was elevated in mesangium cells of streptozotocin-induced diabetic rats. In conclusion, HG increased the O-GlcNAcylated ChREBP level, which resulted in lipid accumulation and up-regulation of fibrotic proteins in mesangial cells. These effects may lead mesangial cells to an ultimately pathological state.

Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.

Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the 'fed' condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology.

Spinal sigma-1 receptor activation increases the production of D-serine in astrocytes which contributes to the development of mechanical allodynia in a mouse model of neuropathic pain.

We have previously demonstrated that activation of the spinal sigma-1 receptor (Sig-1R) plays an important role in the development of mechanical allodynia (MA) via secondary activation of the N-methyl-d-aspartate (NMDA) receptor. Sig-1Rs have been shown to localize to astrocytes, and blockade of Sig-1Rs inhibits the pathologic activation of astrocytes in neuropathic mice. However, the mechanism by which Sig-1R activation in astrocytes modulates NMDA receptors in neurons is currently unknown. d-serine, synthesized from l-serine by serine racemase (Srr) in astrocytes, is an endogenous co-agonist for the NMDA receptor glycine site and can control NMDA receptor activity. Here, we investigated the role of d-serine in the development of MA induced by spinal Sig-1R activation in chronic constriction injury (CCI) mice. The production of d-serine and Srr expression were both significantly increased in the spinal cord dorsal horn post-CCI surgery. Srr and d-serine were only localized to astrocytes in the superficial dorsal horn, while d-serine was also localized to neurons in the deep dorsal horn. Moreover, we found that Srr exists in astrocytes that express Sig-1Rs. The CCI-induced increase in the levels of d-serine and Srr was attenuated by sustained intrathecal treatment with the Sig-1R antagonist, BD-1047 during the induction phase of neuropathic pain. In behavioral experiments, degradation of endogenous d-serine with DAAO, or selective blockade of Srr by LSOS, effectively reduced the development of MA, but not thermal hyperalgesia in CCI mice. Finally, BD-1047 administration inhibited the development of MA and this inhibition was reversed by intrathecal treatment with exogenous d-serine. These findings demonstrate for the first time that the activation of Sig-1Rs increases the expression of Srr and d-serine in astrocytes. The increased production of d-serine induced by CCI ultimately affects dorsal horn neurons that are involved in the development of MA in neuropathic mice.

Acid evoked thermal hyperalgesia involves peripheral P2Y1 receptor mediated TRPV1 phosphorylation in a rodent model of thrombus induced ischemic pain.

BACKGROUND: We previously developed a thrombus-induced ischemic pain (TIIP) animal model, which was characterized by chronic bilateral mechanical allodynia without thermal hyperalgesia (TH). On the other hand we had shown that intraplantar injection of acidic saline facilitated ATP-induced pain, which did result in the induction of TH in normal rats. Because acidic pH and increased ATP are closely associated with ischemic conditions, this study is designed to: (1) examine whether acidic saline injection into the hind paw causes the development of TH in TIIP, but not control, animals; and (2) determine which peripheral mechanisms are involved in the development of this TH. RESULTS: Repeated intraplantar injection of pH 4.0 saline, but not pH 5.5 and 7.0 saline, for 3 days following TIIP surgery resulted in the development of TH. After pH 4.0 saline injections, protein levels of hypoxia inducible factor-1α (HIF-1α) and carbonic anhydrase II (CA II) were elevated in the plantar muscle indicating that acidic stimulation intensified ischemic insults with decreased tissue acidity. At the same time point, there were no changes in the expression of TRPV1 in hind paw skin, whereas a significant increase in TRPV1 phosphorylation (pTRPV1) was shown in acidic saline (pH 4.0) injected TIIP (AS-TIIP) animals. Moreover, intraplantar injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1 antagonist) effectively alleviated the established TH. In order to investigate which proton- or ATP-sensing receptors contributed to the development of TH, amiloride (an ASICs blocker), AMG9810, TNP-ATP (a P2Xs antagonist) or MRS2179 (a P2Y1 antagonist) were pre-injected before the pH 4.0 saline. Only MRS2179 significantly prevented the induction of TH, and the increased pTRPV1 ratio was also blocked in MRS2179 injected animals. CONCLUSION: Collectively these data show that maintenance of an acidic environment in the ischemic hind paw of TIIP rats results in the phosphorylation of TRPV1 receptors via a PKC-dependent pathway, which leads to the development of TH mimicking what occurs in chronic ischemic patients with severe acidosis. More importantly, peripheral P2Y1 receptors play a pivotal role in this process, suggesting a novel peripheral mechanism underlying the development of TH in these patients.

