Food allergy is defined as "a phenomenon in which adverse reactions are caused through antigen-specific immunological mechanisms after exposure to a given food". The number of patients with food allergy is increasing in the last decades. Food allergy develops by failure of acquiring oral tolerance for the foods. However the reasons that acquiring oral tolerance is inhibited are unclear. Previously, we made the models of murine food allergy and oral tolerance for the food. In the models, we used ovalbumin (OVA) as a food antigen. OVA mixed with alum (OVA/alum) was injected into mice intraperitoneally for sensitizations, and the mice were challenged by oral treatment with OVA in the food allergy model. As indexes for food allergy, drop of body temperature, fecal condition, and the level of OVA-specific IgE in blood were estimated. In oral tolerance model, prior oral treatment with OVA prevented the development of food allergy. In recent years, it is reported that food allergy is associated with exposure of food antigens via skin. In this study, we made the model of food allergy sensitized via skin. Additionally, we researched whether prior oral treatment with OVA prevented the development of food allergy. As results, pre-treatment with OVA in the model of food allergy by skin exposure to OVA was able to suppress the indexes of food allergy partially.

As the expression level of a disease-sensitive gene is correlated with the symptom severity, suppression of its gene expression should be good therapeutics. We demonstrated that histamine H₁ receptor (H1R) gene is an allergic rhinitis (AR)-sensitive gene. We isolated maackiain (MCN) from Kujin that suppresses H1R gene up-regulation. We also showed that MCN bound to Hsp90 and disrupts the interaction between PKCδ and Hsp90, suggesting MCN modulates steroid signaling. Western blot analysis showed that MCN completely inhibited ERK phosphorylation although the inhibition of PKC dphosphorylation was partial, suggesting that MCN suppresses H1R gene expression by the additional mechanism. MCN enhanced dexamethasone-activated GRE promoter activity. MCN also enhanced dexamethasone-induced gene up-regulation for dual-specificity phosphatase 1 (DUSP1), that dephosphorylizes ERK. On the other hand, dexamethasone suppressed H1R gene up-regulation. These findings suggest that MCN suppresses H1R gene expression through not only the disruption of interaction between PKCδ and Hsp90 but also the activation of ERK dephosphorylation by the enhancement of dexamethasone-induced DUSP1 gene expression.

Mast cells migrate toward histamine through H₄ receptor (H₄R), which involves the activation of Rac1 and Rac2. Transglutaminase catalyzes the incorporation of histamine into cellular proteins, the process called histaminylation. Although several reports suggest possible roles of histaminlylation in mast cell signaling, there is no direct evidence showing its significance in cellular function. To explore the functional significance of histaminylation, we first investigated the effect of cystamine, a transglutaminase inhibitor, on H₄R-Rac pathway in mast cell. Cystamine attenuated histamine-induced migration and Rac activation, suggesting that H₄R-Rac signaling requires transglutaminase. Organic cation transporter 3 (OCT3) transports histamine into cytoplasm from extracellular space. Given the histaminylation occurs and regulates signaling in mast cells, we next examined if OCT3-mediated transport of histamine plays any role in H₄R signaling. Decynium-22, an inhibitor of OCT3, suppressed histamine-induced migration and Rac activation, suggesting the involvement of OCT3 in H₄R-Rac signaling. Our findings support the idea that transported histamine and transglutaminase activity modulate H₄R-Rac pathway.

Disseminated intracellular coagulation (DIC) is a serious, life-threatening disorder characterized by systemic activation of the blood coagulation pathway, which leads to generation and deposition of fibrin, resulting in microvascular thrombi in various organs and contributing to multiple organ dysfunction syndrome. Sepsis is frequently complicated by coagulopathy and, in about 35 % of severe cases, by DIC. The development and severity of DIC correlate with mortality in severe sepsis. We examined whether overt DIC is found in mice with cecal ligation and puncture (CLP)-induced sepsis, which serve as an animal model that has high clinical relevance to humans. We found that the number of blood platelets strikingly declined in CLP-induced septic mice as compared with sham-operated controls. Plasminogen activator inhibitor-1, a critical regulator of the fibrinolytic system, was markedly increased in all of major organs after CLP, and tissue factor, which has a pivotal role in initiating the extrinsic pathway of blood coagulation, was significantly elevated in lungs and kidneys after CLP. Finally, CLP mice exhibited an abnormal prothrombin time, suggesting that they represent an appropriate model for investigating sepsis-induced DIC.

The cochlea of the inner ear converts sounds into electrical signals. This process is triggered by sound-evoked nanoscale vibrations in the sensory epithelium inside the organ. The epithelium contains outer hair cells that have mechanosensory hair bundles at the apical surface. The deflection of the bundles enters cation through ion channels. The epithelial vibrations are modulated by cation-induced elastic motions in the cell bodies. How the vibrations are regulated in vivo has not yet fully elucidated. Here we develop an advanced laser interferometry that precisely detects the vibrations. When a live guinea pig was exposed to acoustic stimuli, the interferometer quantitatively recorded the vibration amplitude of the sensory epithelium as described elsewhere. Additionally, an upward baseline shift of several nanometers was also detected. This motion was observed with loud sounds of >70 dB, and it was negligible when the animal was dead or under pharmacological perturbation of hair-bundles or cell body motions. A theoretical approach further suggested that the shift protects the epithelium from injury induced by strong stimuli.

It is well-known that cochlear outer hair cell (OHC) loss concomitant with permanent hearing loss induced by intense noise. Our earlier studies demonstrated the production of hydroxynonenal and peroxynitrite, as well as the disruption of gap junction-mediated intercellular communication (GJIC), in the cochlear spiral ligament prior to noise-induced hearing loss. The purpose of this study was to evaluate the mechanism underlying cochlear OHC loss induced by intense noise exposure. In organ of Corti explant cultures from mice, no significant OHC loss was observed after exposure to 4-hydroxynonenal (4-HNE, a product of lipid peroxidation), SIN-1(peroxynitrite generator), and carbenoxolone (a GJ inhibitor). In vivo intracochlear carbenoxolone injection through the posterior semicircular canal caused marked OHC and hearing loss, as well as the disruption of GJIC in the cochlear spiral ligament. However, no significant OHC loss was observed in vivo in mice treated with 4-HNE and SIN-1. In conclusion, our data suggest that disruption of GJIC in the cochlear spiral ligament is an important cause of cochlear OHC loss in models of noise-induced hearing loss.

Delta-opioid receptors (DOR) are highly expressed in the insular cortex, which regulates various autonomic functions including cardiovascular responses. However, the functional roles of DOR in the insular cortex are yet unknown. In the present study we investigated the effects of KNT-127, an agonist of DOR, on the heart rate and neuronal activities in the insular cortex of mice. ddY male mice (5-9 weeks) were used. The electrocardiogram was recorded using wire electrodes inserted subcutaneously in freely moving mice. The extracellular neuronal activities were recorded in the insular cortex in anesthetized mice. The field excitatory postsynaptic potentials (fEPSP) were recorded in the acute insular cortex slices. Microinjection of KNT-127 (46 ng /mouse) in the right, but not left, side of the insular cortex significantly decreased the heart rate. The firing frequency of insular neurons was reduced by intracerebroventricular administration of KNT-127 (230 ng /mouse). Moreover, the fEPSP amplitude was reduced by bath-application of KNT-127 (1-100 μM). Together, activation of DOR in the insular cortex modulates the heart rate via the suppression of neuronal activities.

