Brain-enriched guanylate kinase-associated protein, a component of the post-synaptic density protein complexes, contributes to learning and memory.

Brain-enriched guanylate kinase-associated protein (BEGAIN) is highly enriched in the post-synaptic density (PSD) fraction and was identified in our previous study as a protein associated with neuropathic pain in the spinal dorsal horn. PSD protein complexes containing N-methyl-D-aspartate receptors are known to be involved in neuropathic pain. Since these PSD proteins also participate in learning and memory, BEGAIN is also expected to play a crucial role in this behavior. To verify this, we first examined the distribution of BEGAIN in the brain. We found that BEGAIN was widely distributed in the brain and highly expressed in the dendritic regions of the hippocampus. Moreover, we found that BEGAIN was concentrated in the PSD fraction of the hippocampus. Furthermore, immunoelectron microscopy confirmed that BEGAIN was localized at the asymmetric synapses. Behavioral tests were performed using BEGAIN-knockout (KO) mice to determine the contribution of BEGAIN toward learning and memory. Spatial reference memory and reversal learning in the Barns circular maze test along with contextual fear and cued fear memory in the contextual and cued fear conditioning test were significantly impaired in BEGAIN-KO mice compared to with those in wild-type mice. Thus, this study reveals that BEGAIN is a component of the post-synaptic compartment of excitatory synapses involved in learning and memory.

Efficacy of Nanofiber Sheets Incorporating Lenvatinib in a Hepatocellular Carcinoma Xenograft Model.

Lenvatinib has a high response rate in unresectable advanced hepatocellular carcinoma (HCC). In this study, we investigated whether lenvatinib-incorporating poly(ε-caprolactone) sheets (lenvatinib sheets) as a drug delivery system (DDS) exerted antitumor effects in a murine HCC model. The lenvatinib sheets were designed for sustained release of approximately 1 mg lenvatinib for 14 days. For 14 days, 1 mg lenvatinib was orally administered to mice. Then, we compared the antitumor effects of lenvatinib sheets with those of oral lenvatinib. The tumor volume, body weight, and serum lenvatinib level were measured for 14 days. A peritoneal dissemination model was established to examine the survival prolongation effect of the lenvatinib sheets. Tumor growth was significantly inhibited in the lenvatinib sheet group compared with that in the no treatment and oral groups. The antitumor effect was significantly higher in the lenvatinib sheet group. Regardless of the insertion site, the serum lenvatinib levels were maintained and showed similar antitumor effects. The mitotic index was significantly inhibited in the lenvatinib sheet group compared with that in the control group. Furthermore, lenvatinib sheets improved the 30-day survival. Lenvatinib sheets showed sufficient antitumor effects and may serve as an effective novel DDS for advanced HCC.

Combination therapy with lenvatinib and radiation significantly inhibits thyroid cancer growth by uptake of tyrosine kinase inhibitor.

Although surgical treatment cures >90% of differentiated thyroid cancer (DTC) patients, the remaining patients, including advanced DTC cases, have poor clinical outcomes. These patients with inoperable disease have only two choices of radioactive iodine therapy and tyrosine kinase inhibitors such as lenvatinib, which have a high incidence of treatment-related adverse events and can only prolong progression free survival by approximately 5-15 months. In this study, we investigated the antitumor effects of combination therapy with lenvatinib and radiation (CTLR) for DTC. CTLR synergistically inhibited cell replication and colony formation in vitro and tumor growth in nude mice without apparent toxicities and suppressed the expression of proliferation marker (Ki-67). CTLR also induced apoptosis and G2/M phase cell cycle arrest. Moreover, quantitative analysis of the intracellular uptake of lenvatinib using liquid chromatography and mass spectrometry demonstrated that intracellular uptake of lenvatinib was significantly increased 48 h following irradiation. These data suggest that increased membrane permeability caused by irradiation increases the intracellular concentration of levatinib, contributing to the synergistic effect. This mechanism-based potential of combination therapy suggests a powerful new therapeutic strategy for advanced thyroid cancer with fewer side effects and might be a milestone for developing a regimen in clinical practice.

Diabetic neuropathy research: from mouse models to targets for treatment.

Diabetic neuropathy is one of the most serious complications of diabetes, and its increase shows no sign of stopping. Furthermore, current clinical treatments do not yet approach the best effectiveness. Thus, the development of better strategies for treating diabetic neuropathy is an urgent matter. In this review, we first discuss the advantages and disadvantages of some major mouse models of diabetic neuropathy and then address the targets for mechanism-based treatment that have been studied. We also introduce our studies on each part. Using stem cells as a source of neurotrophic factors to target extrinsic factors of diabetic neuropathy, we found that they present a promising treatment.