Thioredoxin-interacting protein mediates hepatic lipogenesis and inflammation via PRMT1 and PGC-1α regulation in vitro and in vivo.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and type 2 diabetes. Thioredoxin-interacting protein (TXNIP) regulates the cellular redox state and metabolism and has been linked to many diseases, including diabetes. Therefore, we examined the role of TXNIP in hepatic steatosis in vitro and in vivo. METHODS: Lipogenic and inflammatory proteins produced by hepatocytes treated with palmitic acid (PA) or transfected with TXNIP or Txnip siRNA were measured by Western blotting. Lipid accumulation was assessed using Oil Red O staining. Protein interactions were assessed by immunoprecipitation and proximity ligation assay. Hepatic protein levels were measured by Western blotting from wild type or Txnip(-/-) mice fed a high-fat diet (HFD) or chow diet. Livers from NAFLD patients were compared with normal liver by immunohistochemistry. RESULTS: PA increased TXNIP, and inflammatory and lipogenic proteins in both AML12 and H4IIE cells. It also increased the peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α), which mediated the expression of lipogenic markers and lipid accumulation. In addition, PA increased protein arginine methyltransferase-1 (PRMT1) and PRMT1 siRNA abolished the increase in lipogenic markers with PGC-1α. Furthermore, TXNIP interacted with PRMT1 in PA-treated hepatocytes. In vivo, levels of lipogenic proteins, inflammatory molecules, PGC-1α, and PRMT1 were increased in the livers of HFD mice compared with those fed a chow diet, and were ameliorated in HFD Txnip(-/-) mice. Moreover, TXNIP, PRMT1, and PGC-1α were elevated in the livers of human NAFLD patients. CONCLUSIONS: TXNIP mediates hepatic lipogenesis via PRMT1 and PGC-1α regulation and inflammation in vitro and in vivo, implying that targeting TXNIP and PRMT1 is a potential therapeutic approach for treatment of NAFLD.

High-glucose-induced CARM1 expression regulates apoptosis of human retinal pigment epithelial cells via histone 3 arginine 17 dimethylation: role in diabetic retinopathy.

Hyperglycemia-induced apoptosis of retinal pigment epithelial (RPE) cells is considered to be involved in the progression of diabetic retinopathy. Histone arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has emerged as an important histone modification involved in gene regulation. However, the role of PRMTs in diabetic retinopathy has not been elucidated. Here, we found that expression of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) was increased in the high-glucose treated human RPE cell line ARPE-19 and in the RPE layer of streptozotocin-treated rats. In addition, high-glucose induced apoptosis in ARPE-19 cells. To determine the function of CARM1 on RPE cell apoptosis, we performed gain- and loss-of-function studies. CARM1 overexpression increased apoptosis of RPE cells. In contrast, silencing of CARM1 expression by siRNA and pharmacological inhibition of CARM1 activity abolished high-glucose-induced RPE cell apoptosis. Furthermore, we found that inhibition of histone 3 arginine 17 (H3R17) asymmetric dimethylation attenuates both CARM1- and high-glucose-induced apoptosis in RPE cells. Together, these results show that high-glucose-induced CARM1 expression increases RPE cell apoptosis via H3R17 asymmetric dimethylation. Strategies to reduce CARM1 expression or enzymatic activity could be used to prevent apoptosis of RPE cells in the progression of diabetic retinopathy.

Transformation of somatic cells into stem cell-like cells under a stromal niche.