(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for phenylalanine-catabolism, and production of monoamines and nitric oxide (NO). Sepiapterin reductase (SPR) catalysis the final step of BH4 biosynthesis. Previously, we established SPR gene disrupted (Spr^-/-) mice (OYC32, Lexicon Pharmaceuticals Inc.) and reported that they exhibited dystonic posture, low body weight, hyperphenylalaninemia and unstable hypertension with bradycardia. Recently, we found that Spr^-/- mice suffered from severe priapism (persistent erection) at high incidence (69.2%) and analyzed their penile tissues to reveal the mechanism. The content of BH4 and noradrenaline in the penile homogenate of Spr^-/- mice significantly decreased compared to those of wild type (Spr^+/+) mice, which were 0.69% and 17.4%, respectively. There was no significant difference in the protein amount of eNOS, PDE5A, p-PDE5A between two genotypes. Tyrosine hydroxylase significantly decreased and, on the contrary, that of nNOS significantly increased in the penis of Spr^-/- mice. NO metabolites significantly increased in the penis of Spr^-/- also. Thus, we concluded that sympathetic nerve failure and up-regulation of NO production contribute to the severe priapism of Spr^-/- mice.

For treating acute cerebral infarction, we developed a laser thrombolytic system with the second harmonic generation of microsecond Nd:YAG laser, and investigated its effectiveness, safety and mechanisms in animal thrombosis models. The dynamics of laser-induced thrombolysis in a gelatin phantom model was investigated with a high speed camera. The observation revealed that laser irradiation generated a bubble in the gelatin phantom. In vivo thrombolytic efficacy was investigated using animal thrombosis models. Thrombi in the vena cava inferior of rats or in the carotid artery of rabbits were induced by an application of ferric chloride (FeCl₃). Laser irradiation was then carried out through an optical fiber inserted from the femoral vein or artery. Laser irradiation induced significant thrombolysis in the rat thrombosis model. Laser irradiation also resulted in recanalization in the rabbit thrombosis model. One day after the recanalization, neurological disorders, cerebral ischemia and cerebral hemorrhage were not observed. No vascular endothelial damage evaluated by Evans blue staining after laser irradiation was observed. The irradiation of the pulsed green laser can induce bubbles that fragment thrombi without vessel or brain damage.

[Background] Voltage dependent Ca²⁺ channels are divided to L-, T-, N-, P/Q-, and R- types, and N-type Ca²⁺ channel (NCC) are mainly expressed in nerve terminal. Recently, NCC has been reported to express in adrenal gland and renal tubular cells. We examined whether NCC is expressed in cardiac myocytes and if so, the roles of this channel. [Methods and Results] NCC mRNA and protein are expressed in neonatal rat cardiac myocytes, using real time PCR and Western blot analysis. Immunocytochemistry showed this channel was expressed on myocyte plasma membrane. After birth the expression level of this channel in cardiac tissue was gradually decreased within 2 weeks. In pathological condition, such as 5 hours of hypoxia followed by 30 minutes of reoxygenation (H/R) and norepinephrine (10^-5 mol/L, 24 hours) increased NCC expression in neonatal cultured myocytes. In addition, in adult rats (12 weeks) mRNA level of this channel was also increased in non-infarcted myocardium after 4 weeks of myocardial infarction. Furthermore, to clarify the role of NCC in myocyte, we examine the effect of ω-conotoxin, a selective NCC blocker. ω-conotoxin significantly suppressed H/R- and norepinephrine-induced lethal myocytes injury, assessed by LDH activity in cultured medium and caspase 3 activity in myocytes. [Conclusion] NCC is expressed in neonatal and pathological cardiac tissue, and augmented myocyte lethal injury.

Since information is lacking regarding how the enhancement of net sarcoplasmic reticulum (SR) Ca²⁺ uptake may affect cardiac electrophysiological properties in vivo, we analyzed it with caldaret which can decrease SR Ca²⁺ leak, enhance SR Ca²⁺ reuptake and inhibit reverse-mode Na⁺/Ca²⁺ exchanger. Caldaret in doses of 0.5, 5 and 50 µg/kg was intravenously administered over 10 min to the halothane-anesthetized beagle dogs (n=4), possibly providing pharmacologically effective plasma concentration. The low and middle doses increased the ventricular contraction, which can be explained by its on-target pharmacological activities. The high dose enhanced the sinus automaticity followed by its suppression in addition to the increase of the total peripheral resistance. The low and middle doses enhanced the atrioventricular conduction. The middle and high doses prolonged the ventricular effective refractory period without altering the intraventricular conduction or repolarization period. Thus, modulation of intracellular Ca²⁺ handling by caldaret can induce not only inotropic effect, but also various electrophysiological actions on the in situ heart.

Objective: Atrial natriuretic peptide (ANP) functions via guanylyl cyclase (GC)-A, a single transmembrane receptor, resulting in diuresis, natriuresis, and lowering of blood pressure. However, molecular mechanism of hypotensive effect of ANP is not well understood. Results: Immunohistochemistry indicated that GC-A is abundantly expressed in endothelial cells. Intravenous infusion of ANP in wild-type mice significantly lowed systolic blood pressure. ANP failed to lowering systolic blood pressure in endothelial cell-specific GC-A knockout mice. In endothelial nitric oxide synthase knockout mice, ANP also significantly lowed systolic blood pressure. In in vitro study, ANP treatment significantly hyperpolarized cultured human umbilical vein endothelial cells (HUVECs), but not changed intracellular Ca²⁺ concentration in HUVECs. ANP-induced hyperpolarize of HUVECs was dependent of cyclic GMP-Protein Kinase G and regulator of G-protein signaling2 (RGS2) pathway but not dependent of Ca²-activated K⁺ channels. Conclusions: These results suggest that hypotensive effect of acute ANP administration can be caused by endothelial GC-A-mediated RGS2 pathway-dependent endothelial hyperpolarization.

Hypertensive disorders of pregnancy (HDP) is a pathological condition with hypertension and vascular endothelial cell dysfunction (inflammation) during the gestational period. Recently, it was reported that reduction level of plasma histidine-rich glycoprotein (HRG) in human pregnant patients was correlated with the HDP seriousness. HRG is an anti-inflammatory factor that controls the progression of systemic inflammatory pathology such as sepsis syndrome. However, HRG functions in HDP pathology and perinatal physiology have not been clarified yet. In this study, we examined the pregnancy phenotype of HRG gene-deficient (HRG KO) mice to clarify the involvement of plasma HRG on placental formation and hypertensive event during the gestational period. Pregnant HRG KO mice had fetal growth disorder and placental hyperplasia. There was no change in the expression levels of inflammatory factors (IL-1beta, TNF-alpha) in the placenta. On the other hand, the expression level of angiogenic factors (VEGF, PlGF) in placenta increased. In addition, pregnant HRG KO mice had hypertension. These results suggest that HRG may have a physiological function in the gestational period. Pregnant HRG KO mice may be a HDP-like pathological model that causes abnormalities in the placental formation mechanism controlled by HRG.