Distribution of Caskin1 protein and phenotypic characterization of its knockout mice using a comprehensive behavioral test battery.

Calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1 (Caskin1) is a direct binding partner of the synaptic adaptor protein CASK. Because Caskin1 forms homo-multimers and binds not only CASK but also other neuronal proteins in vitro, it is anticipated to have neural functions; but its exact role in mammals remains unclear. Previously, we showed that the concentration of Caskin1 in the spinal dorsal horn increases under chronic pain. To characterize this protein, we generated Caskin1-knockout (Caskin1-KO) mice and specific anti-Caskin1 antibodies. Biochemical and immunohistochemical analyses demonstrated that Caskin1 was broadly distributed in the whole brain and spinal cord, and that it primarily localized at synapses. To elucidate the neural function of Caskin1 in vivo, we subjected Caskin1-KO mice to comprehensive behavioral analysis. The mutant mice exhibited differences in gait, enhanced nociception, and anxiety-like behavior relative to their wild-type littermates. In addition, the knockouts exhibited strong freezing responses, with or without a cue tone, in contextual and cued-fear conditioning tests as well as low memory retention in the Barnes Maze test. Taken together, these results suggest that Caskin1 contributes to a wide spectrum of behavioral phenotypes, including gait, nociception, memory, and stress response, in broad regions of the central nervous system.

Two isoforms of cyclic GMP-dependent kinase-I exhibit distinct expression patterns in the adult mouse dorsal root ganglion.

cGMP-dependent kinase-I (cGKI) is known to regulate spinal pain processing. This enzyme consists of two isoforms (cGKIα and cGKIß) that show distinct substrate specificity and tissue distribution. It has long been believed that the α isoform is exclusively expressed in the adult dorsal root ganglion. The aim of the present study was to reexamine the expression of cGKI isoforms in the adult mouse dorsal root ganglion using isoform-specific cGKI antibodies whose specificities had been validated in the previous studies. Immunoblot and immunohistochemical analyses revealed the presence of both isoforms in the dorsal root ganglion. Moreover, cGKIα was found to be mainly expressed within the cytoplasm of small- to medium-sized peptidergic and nonpeptidegic C-fibers, whereas cGKIß was located within the nuclei of a wide range of dorsal root ganglion neurons. In addition, glutamine synthetase-positive satellite glial cells expressed both isoforms to varying degrees. Finally, using an experimental model for neuropathic pain produced by L5 spinal nerve transection, we found that cGKIα expression was downregulated in the injured, but not in the uninjured, dorsal root ganglion. In contrast, cGKIß expression was upregulated in both the injured and uninjured dorsal root ganglions. Also, injury-induced cGKIß upregulation was found to occur in small-to-medium-diameter dorsal root ganglion neurons. These data thus demonstrate the existence of two differently distributed cGKI isoforms in the dorsal root ganglion, and may provide insight into the cellular and molecular mechanisms of pain.

Impaired peripheral nerve regeneration in type-2 diabetic mouse model.

Peripheral neuropathy is one of the most common and serious complications of type-2 diabetes. Diabetic neuropathy is characterized by a distal symmetrical sensorimotor polyneuropathy, and its incidence increases in patients 40 years of age or older. In spite of extensive research over decades, there are few effective treatments for diabetic neuropathy besides glucose control and improved lifestyle. The earliest changes in diabetic neuropathy occur in sensory nerve fibers, with initial degeneration and regeneration resulting in pain. To seek its effective treatment, here we prepared a type-2 diabetic mouse model by giving mice 2 injections of streptozotocin and nicotinamide and examining the ability for nerve regeneration by using a sciatic nerve transection-regeneration model previously established by us. Seventeen weeks after the last injection, the mice exhibited symptoms of type-2 diabetes, that is, impaired glucose tolerance, decreased insulin level, mechanical hyperalgesia, and impaired sensory nerve fibers in the plantar skin. These mice showed delayed functional recovery and nerve regeneration by 2 weeks compared with young healthy mice and by 1 week compared with age-matched non-diabetic mice after axotomy. Furthermore, type-2 diabetic mice displayed increased expression of PTEN in their DRG neurons. Administration of a PTEN inhibitor at the cutting site of the nerve for 4 weeks promoted the axonal transport and functional recovery remarkably. This study demonstrates that peripheral nerve regeneration was impaired in type-2 diabetic model and that its combination with sciatic nerve transection is suitable for the study of the pathogenesis and treatment of early diabetic neuropathy.