To study the genomic plasticity of somatic cells without ectopic genetic manipulation, we cultured mouse fibroblasts with ovarian cells, embryonic fibroblasts of different strains, and parthenogenetic embryonic stem cells (ESCs). Of 41 trials, cell aggregation resembling nascent ESC colony from inner cell mass was detected in 9 cases (22%), and 6 cases (67%) yielded fibroblast-derived colonies with ESC morphology. Cells used in coculture provided the critical (P=0.0061) inducing factor for the aggregation. These colony-forming fibroblasts (CFFs) showed similar characteristics to those in ESCs and induced pluripotent stem cells (iPSCs), including pluripotency gene expression, in vitro differentiation, and teratoma formation. Furthermore, CFFs produced somatic chimera, although none showed germline chimerism. CFFs had a tetraploid-like karyotype, and their imprinting patterns differed from parthenogenetic ESCs, thereby confirming their nongermline transmissibility. We observed dysregulation of cell cycle-related proteins, as well as both homologous and heterologous recombination of genomic single-nucleotide polymorphisms in CFFs. Our observations provide information on somatic cell plasticity, resulting in stemness or tumorigenesis, regardless of colony-forming cell progenitors in the fibroblast population. The plasticity of somatic genomes under environmental influences, as well as acquisition of pluripotency by cell fusion, is also implicated.

Spinal sigma-1 receptors activate NADPH oxidase 2 leading to the induction of pain hypersensitivity in mice and mechanical allodynia in neuropathic rats.

We have recently demonstrated that spinal sigma-1 receptors (Sig-1Rs) mediate pain hypersensitivity in mice and neuropathic pain in rats. In this study, we examine the role of NADPH oxidase 2 (Nox2)-induced reactive oxygen species (ROS) on Sig-1R-induced pain hypersensitivity and the induction of chronic neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. Mechanical allodynia and thermal hyperalgesia were evaluated in mice and CCI-rats. Western blotting and dihydroethidium (DHE) staining were performed to assess the changes in Nox2 activation and ROS production in spinal cord, respectively. Direct activation of spinal Sig-1Rs with the Sig-1R agonist, PRE084 induced mechanical allodynia and thermal hyperalgesia, which were dose-dependently attenuated by pretreatment with the ROS scavenger, NAC or the Nox inhibitor, apocynin. PRE084 also induced an increase in Nox2 activation and ROS production, which were attenuated by pretreatment with the Sig-1R antagonist, BD1047 or apocynin. CCI-induced nerve injury produced an increase in Nox2 activation and ROS production in the spinal cord, all of which were attenuated by intrathecal administration with BD1047 during the induction phase of neuropathic pain. Furthermore, administration with BD1047 or apocynin reversed CCI-induced mechanical allodynia during the induction phase, but not the maintenance phase. These findings demonstrate that spinal Sig-1Rs modulate Nox2 activation and ROS production in the spinal cord, and ultimately contribute to the Sig-1R-induced pain hypersensitivity and the peripheral nerve injury-induced induction of chronic neuropathic pain.

Statins Enhance the Molecular Response in Chronic Myeloid Leukemia when Combined with Tyrosine Kinase Inhibitors.

Previous studies have suggested that statins can be repurposed for cancer treatment. However, the therapeutic efficacy of statins in chronic myeloid leukemia (CML) has not yet been demonstrated. In this study, we retrospectively evaluated the outcomes of 408 CML patients who underwent imatinib therapy. The deep molecular response rates in patients treated with the statin/TKI combination were significantly higher than those in patients treated with TKI alone (p = 0.0016). The statin/TKI combination exerted potent cytotoxic effects against wild-type and ABL1 mutant CML, BaF3, and K562/T315I mutant cells. Furthermore, the statin/TKI combination additively inhibited the colony-forming capacity of murine CML-KLS+ cells in vitro. In addition, we examined the additive growth-inhibitory effects of the statin/tyrosine kinase inhibitor (TKI) combination against CML patient-derived CD34+ cells. The growth-inhibitory effects of the statin/imatinib combination against CD34+/CML primary cells were higher than those against CD34+/Norm cells (p = 0.005), suggesting that the combination of rosuvastatin and imatinib exerted growth-inhibitory effects against CML CD34+ cells, but not against normal CD34+ cells. Furthermore, results from RNA sequencing of control and statin-treated cells suggested that statins inhibited c-Myc-mediated and hematopoietic cell differentiation pathways. Thus, statins can be potentially repurposed to improve treatment outcomes in CML patients when combined with TKI therapy.