Pulmonary hypertension (PH) is a heterogeneous disorder associated with a progressive increase in pulmonary artery resistance and pressure. Although various therapies have been developed, the 5-year survival rate of PH patients still remains low. There is thus an important need to identify novel molecular networks involved in the pathogenesis of PH. In this study, we performed comparative transcriptome analysis of two mammalian PH models. In the one model, PH was caused by chronic hypoxia (Hx model). In the other model, PH was caused by the combination of a vascular endothelial growth factor receptor antagonist Sugen 5416 and chronic hypoxia (SuHx model). Intima and media proliferation and its consequent pulmonary vascular obstruction were prominent in SuHx model. Comparative transcriptome analysis revealed that five genes were significantly dysregulated in SuHx model and these genes might be regulated by Fos-related antigen-2（Fra-2）. The immunohistochemal analysis confirmed that the expression of Fra-2 were induced and localized within the cell nucleus in the pulmonary vascular lesions in SuHx model but not Hx model. These results suggest that Fra-2 may be involved in the pathophysiology of PH and a novel therapeutic target for the treatment of PH.

Activation of Stat3, a member of the Stat family of transcription factors, plays a pivotal role in induction of reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) increases in brain disorders and promotes astrocytic proliferation through ET_B receptors. In this study, to clarify mechanisms underlying astrocytic proliferation, the effects of ET-1 on Stat3 were examined in rat cultured astrocytes. Treatment with ET-1 stimulated Ser727 phosphorylation of Stat3 in cultured astrocytes, although Tyr705 phosphorylation was not affected. ET-1 stimulated the binding of Stat3 protein to its consensus DNA fragments. ET-induced BrdU incorporation was reduced by Stat3 inhibitors and Stat3 siRNA. ET-1 increased the expression of cyclin D1 and Skp2 in cultured astrocytes. The effects of ET-1 on cyclin D1 and Skp2 expression were reduced by stattic, 5,15-DPP and Stat3 siRNA. ChIP-PCR analysis showed that ET-1 promoted the binding of Stat3 to 5'-flanking regions of rat cyclin D1 and Skp2 genes. These results suggest that cyclin D1 and Skp2 expression through Stat3-mediated mechanisms underlies ET-induced astrocytic proliferation.

Astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Ca²⁺ release from the endoplasmic reticulum (ER) is considered to be crucial for the regulation of astrocytic functions. Inositol 1,4,5-trisphosphate receptor type 2 (IP₃R2)-knockout (KO) mice are reportedly devoid of astrocytic Ca²⁺ signaling, and thus widely used to explore the significance of astrocytic Ca²⁺ signaling. However, functional deficits in IP₃R2-KO mice have been found in some reports, but not in others. To address this controversy, we re-evaluated the assumption that Ca²⁺ release from the ER is abolished in IP₃R2-KO astrocytes. We expressed the ER luminal Ca²⁺ indicator G-CEPIA1er in astrocytes to directly visualize Ca²⁺ release from the ER. We found attenuated but significant Ca²⁺ release in response to application of norepinephrine to IP₃R2-KO astrocytes. This IP₃R2-independent Ca²⁺ release induced only minimal cytosolic Ca²⁺ transients but induced robust Ca²⁺ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca²⁺ release is retained and is sufficient to increase the Ca²⁺ concentration in close proximity to the ER in IP₃R2-KO astrocytes.

[Aim] Extracellular zinc enhances pro-inflammatory cytokines secretion from M1 microglia via reactive oxygen species (ROS) generation. Here, we examined the effect of peridinin, a carotenoid in dinoflagellates, on the zinc-enhanced inflammatory M1 phenotype.

[Methods] M1 polarization of mouse microglia prepared was induced by lipopolysaccharide after ZnCl₂ treatment in the presence of peridinin, and cytokines secretion were assessed by ELISA. In addition, ROS detection assay and HPLC analysis were performed. Transient ischemia in mice was induced 5 min after peridinin injection. The levels of cytokines and M1 marker were examined by qPCR and immunostaining, respectively. Spatial memory was assessed by Y-maze test.

[Results] Peridinin prevented the zinc-induced aggravation of cytokines secretion from M1 microglia and increase in the microglial ROS levels. No shift in the absorption maximum of peridinin reacted with zinc was observed. Injection of peridinin suppressed ischemia-induced expression of cytokines and M1 marker, and memory impairment.

[Conclusion] These results suggest that peridinin exerts neuroprotective effects against the drastic post-ischemic inflammation by inhibiting the zinc-induced increase in the microglial ROS levels.

The onset and progression of Alzheimer's disease (AD) correlate with neuroinflammatory processes, and inflammatory microglia (MG) are associated with AD-like pathology in a transgenic mouse model. However, the distinct role of MG subtypes in AD brain remains unclear. We recently developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG, and identified the antigen molecule for 9F5: truncated form of rat GPNMB/osteoactivin (Kawahara et al., GLIA, 2016). In neonatal rat brain, GPNMB⁺Iba1⁺ MG were a portion of Iba1⁺ MG, and were observed in specific brain areas including corpus callosum. However, the distribution and function of GPNMB⁺ type 1 MG in AD brain are largely unknown. In the present study, we observed GPNMB⁺Iba1⁺ MG surrounding Aβ plaque in neocortex of amyloid precursor protein (APP23) transgenic mice. In addition, GPNMB⁺Iba1⁺ MG were observed in non-plaque areas of hippocampus of APP23 mice. We generated Gpnmb knockout mice to investigate the functional relevance of GPNMB for microglia in vivo. Homozygous Gpnmb-KO mice did not show any growth retardation including body weight loss, and the fertility was normal. We observed that AD-related brain dysfunction in APP23 mice were regulated by Gpnmb gene dosage. These finding suggest that GPNMB⁺ type 1 MG may play a role in regulation of neuropathological process of AD.

Tissue macrophages in the central nervous system (CNS), including parenchymal microglia and non-parenchymal meningeal, perivascular and choroid-plexus macrophages, are important for the development and homeostasis of the healthy CNS. Also, there is a growing body of evidence that microglia plays a key role in neurological and psychiatric disorders. The aim of our study was to understand the spatial arrangement of these cells in the CNS and its change after peripheral inflammation, by using a genetic tool that can label Iba1-positive CNS macrophages (Nakayama et al., Nat Commun, 2018). Tissue clearing and 3D-imaging technique (CUBIC) clarified an activation of microglia in the spinal cord and brain region after peripheral inflammation.

Background: Secreted protein acidic and rich in cysteine (SPARC), one of the ECMs, promotes inflammation in aging hearts. Pro-inflammatory and -fibrotic properties of ADAMTS1 is reported, and SPARC promotes collagen production via ADAMTS1 upregulation. This study investigated the roles of SPARC in hypertensive renal injury.

Methods: Uninephrectomized rats were treated with DOCA and salt for 0, 1, 2, or 3 weeks (DOCA-salt) with/without losartan (30 mg/kg). Blood pressure, proteinuria, CCr, and renal NADPH oxidase activity was measured. Fibrillar collagens and macrophages were detected by Masson's trichrome staining and immunohistochemistry, respectively. The protein levels of MCP-1, TGF-beta, collagen I, SPARC, and ADAMTS1 were examined by immunoblotting.