Na+ /K+ -ATPase coupled to endothelin receptor type B stimulates peripheral nerve regeneration via lactate signalling.

We have recently demonstrated that endothelin (ET) is functionally coupled to Nax , a Na+ concentration-sensitive Na+ channel for lactate release via ET receptor type B (ETB R) and is involved in peripheral nerve regeneration in a sciatic nerve transection-regeneration mouse model. Nax is known to interact directly with Na+ /K+ -ATPase, leading to lactate production in the brain. To investigate the role of Na+ /K+ -ATPase in peripheral nerve regeneration, in this study, we applied ouabain, a Na+ /K+ -ATPase inhibitor, to the cut site for 4 weeks with an osmotic pump. While functional recovery and nerve reinnervation to the toe started at 5 weeks after axotomy and were completed by 7 weeks, ouabain delayed them by 2 weeks. The delay by ouabain was improved by lactate, and its effect was blocked by α-cyano-4-hydroxy-cinnamic acid (CIN), a broad monocarboxylate transporter (MCT) inhibitor. In primary cultures of dorsal root ganglia, neurite outgrowth of neurons and lactate release into the culture medium was inhibited by ouabain. Conversely, lactate enhanced the neurite outgrowth, which was blocked by CIN, but not by AR-C155858, a MCT1/2-selective inhibitor. ET-1 and ET-3 increased neurite outgrowth of neurons, which was attenuated by an ETB R antagonist, ouabain and 2 protein kinase C inhibitors. Taken together with the finding that ETB R was expressed in Schwann cells, these results demonstrate that ET enhanced neurite outgrowth of neurons mediated by Na+ /K+ -ATPase via ETB R in Schwann cells. This study suggests that Na+ /K+ -ATPase coupled to the ET-ETB R system plays a critical role in peripheral nerve regeneration via lactate signalling.

Involvement of Brain-Enriched Guanylate Kinase-Associated Protein (BEGAIN) in Chronic Pain after Peripheral Nerve Injury.

Maintenance of neuropathic pain caused by peripheral nerve injury crucially depends on the phosphorylation of GluN2B, a subunit of the N-methyl-d-aspartate (NMDA) receptor, at Tyr1472 (Y1472) and subsequent formation of a postsynaptic density (PSD) complex of superficial spinal dorsal horn neurons. Here we took advantage of comparative proteomic analysis based on isobaric stable isotope tags (iTRAQ) between wild-type and knock-in mice with a mutation of Y1472 to Phe of GluN2B (Y1472F-KI) to search for PSD proteins in the spinal dorsal horn that mediate the signaling downstream of phosphorylated Y1472 GluN2B. Among several candidate proteins, we focused on brain-enriched guanylate kinase-associated protein (BEGAIN), which was specifically up-regulated in wild-type mice after spared nerve injury (SNI). Immunohistochemical analysis using the generated antibody demonstrated that BEGAIN was highly localized at the synapse of inner lamina II in the spinal dorsal horn and that its expression was up-regulated after SNI in wild-type, but not in Y1472F-KI, mice. In addition, alteration of the kinetics of evoked excitatory postsynaptic currents for NMDA but not those for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in spinal lamina II was demonstrated by BEGAIN deletion. We demonstrated that mechanical allodynia, a condition of abnormal pain induced by innocuous stimuli, in the SNI model was significantly attenuated in BEGAIN-deficient mice. However, there was no significant difference between naive wild-type and BEGAIN-knockout mice in terms of physiological threshold for mechanical stimuli. These results suggest that BEGAIN was involved in pathological pain transmission through NMDA receptor activation by the phosphorylation of GluN2B at Y1472 in spinal inner lamina II.

Role of c-Jun N-terminal kinase in late nerve regeneration monitored by in vivo imaging of thy1-yellow fluorescent protein transgenic mice.