Natural Products in the Prevention of Metabolic Diseases: Lessons Learned from the 20th KAST Frontier Scientists Workshop.

The incidence of metabolic and chronic diseases including cancer, obesity, inflammation-related diseases sharply increased in the 21st century. Major underlying causes for these diseases are inflammation and oxidative stress. Accordingly, natural products and their bioactive components are obvious therapeutic agents for these diseases, given their antioxidant and anti-inflammatory properties. Research in this area has been significantly expanded to include chemical identification of these compounds using advanced analytical techniques, determining their mechanism of action, food fortification and supplement development, and enhancing their bioavailability and bioactivity using nanotechnology. These timely topics were discussed at the 20th Frontier Scientists Workshop sponsored by the Korean Academy of Science and Technology, held at the University of Hawaii at Manoa on 23 November 2019. Scientists from South Korea and the U.S. shared their recent research under the overarching theme of Bioactive Compounds, Nanoparticles, and Disease Prevention. This review summarizes presentations at the workshop to provide current knowledge of the role of natural products in the prevention and treatment of metabolic diseases.

Glypican 3 binds to GLUT1 and decreases glucose transport activity in hepatocellular carcinoma cells.

Glypican 3 (GPC3), a member of heparin sulfate proteoglycans, is attached to the cell surface by a glycosylphosphatidylinositol anchor and is reported to be overexpressed in liver cancers. In order to identify GPC3 binding proteins on the cell surface, we constructed a cDNA containing the C-terminal cell surface-attached form of GPC3 (GPC3c) in a baculoviral vector. The GPC3c bait protein was produced by expressing the construct in Sf21 insect cells and double purified using a His column and Flag immunoprecipitation. Purified GPC3c was used to uncover GPC3c-interacting proteins. Using an LC-MS/MS proteomics strategy, we identified glucose transporter 1 (GLUT1) as a novel GPC3 interacting protein from the HepG2 hepatoma cell lysates. The interaction was confirmed by immunoprecipitation (IP)-WB analysis and surface plasmon resonance (SPR). SPR result showed the interaction of GLUT1 to GPC3c with equilibrium dissociation constants (K(D) ) of 1.61 nM. Moreover, both incubation with GPC3c protein and transfection of Gpc3c cDNA into HepG2 cells resulted in reduced glucose uptake activity. Our results indicate that GPC3 plays a role in glucose transport by interacting with GLUT1.

Activation of spinal alpha-2 adrenoceptors, but not mu-opioid receptors, reduces the intrathecal N-methyl-D-aspartate-induced increase in spinal NR1 subunit phosphorylation and nociceptive behaviors in the rat.