Results: Blood pressure increased time-dependently from 2w. Proteinuria increased from 2w and CCr decreased at 3w. NADPH oxidase activity, macrophage numbers, and MCP-1 increased in DOCA-salt. TGF-beta, tubulointerstitial fibrosis, and glomerulosclerosis increased time-dependently from 2w. The collagen I protein, in particular collagen I with larger molecular weight, increased in 3w. SPARC increased and peaked at 2w and reversed to the control levels at 3 weeks, and ADAMTS1 gradually increased until 3w, both of which were suppressed by Losartan.

Conclusions: Renal renin-angiotensin system-induced SPARC upregulation and subsequent ADAMTS1 production may mediate renal injury.

Herein, we studied the effects of the novel nonsteroidal selective mineralocorticoid receptor (MR) blocker, esaxerenone, on blood pressure and renal injury in Dahl salt-sensitive (DSS) rats. We also monitored the urinary intact and total angiotensinogen (AGT). DSS rats were given a normal salt diet (NS: 0.4% NaCl, n = 10), high salt diet (HS: 8% NaCl), HS + esaxerenone (1 mg/kg/day, p.o.), or HS + losartan (angiotensin II receptor blocker, 10 mg/kg/day, p.o.) for 6 weeks. HS-treated DSS rats developed hypertension, albuminuria and glomerular injury, which were associated with increased glomerular desmin staining and reduced mRNA levels of glomerular podocin and nephrin. HS-treated DSS rats also showed tubulointerstitial fibrosis with an increase in renal oxidative stress (4-hydroxynonenal staining). The urinary (total AGT – intact AGT)/intact AGT ratio, an indicator of intrarenal renin activity, was significantly suppressed in HS-treated DSS rats. Treatment with esaxerenone significantly decreased blood pressure, while losartan did not. Furthermore, esaxerenone attenuated the development of albuminuria, glomerular injury and tubulointerstitial fibrosis more than losartan did, and this was associated with reduced renal oxidative stress. These data indicate that esaxerenone induces antihypertensive and renal protective effects in salt-dependent hypertensive rats with suppressed intrarenal renin activity, as indicated by low levels of the urinary (total AGT – intact-AGT)/intact AGT ratio.

We previously reported by using intravital imaging technique that lipopolysaccharide (LPS) slowed proximal tubular flow rate in an early phase of endotoxemia. Hereby, we hypothesized that LPS disrupts tight junction in proximal tubular cells and induce leakage of filtrate through a Toll-like receptor 4 (TLR4)-dependent mechanism. LPS at 5 mg/kg did not change glomerular filtration rate (GFR), and significantly reduced the washout rate of tubular fluid from the proximal tubules and urine output in the early phase, reflecting slowing down of tubular flow rate in the proximal tubules during oliguria. LPS at 15 mg/kg reduced both GFR and urine output. LPS (5 mg/kg) induced paracellular leakage of FITC-inulin and reduced tight junction mRNA expression (occludin and cldn2). LPS also increased water content, interstitial hydrostatic pressure and Na⁺/K⁺ ratio in the kidney, indicating the accumulation of extracellular fluid in the interstitium. The mice lacking TLR4 in proximal tubules showed markedly blunted aforementioned responses and an increased sensitivity to the fluid resuscitation. Our results suggest that LPS disrupted tight junction of proximal tubular cells via a TLR4-dependent mechanism, resulting in paracellular leakage of filtrate to interstitium, which blunted fluid sensitivity, during endotoxemia in mice.

L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of catecholamines, has been believed to be a pharmacologically inert amino acid. We previously showed that depressor and bradycardic responses to L-DOPA-microinjected into the nucleus tractus solitarii (NTS) were suppressed by shRNA knockdown of GPR143. The specific binding of [3H]-L-DOPA was detected in CHO cells expressing GPR143, which was displaced by L-DOPA CHE, an antagonist of L-DOPA. These findings suggest that L-DOPA itself can exert some of its actions through GPR143. GPR143 is expressed in both neuronal and non-neuronal organs including kidney. Although we reported that L-DOPA sensitize the vascular alpha 1 adrenergic receptor through activation of GPR143, peripheral functions of GPR143 are largely unknown. Here we focused on roles of GPR143 in adenine-induced chronic kidney disease model. Gpr143 gene-deficient mice (GPR143-KO) and control mice were fed diets supplemented with 0.2% adenine. Although blood urea nitrogen and serum creatinine levels were similar, Gpr143-KO showed significant body weight loss of adenine diet than did wild type animals. Serum amyloid A gene expression was higher in Gpr143-KO of adenine diet, thereby suggesting that GPR143 may be involved in inflammation.

SGLT2 is a sodium-coupled glucose cotransporter localized at the apical membrane of renal proximal tubule that uptakes glucose from urine into the cells in a Na⁺-dependent manner. Although SGLT2 inhibitors are clinically used to reduce blood sugar, they exhibit uric acid lowering action at the same time. However, its molecular mechanism is still unknown. This study aims to elucidate its molecular mechanism behind the interaction between SGLT2 inhibitor and renal tubular transporters. cRNAs of URAT1 (SLC22A12), OAT10 (SLC22A13) and URATv1 (SLC2A9) were injected into Xenopus oocytes. 2-3 days later, we measured urate and glucose transport activities, the inhibition by glucose and SGLT2 inhibitor tofogliflozin (tofo) on uric acid transport, and the inhibition by urate on glucose transport. Urate transport by three was not inhibited by glucose. It was newly found that glucose is significantly transported by all, but since its transport inhibition by urate was not observed, any transporter has different substrate recognition sites for glucose and uric acid. In addition, urate transport by three was not inhibited by tofo.

Thus, urate-lowering action by SGLT2 inhibitor was considered unlikely to be due to the interaction with the existing major renal tubular urate transporter.

Intestinal epithelium undergoes a continuous self-renewal to maintain the intestinal homeostasis. Recently, we identified ZIP7 as a novel zinc transporter, securing of vigorous proliferation of immature intestinal epithelial cells that are called transit-amplifying (TA) cells by resolving endoplasmic reticulum (ER) stress. Dysregulation of cell proliferation machinery has been implicated in tumorigenesis. In this study, we investigated the role of ZIP7 in colorectal cancer development. siRNA or shRNA knockdown of ZIP7 suppressed cell proliferation, which was assessed by cell counting assay or crystal violet analysis, in human colorectal cancer cell lines. Apoptotic cell death was caused by siRNA knockdown of ZIP7, suggesting that ZIP7 is required for survival of colon cancer cells. Xenograft assays in immunodeficient mice showed that tumors generated by ZIP7-depleted cells were smaller than those generated by control cells, showing the requirement of ZIP7 expression in cancer cells for tumorigenicity in vivo. Furthermore, we found that overexpression of ZIP7 substantially increased volumes and weights of the xenograft tumors. These findings suggest that ZIP7 not only is required for tumor formation but also promotes tumorigenesis and thus, represents an attractive therapeutic target for colorectal cancer.