The restoration of function to injured peripheral nerves separated by a gap requires regeneration across it and reinnervation to target organs. To elucidate these processes, we have established an in vivo monitoring system of nerve regeneration in thy1-yellow fluorescent protein transgenic mice expressing a fluorescent protein in their nervous system. Here we demonstrated that motor and sensory nerves were regenerated in a coordinated fashion across the gap and that the functional recovery of the response to mechanical stimuli correlated well with sensory innervation to the foot. Among the mitogen-activated protein kinase inhibitors examined, only the c-Jun N-terminal kinase (JNK) inhibitors delayed functional recovery. Although it did not affect the reinnervation of the muscle, the JNK inhibitor delayed sensory nerve innervation to the skin for over 8 weeks and increased the expression of activatng transcription factor 3 (ATF3), a neuronal injury marker, in the dorsal root ganglion over the same time period. Antibodies against nerve growth factor, glia-derived neurotrophic factor, and brain-derived neurotrophic factor applied to the transection site delayed the functional recovery in this order of potency. These neurotrophic factors enhanced neurite outgrowth from cultured dorsal root ganglion neurons, and the JNK inhibitor reversed their stimulatory effects. These results suggest that JNK played roles in nerve regeneration at both early and late phases. Taken together, the present study demonstrated that neurotrophic factors released from the distal nerve may accelerate motor and sensory nerve regeneration across the gap in a coordinated fashion and reinnervation of the target organs independently. The model characterized here has the advantage of in vivo monitoring of the evaluation of morphological and functional recovery in the same mice for a long period of time.

Involvement of Nax sodium channel in peripheral nerve regeneration via lactate signaling.

Na(x), a sodium concentration-sensitive sodium channel, is expressed in non-myelinating Schwann cells of the adult peripheral nervous system, but the pathophysiological role remains unclear. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Na(x) knockout (Na(x)⁻/⁻) mice. Histological analyses showed a decrease in the number of regenerated myelinated axons in (Na(x)⁻/⁻) sciatic nerves. The delay in the recovery in Na(x)⁻/⁻ mice was improved by lactate and inhibited by a monocarboxylate transporter inhibitor. In vitro experiments using cultured Schwann cells showed that lactate release was enhanced by endothelin (ET)-1 and blocked by an ET receptor type B antagonist. Here, it is conceivable that Na(x) was activated by ET-1. The amount of lactate release by ET-1 was lower in Na(x)⁻/⁻ mice than in wild-type mice. These results indicated that Na(x) is functionally coupled to ET for lactate release via ET receptor type B and is involved in peripheral nerve regeneration.

Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry.

The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 µM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.

Proteomic analysis of cerebrospinal fluid before and after intrathecal injection of steroid into patients with postherpetic pain.

Postherpetic neuralgia (PHN) is the most frequent complication of herpes zoster, and the risk of it increases with age. By comparing proteomes of the cerebrospinal fluid (CSF) before and after the treatment, it may be possible to identify proteins that play a role in PHN and to predict responses to various treatments. To address this issue, we enrolled eight outpatients with PHN over 55 years of age and treated them with intrathecal methylprednisolone and lidocaine four times every week, collecting CSF samples before the treatment at each visit. We used 2D DIGE to investigate differentially expressed proteins in the CSF before and after repetitive treatments individually. Of 145 differentially expressed spots, the levels of nine proteins were decreased by the treatment including lipocalin-type prostaglandin D synthase (L-PGDS), and five were increased by it. The time course of alterations in the L-PGDS concentration in the CSF of each patient, detected by a pairwise and sandwich ELISA by SPR constructed here was well correlated with that by 1DE Western blots with anti-L-PGDS antibody, but was not related with that of the pain relief. The present study demonstrates that the real-time ELISA was precise and sensitive enough to measure L-PGDS in the CSF and that the steroid treatment decreased the L-PGDS concentration in CSF.

Involvement of Tyr1472 phosphorylation of NMDA receptor NR2B subunit in postherpetic neuralgia in model mice.