BACKGROUND: A previous study from our laboratories showed that a significant reduction in spinal N-methyl-D-aspartate (NMDA) receptor NR1 subunit phosphorylation (pNR1) is associated with the antiallodynic effect produced by intrathecal (IT) injection of the alpha-2 adrenoceptor agonist, clonidine, in neuropathic rats. In this study, we determined whether the spontaneous pain and increased pNR1 expression induced by NMDA injection are reduced by IT injection of either clonidine or the mu-opioid receptor agonist, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO). METHODS: We examined the effect of clonidine (20 microg/rat) or DAMGO (1 microg/rat) injection on IT NMDA-induced spontaneous nociceptive behavior and pNR1 expression in the spinal dorsal horn. We also determined whether the effect of clonidine is mediated by alpha-2A or alpha-2C adrenoceptors. Finally, rat spinal cords were immunohistochemically processed for double staining of pNR1 and alpha-2A or alpha-2C adrenoceptors or mu-opioid receptors. RESULTS: The NMDA-induced increase in both pNR1 expression and nociceptive behavior was significantly reduced by IT clonidine but not DAMGO. This analgesic effect of clonidine was blocked by administration of either an alpha-2A (BRL44408, 30 microg/rat) or an alpha-2C (JP-1302, 50 microg/rat) adrenoceptor antagonist. In addition, immunocytochemistry revealed that spinal pNR1 immunoreactive cells co-contain alpha-2A and alpha-2C adrenoceptors. CONCLUSIONS: These results demonstrate that the IT NMDA-induced increase in pNR1 expression and nociceptive behavior is significantly reduced by activation of alpha-2 adrenoceptors, but not mu-opioid receptors, in the spinal cord dorsal horn. Furthermore, these findings suggest that the modulation of spinal NR1 phosphorylation is linked to the effect of IT clonidine on postsynaptic neuronal activity.

Spinal Interleukin-1ß Inhibits Astrocyte Cytochrome P450c17 Expression Which Controls the Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain.

We have recently demonstrated that sciatic nerve injury increases the expression of spinal cytochrome P450c17, a key neurosteroidogenic enzyme, which plays a critical role in the development of peripheral neuropathic pain. However, the modulatory mechanisms responsible for the expression of spinal P450c17 have yet to be examined. Here we investigated the possible involvement of interleukin-1ß (IL-1ß) in altering P450c17 expression during the induction phase of neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in mice and mechanical allodynia was evaluated in the hind paws using a von-Frey filament (0.16 g). Western blotting and immunohistochemistry were performed to assess the expression of spinal IL-1ß, interleukin-1 receptor type 1 (IL-1R1), P450c17, and GFAP. Spinal IL-1ß was significantly increased on day 1 post-surgery and its receptor, IL-1R1 was expressed in GFAP-positive astrocytes. Intrathecal administration of the recombinant interleukin-1 receptor antagonist (IL-1ra, 20 ng) on days 0 and 1 post-surgery enhanced GFAP expression on day 1 post-surgery and induced an early increase in P450c17 expression in astrocytes, but not in neurons. Administration of IL-1ß (10 ng) on days 0 and 1 post-surgery blocked the enhancement of both spinal P450c17 and GFAP expression induced by IL-1ra (20 ng) administration. Intrathecal administration of IL-1ra (20 ng) on days 0 to 3 post-surgery also facilitated the CCI-induced development of mechanical allodynia, and this early developed pain was dose-dependently attenuated by the administration of the P450c17 inhibitor, ketoconazole (1, 3, or 10 nmol) or the astrocyte metabolic inhibitor, fluorocitrate (0.01, 0.03, or 0.1 nmol). These results demonstrate that early increases in spinal IL-1ß temporally inhibit astrocyte P450c17 expression and astrocyte activation ultimately controlling the development of mechanical allodynia induced by peripheral nerve injury. These findings imply that spinal IL-1ß plays an important role as an early, but transient, control mechanism in the development of peripheral neuropathic pain via the inhibition of astrocyte P450c17 expression and astrocyte activation.

nNOS-PSD95 interactions activate the PKC-ε isoform leading to increased GluN1 phosphorylation and the development of neuropathic mechanical allodynia in mice.

It has been suggested that interactions of neuronal nitric oxide synthase (nNOS) with postsynaptic density 95 (PSD95) play important roles in the development of chronic neuropathic pain. Here we examine the possible role of nNOS-PSD95 interactions in central sensitization as represented by phosphorylation of the NMDA receptor GluN1 subunit (pGluN1) in mice with chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the nNOS-PSD95 interactions inhibitor, IC87201 on post-operative days 0-3 significantly reduced the CCI-induced increase in total NO levels in the lumbar spinal cord dorsal horn. IC87201 administration on post-operative days 0-3 also attenuated the CCI-induced development of mechanical allodynia (MA) and PKC-dependent (Ser896) pGluN1. Sciatic nerve injury elicited a significant translocation of the PKC-ε isoform from the cytosol to the membrane fraction in the lumbar spinal cord dorsal horn on day 3 post-CCI surgery. Administration of IC87201 significantly inhibited this translocation of PKC-ε, while the expression of PKC-α and -ξ in the cytosol and membrane fractions was unaffected by sciatic nerve injury or injection of IC87201. Furthermore, administration of the PKC-ε inhibitor, εV1-2 on post-operative days 0-3 attenuated the CCI-induced development of MA and pGluN1. Collectively these results demonstrate that spinal nNOS-PSD95 interactions play an important role in PKC-dependent GluN1 phosphorylation via activation of the PKC-ε isoform, and ultimately contributes to the development of MA in peripheral neuropathy.