Although radiation therapy is one of the choices to treat cancers and is an excellent local treatment method, existence of radiation resistant cell is a major problem. We established clinically relevant radioresistant (CRR) cells that can survive exposing to 2 Gy/day X-rays for more than 30 days. However, the mechanism to obtain resistance has not been elucidated yet. We investigated the relationships between resistant mechanism to hydrogen peroxide (H₂O₂) and plasma membrane state in CRR cells.

　CRR cells showed resistance to H₂O₂, but catalase enzyme activity was down-regulated. Plasma membrane potential were low, no internal H₂O₂ increase and no lipid peroxidation were seen even after 2 hours of H₂O₂ treatment in CRR cells. Administration of oxidized lipid led to further cell death after H₂O₂ treatment in CRR cells. The lipoxygenase (ALOX) gene and protein expressions were down-regulated in CRR cells. We also established stress-sensitive ρ⁰ cells that lack mitochondrial DNA. Gene and protein expressions of ALOX were up-regulated in ρ⁰ cells. Expression of cyclooxygenase-2 does not seem to be involved in this mechanism.

　These results suggest that the involvement of ALOX in resistance or sensitivity of cancer treatment.

Melanoma is one of the most deadly malignant diseases with the highest increase in incidence rate over the past 50 years. Even with new drug applications, its death rates have been stable over this decade. To discover new anti-melanoma drugs, we performed chemical screening using zebrafish melanoma allograft model.  For preparing melanoma model of zebrafish, human BRAF mutation (BRAF[V600E]) -driven zebrafish melanoma cells were injected into the circulation of young zebrafish (48 hours-post-fertilization). The melanoma cells increased about 10-times on 6 days after implantation. We tested 2320 chemicals using this allograft zebrafish in combination with the fluorescence plate reader, and found that several chemicals can suppressed melanoma proliferation in vivo. Of these, flubendazole (FLBZ)  also suppressed A375 melanoma cell proliferation in mouse xenografts. We further identified that the anti-melanoma function of FLBZ was responsible to inhibition of epithelial-to-mesenchymal transition, not to BRAF mutation or autophagy induction as previously reported. In summary, integrated cross-species analysis using zebrafish, mice and human cells revealed that FLBZ should be one of strong candidates for anti-melanoma drug.

Purpose: Podoplanin (PDPN) is involved in cancer malignancy. PDPN is highly expressed in both cancer and normal cells. We aimed to develop cancer-specific anti-PDPN mAbs.

Methods: We immunized mice with PDPN-expressing cancer cells, and produced cancer-specific anti-PDPN monoclonal antibodies (mAbs). We characterized mAbs using flow cytometry (FCM) and immmunohistochemistry (IHC). Anti-tumor activities by a mouse-human chimeric mAb was examined using mouse xenograft models of oral cancer cells. We further investigated the possibility of PDPN as a diagnostic marker of oral cancers.

Results: One of established mAbs, LpMab-23 reacted with PDPN-expressing cancer cells, not with normal cells by FCM and IHC using oral cancers. LpMab-23 recognized a cancer-specific glycopeptide including Thr55/Ser56. chLpMab-23 revealed high ADCC and anti-tumor activities against oral cancers. We further showed that LpMab-23 is a prognostic marker of oral cancer. The Kaplan-Meier curves of the five-year new metastasis-free survival rate (nMFS) were significantly lower in LpMab-23-positive patients than in LpMab-23-negative ones.

Conclusion: LpMab-23 has antitumor activities, and LpMab-23-positive cases could be a useful predictor of poor prognosis for oral cancer.

【Introduction】Bladder cancer (BC) is the most common neoplasmaffecting the urinary tract of dogs. Stem cell-derived 3D organoid culture couldrecapitulate organ structure and physiology. In our previous study, urinesample-derived dog prostate cancer organoids have been established. However,urine-derived dog BC organoids have never been developed. We therefore generateddog BC organoids using the urine samples.

【Methods and Results】After dogs were clinically diagnosed withbladder tumor, urine samples were collected by catheterization and used for theorganoid culture. Organoids from each BC dog were successfully generated.Expression of an epithelial cell marker (E-cadherin) and a myofibroblast marker(α-smooth muscle actin, (SMA)) was confirmed in the organoids. The organoidsalso expressed a stem cell marker (CD44). Injection of the BC organoids intoimmunodeficiency mice successfully generated tumor. While treatment withcisplatin and vinblastine decreased cell viability of organoids in adose-dependent manner, treatment with gemcitabine and piroxicam had littleeffect on the cell viability of the organoids.

【Conclusions】These findings revealed that BC organoids derived fromurine stem cells might become a useful tool to investigate the mechanisms ofpathogenesis and treatment of dog BC

Higher impulsivity is a risk factor for substance abuse and suicide, but only a few anti-impulsive drugs are clinically available. We recently proposed a strategy for identifying anti-impulsive drugs by investigating drugs that increase extracellular dopamine levels in the medial prefrontal cortex (mPFC) and stimulate dopamine D₁-like receptors, but not in the nucleus accumbens (NAc). To determine whether this strategy is promising, we examined the effects of duloxetine, a serotonin-noradrenaline reuptake inhibitor that might meet these criteria, on impulsive action in adult male Wistar/ST rats using a 3-choice serial reaction time task. The effects of duloxetine on the dopamine levels in the areas were evaluated using in vivo microdialysis, as the noradrenaline transporter mediates dopamine reuptake in some brain regions. We found that the administration of duloxetine reduced impulsive actions and increased dopamine levels in the mPFC but not in the NAc. Microinjection of a D₁-like receptor antagonist into the ventromedial prefrontal cortex blocked the suppression of impulsive action by duloxetine. These results support our proposed strategy for identifying and developing anti-impulsive drugs.

Acute psychological stress dynamically changes functional brain networks and induces negative emotional states including anxiety through altered release of neuromodulators such as catecholamines. The underlying neuronal mechanisms of stress responses have been extensively investigated, however they remain unclear. Recently, we found that the neurons in the claustrum are critically involved in stress-induced anxiety-like behaviors as determined by whole-brain activity mapping and DREADD-based pharmacogenetic manipulation of these neurons. Here, we show that pharmacogenetic activation of glutamatergic neurons in a subregion of the claustrum induces anxiety-like behaviors and leads to activation of several nuclei involved in stress response. Activation of the claustral glutamatergic neurons induced a tonic release of dopamine and noradrenaline in the medial prefrontal cortex. Pharmacological inhibition of the catecholamine signal attenuated the claustral activation-induced anxiety-like behaviors.　The present results suggest that glutamatergic neurons in the claustrum mediate stress-induced anxiety-like behaviors through modulating catecholamine release.

SSRIs are widely used as anxiolytics. Previously, we demonstrated that local injection of an SSRI into the basolateral nucleus of the amygdala (BLA) had anxiolytic effect in rats. In the present study, we investigated the effect of local co-administration of an SSRI and 5-HT_1A or 5-HT_2A antagonists into the BLA on conditioned fear in Wistar/ST rats, and indicated the expression of 5-HT_1A and 5-HT_2A receptor mRNAs in the BLA by in situ hybridization in C57BL/6 mice.