BACKGROUND: Postherpetic neuralgia is spontaneous pain and allodynia that persist long after the disappearance of the cutaneous lesions caused by herpes zoster. Inoculation of mice with herpes simplex virus-1 causes herpes zoster-like skin lesions and herpetic and postherpetic pain. Although NMDA receptors have been suggested to be involved in postherpetic pain as in other types of neuropathic pain, the neural mechanism remains unclear. NMDA receptor NR2B subunit is the most tyrosine-phosphorylated protein in the brain, and Tyr1472 is the major phosphorylation site of this subunit. RESULTS: To elucidate the role of Tyr1472 phosphorylation of the NR2B subunit in herpetic and postherpetic allodynia, we inoculated herpes simplex virus-1 into the unilateral hind paw of knock-in mice with a mutation of Tyr1472 of the NR2B subunit to Phe (Y1472F-KI). On day 7 post-inoculation, acute herpetic allodynia was observed in more than 80% of the inoculated wild-type and Y1472F-KI mice. Y1472F-KI mice showed significantly reduced intensity and incidence of postherpetic allodynia on days 45-50 post-inoculation as compared with wild-type mice. The innervation in the skin at the postherpetic neuralgia phase was retained to a greater extent in the Y1472F-KI mice. The level of activating transcription factor-3 mRNA, a marker of axonal damage, increased much less in the dorsal root ganglia (DRGs) of Y1472F-KI mice than in those of wild-type mice; and the level of nerve growth factor mRNA significantly increased in wild-type mice, but not at all in Y1472F-KI mice on day 7 post-inoculation. Production of nerve growth factor was at the basal level in the skin of both groups of mice on day 50 post-inoculation. Nerve growth factor and glial cell-derived neurotrophic factor stimulated neurite outgrowth of cultured DRG neurons from Y1472F-KI mice, similarly or less so as they did the outgrowth of those from wild-type mice. Wild-type DRG neurons were more susceptible to glutamate neurotoxicity than Y1472F-KI ones. CONCLUSIONS: Taken together, the present data suggest that phosphorylation of the NR2B subunit at its Tyr1472 is involved in the development of postherpetic allodynia due to nerve damage and that the nerve damage at the acute herpetic phase is correlated with the incidence of postherpetic pain.

Restricted expression of Ovol2/MOVO in XY body of mouse spermatocytes at the pachytene stage.

The development of multicellular organisms is controlled by sequential activation of a hierarchy of regulatory genes, which encode transcription factors having DNA-binding motifs. We previously identified a testis-specific zinc finger transcriptional factor, Ovol2/MOVO, as a mouse homologue of Drosophila Ovo. Because mice deficient in Ovol2/Movo die during early embryogenesis, its function in male germ cells has remained unknown. We have recently succeeded in preparing anti-Ovol2/MOVO antiserum for immunohistochemical use. In the present study, we demonstrated that Ovol2/MOVO protein started to be expressed in male germ cells at 2 weeks after birth and that Ovol2/MOVO expression was restricted to the XY body in spermatocytes at the pachytene stage. In a reporter assay, Ovol2/MOVO repressed the histone H1t promoter activity in the spermatogenic cell line GC-2spd. These results suggest that Ovol2/MOVO may play an important role in the XY body during spermatogenesis, possibly in the processes of XY body formation and meiotic sex chromosome inactivation.

Rapid S-nitrosylation of actin by NO-generating donors and in inflammatory pain model mice.

BACKGROUND: S-Nitrosylation, the reversible post-translational modification of reactive cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. We recently demonstrated that actin is a major S-nitrosylated protein in the spinal cord and suggested that NO directly attenuates dopamine release from PC12 cells by causing the breakdown of F-actin. However, the occurrence of S-nitrosylation of actin remained unclarified in animal pain model. Kinetic analysis of S-nitrosylation of actin in the present study was made by using NO-generating donors. The biotin-switch assay and purification on streptavidin-agarose were employed for identification of S-nitrosylated actin. RESULTS: Dopamine release from PC12 cells was markedly attenuated by NOR1 (t1/2 = 1.8 min) and much less by NOR3 (t1/2 = 30 min), but not by S-nitroso-glutathione, an endogenous NO donor. A membrane-permeable cGMP analogue could not substitute for NOR1 as a suppressor nor could inhibitors of soluble guanylate cyclase and cGMP-dependent protein kinase attenuate the suppression. S-Nitrosylated actin was detected by the biotin-switch assay at 5 min after the addition of NOR1. Consistent with the kinetic analysis, actin in the spinal cord was rapidly and maximally S-nitrosylated in an inflammatory pain model at 5 min after the injection of 2% formalin into the hind paws. In vivo patch-clamp recordings of the spinal dorsal horn, NOR3 showed an inhibitory action on inhibitory synaptic transmission in interneurons of the substantia gelatinosa. CONCLUSIONS: The present study demonstrates that rapid S-nitrosylation of actin occurred in vitro in the presence of exogenous NO-generating donors and in vivo in inflammatory pain model mice. Our data suggest that, in addition to the well-known cGMP-dependent protein kinase pathway, S-nitrosylation is involved in pain transmission via disinhibition of inhibitory neurons.

Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, and Ser831-GluR1 in neuropathic pain.