Spinal cytochrome P450c17 plays a key role in the development of neuropathic mechanical allodynia: Involvement of astrocyte sigma-1 receptors.

While evidence indicates that sigma-1 receptors (Sig-1Rs) play an important role in the induction of peripheral neuropathic pain, there is limited understanding of the role that the neurosteroidogenic enzymes, which produce Sig-1R endogenous ligands, play during the development of neuropathic pain. We examined whether sciatic nerve injury upregulates the neurosteroidogenic enzymes, cytochrome P450c17 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which modulate the expression and/or activation of Sig-1Rs leading to the development of peripheral neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of P450c17, but not 3ß-HSD, in the ipsilateral lumbar spinal cord dorsal horn at postoperative day 3. Intrathecal administration of the P450c17 inhibitor, ketoconazole during the induction phase of neuropathic pain (day 0 to day 3 post-surgery) significantly reduced the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw. However, administration of the 3ß-HSD inhibitor, trilostane had no effect on the development of neuropathic pain. Sciatic nerve injury increased astrocyte Sig-1R expression as well as dissociation of Sig-1Rs from BiP in the spinal cord. These increases were suppressed by administration of ketoconazole, but not by administration of trilostane. Co-administration of the Sig-1R agonist, PRE084 restored the development of mechanical allodynia originally suppressed by the ketoconazole administration. However, ketoconazole-induced inhibition of thermal hyperalgesia was not affected by co-administration of PRE084. Collectively these results demonstrate that early activation of P450c17 modulates the expression and activation of astrocyte Sig-1Rs, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.

Intrathecal administration of sigma-1 receptor agonists facilitates nociception: involvement of a protein kinase C-dependent pathway.

Sigma sites, originally proposed as opioid receptor subtypes, are currently thought to represent unique receptors with a specific pattern of drug selectivity, a well-established anatomical distribution and broad range of functional roles including potential involvement in nociceptive mechanisms. We have recently demonstrated that intrathecal (i.t.) treatment with a sigma-1 receptor antagonist reduced formalin-induced pain behavior. In the present study, we investigated the potential role of spinal sigma-1 receptor agonists in peripherally initiated nociception and attempted to elucidate intracellular signaling mechanisms associated with spinal cord sigma-1 receptor activation in mice. The i.t. injection of the sigma-1 receptor agonists PRE-084 (PRE) or carbetapentane (CAR) significantly decreased tail-flick latency (TFL) and increased the frequency of paw withdrawal responses to mechanical stimulation (von Frey filament, 0.6 g) as well as the amount of Fos expression in the spinal cord dorsal horn induced by noxious paw-pinch stimulation. These PRE- or CAR-induced facilitatory effects on nociception were significantly blocked by i.t. pretreatment with the sigma-1 receptor antagonist, BD-1047, the phospholipase C (PLC) inhibitor, U-73,122, the Ca(2+)-ATPase inhibitor, thapsigargin, and the protein kinase C (PKC) inhibitor, chelerythrine. Western blot analysis further revealed that i.t. PRE or CAR injection significantly increased pan-PKC as well as the PKCalpha, epsilon, and zeta isoforms in the dorsal horn. Collectively, these findings demonstrate that calcium-dependent second messenger cascades including PKC are involved in the facilitation of nociception associated with spinal sigma-1 receptor activation.