Local injection of citalopram (SSRI) into the BLA attenuated conditioned freezing, and this effect was blocked by local co-administration of WAY100635 (5-HT_1A antagonist) or MDL11939 (5-HT_2A antagonist). In in situ hybridization, 5-HT_1A mRNA was mainly expressed in GABAergic interneurons expressing somatostatin (SOM) mRNA, and 5-HT_2A mRNA was in those expressing parvalbumin (PV) or SOM mRNA.

From these results, it is speculated that SSRIs exert anxiolytic effect via 5-HT_1A and 5-HT_2A receptors in the BLA. Because PV- and SOM-positive GABAergic interneurons are known to form local neural circuits with glutamatergic pyramidal neurons, the anxiolytic action of SSRIs is likely to be mediated by serotonergic modulation of pyramidal neurons via these interneuron subclasses.

Suicide is a major public health concern and often associated with mental disorders, such as depression, bipolar illness, and schizophrenia. Although psychosocial stressors are major risk factors of suicide behavior, abnormalities in neurobiological mechanisms have been supposed to be another risk factor. In the present study, functional coupling of G(alpha)_q proteins with 5-HT_2A receptor and M₁ muscarinic acetylcholine receptor (mAChR) was determined by means of [³⁵S]GTPγS binding/immunoprecipitation assay in postmortem human brain membranes, and the effects of suicide on these measures were evaluated. Postmortem human brains were obtained from 20 patients with bipolar disorder, 20 depression, 20 schizophrenia, and 20 control subjects, and the patients groups included suicide victims in 14, 17, and 10 subjects, respectively. When these 80 subjects were divided into suicide victims (n = 41) and non-suicide (n = 39), there were no significant differences in %E_max, pEC₅₀, and slope factor values for each of the two measures between them. Our results indicate that 5-HT_2A receptor- and M₁ mAChR-mediated signaling through G(alpha)_q proteins is unaltered in suicide victims.

Recent studies suggest that oligodendrocyte disruptions in the central nervous system can impact potentially to mood regulation in human psychiatric disorders. Leukemia inhibitory factor (LIF) has been shown to be involved in myelination. In the present study, we investigated whether LIF are involved in the formation of stress adaptation. A single exposure to restraint stress for 60 min induced a decrease in head-dipping behavior in the hole-board test. This emotional stress response was not observed in mice that had been exposed to repeated restraint stress for 60 min/day for 14 days, which confirmed the development of stress adaptation. In contrast, mice that were exposed to restraint stress for 240 min/days for 14 days did not develop the stress adaptation, and continued to show a decrease in head-dipping behavior. Major myelin proteins including myelin basic protein and myelin-associated glycoprotein expression were decreased in the hippocampus of stress-maladaptive, but not stress-adaptive, mice. Under these conditions, protein levels of LIF was significantly increased only in the hippocampus of stress-adaptive mice. These results indicate that increased LIF in the hippocampus may contribute to the development of stress adaptation.

Store-operated Ca²⁺ entry (SOCE) plays critical roles in intracellular Ca²⁺ ([Ca²⁺]_i) homeostasis. Recent studies have shown that SOCE is essential for osteoblastic differentiation. In non-excitable cells, K⁺ channels are key regulators of SOCE-mediated Ca²⁺ signaling and control cell proliferation, differentiation, and migration, however, the functional role of K⁺ channels in osteoblast Ca²⁺ signaling remains unknown. In the present study, the contribution of K⁺ channels to the SOCE activity in the osteoblastic cell line MC3T3-E1, established from mouse calvaria was investigated. We found that the expression levels of inward rectifier K⁺ channel Kir2.1 transcripts were up-regulated in the differentiated MC3T3-E1 cells. The application of ML133, a Kir2 inhibitor, significantly reduced the SOCE-mediated [Ca²⁺]_i elevation in differentiated MC3T3-E1 cells, but not in immature MC3T3-E1 cells. In addition, the treatment with ML133 suppressed the expression of the differentiation markers in osteoblasts, and attenuated the endochondral ossification in murine embryonic metatarsals. These results suggest that Kir2.1 channels play essential roles in maintaining the bone homeostasis via modulating osteoblast differentiation.

KCNK9, a member of the two-pore K⁺channel family, is overexpressed in several types of human carcinomas, such as breast cancers and lung cancers, and is generally thought to be an oncogenic channel. However, it has been also reported that overexpression of KCNK9 induces apoptosis in some kinds of cells including cerebellar granular neurons. Thus, a paradox is observed in the effects of KCNK9 overexpression on apoptosis, which is considered to be due to differences in KCNK9 expression levels: low expression is anti-apoptotic and high expression is pro-apoptotic. In this study, we investigated whether it is true in the human pancreatic cancer cell lines, PANC1. For that purpose, KCNK9 was expressed transiently and stably in the cells. Transient overexpression of KCNK9 increased caspase activities in the transfected cells, whereas stable expression of KCNK9 made the cells resistant to hyperosmolarity-induced apoptosis. Although the mechanism is not fully understood, our data suggest that the opposite effect of KCNK9 overexpression on PANC1 apoptosis is not solely due to differential expression levels of the channels.

Neuropathic pain is refractory to conventional analgesics. Thus, the mechanism of neuropathic pain in rats with left L5 spinal nerve transection was reexamined. The patch-clamp technique was used on the isolated bilateral L5 dorsal root ganglion neurons. When rats exhibited established neuropathic pain, only the neurons with the diameter of 40-50 um (Abeta neurons) on the ipsilateral side showed a significantly larger density of an inward current at <–80 mV (I_h current) than that on the contralateral side. Ivabradine—an I_h current inhibitor—inhibited I_h currents in these neurons on both the sides in a similar concentration-dependent manner, with an IC₅₀ of ～3 uM. Moreover, the oral administration of ivabradine significantly alleviated the neuropathic pain on the ipsilateral side. An inhibitor of adenylyl cyclase or an antagonist of prostanoid EP4 receptors (CJ-023423) inhibited ipsilateral, but not contralateral I_h currents in these neurons. Furthermore, the intrathecal administration of CJ-023423 significantly attenuated neuropathic pain on the ipsilateral side. Thus, ivabradine and/or CJ-023423 may be a lead compound for the development of novel therapeutics against neuropathic pain.

GIRK channels regulate membrane excitability dependent on GPCR activity. Gain-of-function mutations in the channels have been identified in patients suffering from several diseases; e.g. primary aldsteronism. Since gene elimination of these channels yielded mild phenotypes of the mice, the channel blockage could be expected as therapeutic strategy. While channel blockers used to bind at a central cavity of ion permeation pathway located at the membrane domain, a loose spatial constraint of the mode of binding leads to a low selectivity of drug-channel interaction. Wide distribution of binding sites of their activators and blockers suggests that the prerequisite of conformational changes of entire molecules for functioning and the possibility to design allosteric modulators that bind to sites out of the central cavity. In an electron density map of a high-resolution crystal structure of Kir3.2 cytoplasmic region, we identified an electron density which was not belonging to the polypeptide chain. A model compound related to the shape of the density inhibited Kir3.2 activity. These results strongly suggested that the electron density and its surrounding area correspond to an allosteric modulator and its binding site.

Type 1 ryanodine receptor (RyR1) is a Ca²⁺ release channel on the sarcoplasmic reticulum in the skeletal muscle. Mutations in RyR1 cause various muscle diseases including malignant hyperthermia (MH) and central core disease (CCD). Although dantrolene is the only therapeutic drugs for MH, it cannot be used for CCD due to its lower solubility and side effects. It is therefore urgent to discover novel RyR1 inhibitors. We have recently identified oxolinic acid as a novel RyR1 inhibitor. However, affinity of oxolinic acid was much lower than that of dantrolene. In this study, we designed and synthesized a series of quinolone derivatives using oxolinic acid as a lead compound. Dose-dependent inhibitory effects were evaluated by ER Ca²⁺ measurement using HEK293 cells expressing R-CEPIA1er, a genetically-encoded ER Ca²⁺ indicator, and RyR1 carrying an MH mutation (R2163C). Compounds bearing a longer alkyl chain at the nitrogen atom of quinolone ring exhibited stronger RyR1 inhibiting activity. Modification at 6 and 7 positions of quinolone ring greatly affected the inhibitory activity. Derivatives of oxolinic acid may be good candidates for treatment of RyR1-related diseases.

Platelet-derived growth factor (PDGF) potently induces migration of vascular smooth muscle cells (VSMC); however, molecular mechanism underlying this phenomenon remains unclear. The migration of rat aorta-derived synthetic VSMCs, A7r5, in response to PDGF was potently inhibited by a Ca_V1.2 channel inhibitor, nifedipine, and a Src family tyrosine kinase inhibitor, bosutinib, in a less-than-additive manner. In contractile VSMCs, the C-terminus of Ca_V1.2 is proteolytically cleaved into proximal and distal C-termini (PCT and DCT, respectively). Clipped DCT is noncovalently reassociated with PCT to autoinhibit the channel activity. Conversely, in synthetic A7r5 cells, full-length Ca_V1.2 (Ca_V1.2FL) is expressed much more abundantly than truncated Ca_V1.2. In a heterologous expression system, c-Src was bound to, phosphorylated Tyr1709 and Tyr1758 in PCT and activated Ca_V1.2 channels composed of Ca_V1.2FL significantly more efficiently than Ca_V1.2 (Ca_V1.2delta1763) or Ca_V1.2delta1763 plus clipped DCT. Therefore, in atherosclerotic lesions, phenotypic switching of VSMCs may facilitate pro-migratory effects of PDGF on VSMCs by suppressing posttranslational Ca_V1.2 modifications.

In smooth muscle cells (SMCs), caveolin (cav)-1, an essential component of caveolae, forms Ca²⁺ microdomain accumulating voltage-dependent Ca²⁺ channels (VDCC) and ryanodine receptors (RyR). The functional coupling between VDCC and RyR (Ca²⁺-induced Ca²⁺ release: CICR) causes SMC contraction, i.e. excitation-contraction (E-C) coupling. On the other hand, Ca²⁺ influx through VDCC activates Ca²⁺/calmodulin-dependent protein kinase, and promotes gene transcription in neurons, i.e. excitation-transcription (E-T) coupling. E-T coupling is known in SMCs, but its structural basis and physiologic function are unknown. Therefore, we examined the relationships between Ca²⁺ microdomain formed by caveolae and E-T coupling in SMCs. When the mesenteric artery was depolarized, the phosphorylation of CREB was detected in the nuclei of SMCs. This response was not observed in tissues from cav-1 KO mice that lack caveolae in SMCs and those in which caveolae were destroyed by methyl beta cyclodextrin. Inhibition of RyR by tetracaine also reduced the CREB phosphorylation. These results suggest that CICR in caveolae is necessary for the E-T coupling in SMCs. Caveolae can control not only SMC contractility but also gene expression by regulating Ca²⁺ signaling.

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. We previously reported that the Ca²⁺-sensing receptor (CaSR) is upregulated in pulmonary arterial smooth muscle cells (PASMCs) from patients with IPAH, contributing to an enhanced Ca²⁺ response and excessive cell proliferation in IPAH-PASMCs. However, the mechanisms underlying the upregulation of CaSR in IPAH-PASMCs have not yet been elucidated. The present results demonstrated that platelet-derived growth factor (PDGF) and its signaling pathway promote the expression of CaSR in PASMCs, thereby facilitating Ca²⁺ responses, cell proliferation, and migration followed by pulmonary vascular remodeling. Therefore, siRNA knockdown of PDGFα/β receptors and STAT1/3 or imatinib (a tyrosine kinase inhibitor including PDGF receptors) blocked the CaSR upregulation and functions in IPAH-PASMCs. The combination of NPS2143 (a CaSR antagonist) and imatinib acted additively to inhibit the development of pulmonary hypertension in monocrotaline-treated rats. In conclusion, the crosslink between CaSR and PDGF signals is a novel pathophysiological mechanism contributing to the development of IPAH.

Inactivation of myosin light chain phosphatase thorough myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation by Rho kinase is important for angiotensin II (Ang II)-induced constriction. However, the mechanism of Rho kinase activation by Ang II is unknown. We investigated whether Ang II-induced constriction in pressure-overloaded rat thoracic aortas is mediated by Rho kinase activation through Src, epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (Erk), and janus kinase (JAK).

The pressure-overload in rat thoracic aortas was produced by suprarenal abdominal aortic coarctation. After 4 weeks, thoracic aortas were excised and performed organ chamber experiments. Protein levels were measured by Western blotting.

Contractile response to Ang II significantly attenuated by inhibitors of Rho kinase, Erk1/2, Src, and EGFR in sham-treated and pressure-overloaded rats. Total and phosphorylated levels of MYPT1 and Src were increased in pressure-overloaded rat thoracic aortas. These data suggest that Ang II-induced constriction is mediated by Rho kinase activation via Src, EGFR, and Erk in pressure-overloaded rat thoracic aortas.

Objectives: Coagulation factor XI (FXI) was reported to contribute to atherogenesis. The underlying mechanism remains unknown. We investigated the cellular effect of FXI and its mechanism in vascular smooth muscle cells.

Methods and main results: Activated FXI (FXIa) induced intracellular Ca²⁺ signaling mainly due to Ca²⁺ influx in the fura-2-loaded A7r5 cells, rat embryonic aorta smooth muscle cells. Pharmacological inhibitor (diltiazem) and genetic knockdown of L-type Ca²⁺ channel abolished the FXIa-induced Ca²⁺ influx. FXIa-induced Ca²⁺ signaling was abolished by atopaxar, a PAR₁ antagonist. FXIa, when pre-incubated with a proteinase inhibitor p-APMSF, failed to elicit Ca²⁺ signaling in A7r5 cells. FXIa failed to induce Ca²⁺ signal in embryonic fibroblasts derived from PAR₁-knockout mice. In vitro digestion assay revealed that FXIa cleaved the extracellular domain of PAR₁ at the same site that thrombin cleaved. FXIa accelerated cell migration of A7r5 cells in a wound healing assay. The acceleration of cell migration was partly inhibited by atopaxar and diltiazem.

Conclusions: We provides the first evidence that FXIa exerts cellular effect via PAR₁ in vascular smooth muscle cells.

Background: Skeletal muscle mass is defined by the homeostatic coordination of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via the ubiquitin-ligase dependent pathway. However, the actionof iron on muscle myogenesis has remained unclear. In the present study, we investigated the effect of iron on skeletal muscle myogenesis.

Methods: We examined muscle regeneration by using cardiotoxin (CTX)-induced muscle injury mice model with or without iron overload in in vivo experiments, and C2C12 mice myoblast cells for in vitro study.

Results: In mice with iron overload, the skeletal muscles exhibited an increase in oxidative stress and a decrease in the expression of satellite cells markers such as Pax-7 and MyoD expression. Following CTX-induced muscle injury, mice with iron overload also exhibited delay in muscle regeneration with the reduced size of regenerating myofibres, decreased expression of myogenin and myosin heavy chain, and less phosphorylation of the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2. Similarly to the findings in the in vivo experiments, iron treatment also inhibited C2C12 myoblast cells differentiation, and it was ameliorated by a superoxide scavenger drug.

Conclusion: Iron accumulation suppresses skeletal muscle myogenesis through inhibiting MAPKs signalling via oxidative stress, causing an imbalance in the skeletal muscle homeostasis.

Heparan sulfate (HS) is a sulfated linear polysaccharide around cell surface. HS is involved in various physiological processes. Recent studies revealed that lack of HS induced autism-like behaviors and hyperglycemia. However, the roles of HS in skeletal muscles remained to be elucidated.

First, we examined the role of HS in the differentiation of muscle cells using C2C12 cells, a mouse myoblast cell line. CRISPR/CAS9 technology was used to delete Ext1, which encodes a heparan sulfate synthase, to generate a HS-deleted C2C12 cell line. HS deletion dramatically impaired myoblast differentiation, demonstrating the essential role of HS in myoblast differentiation. Next, we generated skeletal muscle-specific Ext1 deleted mice (cKO). Muscle weakness of cKO was revealed in treadmill tests and wire hang tests. Histochemical analysis of skeletal muscles revealed that the cross sectional area of each muscle was smaller in cKO. Electromicroscopic observation showed that myofibrils were thinner in cKO. Finally, we examined muscle differentiation after muscle injury Myosin heavy chain expression, one of the marker proteins for muscle differentiation, was significantly decreased in cKO muscles. These results demonstrate that HS plays an important role in skeletal muscle, especially in differentiation.

The relationship between the uses of antiepileptic drugs (AEDs) and the risk of fractures have been reported. While, there is only limited data concerning the effects of the newer AEDs on bone metabolism. In this study, we investigated the effects of gabapentin, a newer antiepileptic drug on bone metabolism. Male SD rats were administrated with gabapentin (30, 150 mg/kg) orally every morning for 12 weeks. Bone histomorphometry was analyzed using a semiautomatic image analyzing system, and bone strength was evaluated using a three-point bending method. Bone mineral density was measured using quantitative computed tomography, and serum biochemical markers (osteocalcin, tartrate-resistant acid phosphatase-5b) were examined. Administrated of gabapentin significantly decreased bone volume and increased trabecular separation, as shown by bone histomorphometric analysis. Moreover, the bone formation parameters, osteoid volume and mineralizing surface, were decreased after gabapentin treatment, whereas the bone resorption parameters, osteoclast surface and number, were increased. These results suggested that gabapentin may act on bone metabolism through the suppression of bone formation and enhancement of bone resorption.

We have reported that Eucommia leaf extract (ELE) influences the early stage of differentiation of both osteoblast and chondrocyte cells. Therefore, in this study, we focused on the bone marrow containing mesenchymal stem cells, which are the origin of both cells, and analyzed protein composition in bone marrow of rat administered with ELE. Femoral bones were extirpated from 2-month-old wistar rats orally administered with 10% ELE for 3 days. Bone marrow was washed out from the diaphysis of femur by lysis buffer and protein was extracted from the obtained bone marrow and fluorescent labeling was performed. Protein separation was then carried out by the immobilized pH gradient method. Subsequently, protein separation was carried out by molecular weight by performing SDS-PAGE. After electrophoresis, a fluorescence image of the gel was captured and image analysis was performed using ImageMsater 2D Platinum 7.0. Proteins were then extracted from significantly changed spots and mass spectrometry was performed by MALDI-TOF-MS. As a result of image analysis, 1,567 spots changed in expression level, and 74 spots were detected in which protein amount was changed more than 2 times. In addition, 20 spots were detected in which the expression was changed at 0.05> P.

Quinonoid dihydropteridine reductase (QDPR) regenerates tetrahydrobiopterin (BH4), an indispensable cofactor for synthesizing catecholamines and serotonin. We evaluated the platelet aggregation of QDPR gene knockout (Qdpr ^-/-) mice. Citrated blood was collected from wild type (Qdpr ^+/+) mice and Qdpr ^-/- mice (OYC35, Lexicon Pharmaceuticals Inc.). Platelet rich plasma (PRP) was obtained by centrifuging the blood, and adjusted platelet count to 250,000/μL. Platelets were stimulated with collagen (3～4 μg/mL) or ADP (5～20 μM) and aggregation was measured by light transmission method for 10 minutes. Intraplatelet serotonin was quantified by high-performance liquid chromatography (HPLC) with electrochemical detection. The Qdpr ^-/- mice significantly decreased the area under the aggregation curve (AUC) stimulated with collagen (Qdpr ^+/+: 6,061±600, Qdpr ^-/-: 3,424±514, p<0.05 by Tukey-Kramer test) and ADP (Qdpr ^+/+: 4,197±295, Qdpr ^-/-: 2,452±266, p<0.05). The intraplatelet serotonin content was significantly decreased in the Qdpr ^-/- mice (Qdpr ^+/+: 48.1±4.0 pmol/10⁶ platelets, Qdpr ^-/-: 22.1±2.8 pmol/10⁶ platelets, p<0.01). These results indicate that the Qdpr ^-/- mice suppresses secondary platelet aggregation via serotonin.

During the course of neuronal development, an axon is specified by the PI3 kinase- and PKA-dependent signaling pathways. However, the up-stream factors that modulate the activity of these kinases remain to be fully elucidated. Recently, we clarified that the subcellular dynamics of Gαs activating G protein-coupled receptor 3 (GPR3) are associated with the local activation of PKA in cerebellar granular neurons. In the current study, we focused on the possible involvement of GPR3 in axon specification. The numbers of neurons with Tau-1-positive neurites significantly decreased at 48–60 h after GPR3 siRNA transfection in rat hippocampal neurons. In contrast, the upregulated expression of GPR3 resulted in an accelerated formation of Tau-1-positive neurites at 24 h after transfection. Similarly, a delayed formation of Tau-1-positive neurites was observed in the hippocampal neurons from GPR3-knockout mice. GPR3-mediated acceleration of axon formation was significantly reduced by the administration of PI3-kinase inhibitor and not by PKA inhibitor. Subsequently, we evaluated whether the GPR3 expression affects the de-phosphorylation of CRMP2, which is downstream in the PI3-kinase signaling pathway. The number of neurons with pCRMP2-negative neurites significantly decreased 60 h after GPR3 siRNA transfection. Furthermore, the staining intensity of pCRMP2 at the neurite tip was significantly stronger in GPR3-knockout mice than in the wild-type mice. Our cumulative results indicate the potential role of GPR3 in the axon specification in cultured hippocampal neurons.