Previously we demonstrated that phosphorylation of NR2B subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor at Tyr1472 is increased in a neuropathic-pain model and that this phosphorylation is required for the maintenance of neuropathic pain by L5-spinal nerve transection. We obtained these results by using a selective NR2B antagonist and mice deficient in Fyn, which is an Src-family tyrosine protein kinase. However, how Tyr1472 phosphorylation of NR2B is involved in the maintenance of neuropathic pain was unclear. Here, we demonstrated that neuropathic pain was markedly attenuated in the spared nerve injury model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of NR2B (Y1472F-KI). While phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) at its Thr286 and that of the GluR1 subunit of the AMPA receptor at its Ser831 was enhanced in the spinal dorsal horn after spared nerve injury in wild-type mice, such phosphorylation was markedly impaired in Y1472F-KI mice. Inhibition of CaMKII by intrathecal injection of KN93, an inhibitor of CaMKII, reduced mechanical allodynia and phosphorylation of CaMKII at its Thr286 and that of GluR1 at its Ser831 in the spinal cord 7 days after spared nerve injury. These results demonstrate that the phosphorylation of CaMKII and GluR1 occurs downstream of the Tyr1472 phosphorylation of NR2B subunits in the spinal cord and give the first suggestion that activation of CaMKII and GluR1-AMPA receptors may be involved in mechanical allodynia caused by peripheral nerve injury.

Reversal of inducible nitric oxide synthase uncoupling unmasks tolerance to ischemia/reperfusion injury in the diabetic rat heart.

The diabetic heart is known to be susceptible to ischemia/reperfusion (I/R) injury by increased oxidative stress. Although oxidative stress upregulates inducible nitric oxide (iNOS), the role of iNOS in I/R injury in the diabetic heart has been poorly understood. Because iNOS-derived nitric oxide (NO) plays a crucial role in cardioprotection against I/R injury, we hypothesized that inhibition of iNOS uncoupling would restore tolerance to I/R injury in the diabetic heart. The present study demonstrated that iNOS-derived superoxide generation was reduced, and that the NO bioavailability was increased, by treatment with the NOS-cofactor, tetrahydrobiopterin (BH4), before I/R in the hearts isolated from diabetic rats. This was associated with a reduction of infarct size and improvement of left ventricular (LV) function after I/R. The cardioprotective effect of BH4 was abrogated by treatment with a thiol reducing agent dithiothreitol (DTT), but not a NO-sensitive guanylyl cyclase inhibitor ODQ, suggesting that iNOS-derived NO-mediated cardioprotection occurs through protein S-nitrosylation but not cGMP-dependent signaling in the diabetic heart. Indeed, protein S-nitrosylation was increased by treatment with BH4 in the diabetic heart and was inhibited by DTT. These results suggest that the inhibition of iNOS uncoupling unmasks tolerance to I/R injury through enhanced protein S-nitrosylation in the diabetic rat heart.

A novel role of prostaglandin E2 in neuropathic pain: blockade of microglial migration in the spinal cord.

Neuropathic pain produced by damage to or dysfunction of the nervous system is a common and severely disabling state that affects millions of people worldwide. Recent evidence indicates that activated microglia are key cellular intermediaries in the pathogenesis of neuropathic pain and that ATP serves as the mediator. However, the in vivo mechanism underlying the retention of activated microglia in the injured region has not yet been completely elucidated. Prostaglandin E(2) (PGE(2)) is the principal proinflammatory prostanoid and plays versatile roles by acting via four PGE receptor subtypes, EP1-EP4. In the present study, we investigated the role of PGE(2) in spinal microglial activation in relation to neuropathic pain by using genetic and pharmacological methods. Mice deficient in microsomal prostaglandin E synthase-1 impaired the activation of microglia and the NMDA-nitric oxide (NO) cascade in spinal neurons in the dorsal horn and did not exhibit mechanical allodynia after peripheral nerve injury. The intrathecal injection of indomethacin, a nonsteroidal anti-inflammatory drug, ONO-8713, a selective EP1 antagonist, or 7-nitroindole, a neuronal NO synthase inhibitor, attenuated mechanical allodynia and the increase in activated microglia observed in the established neuropathic-pain state. We further demonstrated that ATP-induced microglial migration was blocked in vitro by PGE(2) via EP2 and by S-nitrosoglutathione, an NO donor. Taken together, the present study suggests that PGE(2) participated in the maintenance of neuropathic pain in vivo not only by activating spinal neurons, but also by retaining microglia in the central terminals of primary afferent fibers via EP2 subtype and via EP1-mediated NO production.

Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472.

Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N-methyl-d-aspartate (NMDA) receptor-mediated Ca(2+) influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca(2+) response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca(2+) response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase Cγ at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain.