Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Differential regulation of histamine H1 receptor-mediated ERK phosphorylation by Gq proteins and arrestins.

Gq protein-coupled histamine H1 receptors play crucial roles in allergic and inflammatory reactions, in which the phosphorylation of extracellular signal-regulated kinase (ERK) appears to mediate the production of inflammatory cytokines. ERK phosphorylation is regulated by G protein- and arrestin-mediated signal transduction pathways. Here, we aimed to explore how H1 receptor-mediated processes of ERK phosphorylation might be differentially regulated by Gq proteins and arrestins. For this purpose, we evaluated the regulatory mechanism(s) of H1 receptor-mediated ERK phosphorylation in Chinese hamster ovary cells expressing Gq protein- and arrestin-biased mutants of human H1 receptors, S487TR and S487A, in which the Ser487 residue in the C-terminal was truncated and mutated to alanine, respectively. Immunoblotting analysis indicated that histamine-induced ERK phosphorylation was prompt and transient in cells expressing Gq protein-biased S487TR, whereas it was slow and sustained in cells expressing arrestin-biased S487A. Inhibitors of Gq proteins (YM-254890) and protein kinase C (PKC) (GF109203X), and an intracellular Ca2+ chelator (BAPTA-AM) suppressed histamine-induced ERK phosphorylation in cells expressing S487TR, but not those expressing S487A. Conversely, inhibitors of G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), clathrin (hypertonic sucrose), Raf (LY3009120), and MEK (U0126) suppressed histamine-induced ERK phosphorylation in cells expressing S487A, but not those expressing S487TR. These results suggest that H1 receptor-mediated ERK phosphorylation might be differentially regulated by the Gq protein/Ca2+/PKC and GRK/arrestin/clathrin/Raf/MEK pathways to potentially determine the early and late phases of histamine-induced allergic and inflammatory responses, respectively.

Liver-specific overexpression of lipoprotein lipase improves glucose metabolism in high-fat diet-fed mice.

The liver is the main organ that regulates lipid and glucose metabolism. Ectopic lipid accumulation in the liver impairs insulin sensitivity and glucose metabolism. Lipoprotein lipase (LPL), mainly expressed in the adipose tissue and muscle, is a key enzyme that regulates lipid metabolism via the hydrolysis of triglyceride in chylomicrons and very-low-density lipoproteins. Here, we aimed to investigate whether the suppression level of hepatic lipid accumulation via overexpression of LPL in mouse liver leads to improved metabolism. To overexpress LPL in the liver, we generated an LPL-expressing adenovirus (Ad) vector using an improved Ad vector that exhibited considerably lower hepatotoxicity (Ad-LPL). C57BL/6 mice were treated with Ad vectors and simultaneously fed a high-fat diet (HFD). Lipid droplet formation in the liver decreased in Ad-LPL-treated mice relative to that in control Ad vector-treated mice. Glucose tolerance and insulin resistance were remarkably improved in Ad-LPL-treated mice compared to those in control Ad vector-treated mice. The expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, and acyl-CoA oxidase 1, were 1.7-2.0-fold higher in Ad-LPL-treated mouse livers than that in control Ad-vector-treated mouse livers. Furthermore, hepatic LPL overexpression partly maintained mitochondrial content in HFD-fed mice. These results indicate that LPL overexpression in the livers of HFD-fed mice attenuates the accumulation of lipid droplets in the liver and improves glucose metabolism. These findings may enable the development of new drugs to treat metabolic syndromes such as type 2 diabetes mellitus and non-alcoholic fatty liver disease.

ZFAND3 Overexpression in the Mouse Liver Improves Glucose Tolerance and Hepatic Insulin Resistance.

Genome-wide association studies have identified more than 300 loci associated with type 2 diabetes mellitus; however, the mechanisms underlying their role in type 2 diabetes mellitus susceptibility remain largely unknown. Zinc finger AN1-type domain 3 (ZFAND3), known as testis-expressed sequence 27, is a type 2 diabetes mellitus-susceptibility gene. Limited information is available regarding the physiological role of ZFAND3 in vivo. This study aimed to investigate the association between ZFAND3 and type 2 diabetes mellitus. ZFAND3 was significantly upregulated in the liver of diabetic mice compared to wild-type mice. To overexpress ZFAND3, we generated a ZFAND3-expressing adenovirus (Ad) vector using an improved Ad vector exhibiting significantly lower hepatotoxicity (Ad-ZFAND3). Glucose tolerance was significantly improved in Ad-ZFAND3-treated mice compared to the control Ad-treated mice. ZFAND3 overexpression in the mouse liver also improved insulin resistance. Furthermore, gluconeogenic gene expression was significantly lower in primary mouse hepatocytes transduced with Ad-ZFAND3 than those transduced with the control Ad vector. The present results suggest that ZFAND3 improves glucose tolerance by improving insulin resistance and suppressing gluconeogenesis, serving as a potential novel therapeutic target for type 2 diabetes mellitus.

Pharmacological Inhibition of Transient Receptor Potential Vanilloid 4 Reduces Vasogenic Edema after Traumatic Brain Injury in Mice.

Vasogenic edema results from blood-brain barrier (BBB) disruption after traumatic brain injury (TBI), and although it can be fatal, no promising therapeutic drugs have been developed as yet. Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable channel that is sensitive to temperature and osmotic pressure. As TRPV4 is known to be responsible for various pathological conditions following brain injury, we investigated the effects of pharmacological TRPV4 antagonists on TBI-induced vasogenic edema in this study. A TBI model was established by inflicting fluid percussion injury (FPI) in the mouse cerebrum and cultured astrocytes. Vasogenic brain edema and BBB disruption were assessed based on brain water content and Evans blue (EB) extravasation into brain tissue, respectively. After FPI, brain water content and EB extravasation increased. Repeated intracerebroventricular administration of the specific TRPV4 antagonists HC-067047 and RN-1734 dose-dependently reduced brain water content and alleviated EB extravasation in FPI mice. Additionally, real-time PCR analysis indicated that administration of HC-067047 and RN-1734 reversed the FPI-induced increase in mRNA levels of endogenous causal factors for BBB disruption, including matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and endothelin-1 (ET-1). In astrocytes, TRPV4 level was observed to be higher than that in brain microvascular endothelial cells. Treatment with HC-067047 and RN-1734 inhibited the increase in mRNA levels of MMP-9, VEGF-A, and ET-1 in cultured astrocytes subjected to in vitro FPI. These results suggest that pharmacological inhibition of TRPV4 is expected to be a promising therapeutic strategy for treating TBI-induced vasogenic edema.

Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase-associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes.

Brain injury-mediated induction of reactive astrocytes often leads to glial scar formation in damaged brain regions. Activation of signal transducer and activator of transcription 3 (STAT3), a member of the STAT family of transcription factors, plays a pivotal role in inducing reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) is a vasoconstrictor peptide, and its levels increase in brain disorders and promote astrocytic proliferation through ETB receptors. To clarify the mechanisms underlying ET-1-mediated astrocytic proliferation, here we examined its effects on STAT3 in cultured rat astrocytes. ET-1 treatment stimulated Ser-727 phosphorylation of STAT3 in the astrocytes, but Tyr-705 phosphorylation was unaffected, and ET-induced STAT3 Ser-727 phosphorylation was reduced by the ETB antagonist BQ788. ET-1 stimulated STAT3 binding to its consensus DNA-binding motifs. Monitoring G1/S phase cell cycle transition through bromodeoxyuridine (BrdU) incorporation, we found that ET-1 increases BrdU incorporation into the astrocytic nucleus, indicating cell cycle progression. Of note, STAT3 chemical inhibition (with stattic or 5,15-diphenyl-porphine (5,15-DPP)) or siRNA-mediated STAT3 silencing reduced ET-induced BrdU incorporation. Moreover, ET-1 increased astrocytic expression levels of cyclin D1 and S-phase kinase-associated protein 2 (SKP2), which were reduced by stattic, 5,15-DPP, and STAT3 siRNA. ChIP-based PCR analysis revealed that ET-1 promotes the binding of SAT3 to the 5'-flanking regions of rat cyclin D1 and SKP2 genes. Our results suggest that STAT3-mediated regulation of cyclin D1 and SKP2 expression underlies ET-induced astrocytic proliferation.

Effects of irradiation with narrowband-ultraviolet B on up-regulation of histamine H1 receptor mRNA and induction of apoptosis in HeLa cells and nasal mucosa of rats.

Narrowband-ultraviolet B (NB-UVB) phototherapy is used for the treatment of atopic dermatitis. Previously, we reported that irradiation with 200 mJ/cm2 of 310 nm NB-UVB suppressed phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) gene expression without induction of apoptosis in HeLa cells. However, the effect of NB-UVB irradiation on nasal symptoms is still unclear. Here, we show that low dose irradiation with 310 nm NB-UVB alleviates nasal symptoms in toluene 2,4-diisocyanate (TDI)-sensitized allergy model rats. Irradiation with 310 nm NB-UVB suppressed PMA-induced H1R mRNA up-regulation in HeLa cells dose-dependently at doses of 75-200 mJ/cm2 and reversibly at a dose of 150 mJ/cm2 without induction of apoptosis. While, at doses of more than 200 mJ/cm2, irradiation with 310 nm NB-UVB induced apoptosis. Western blot analysis showed that the suppressive effect of NB-UVB irradiation on H1R gene expression was through the inhibition of ERK phosphorylation. In TDI-sensitized rat, intranasal irradiation with 310 nm NB-UVB at an estimated dose of 100 mJ/cm2 once a day for three days suppressed TDI-induced sneezes and up-regulation of H1R mRNA in nasal mucosa without induction of apoptosis. These findings suggest that repeated intranasal irradiation with low dose of NB-UVB could be clinically used as phototherapy of AR.

The Endothelin ETB Receptor Antagonist BQ788 Protects against Brain Edema after Fluid Percussion Injury by Decreasing Vascular Endothelial Growth Factor-A Expression in Mice.

Brain edema is a severe morbid complication of brain injury, characterized by excessive fluid accumulation and an elevation of intracranial pressure. However, effective anti-brain edema drugs are lacking. One of the causes of brain edema is disruption of blood-brain barrier (BBB) function, which results in extravasation of intravascular fluid. After brain damage, astrocytes are activated, and astrocyte-derived vascular endothelial growth factor-A (VEGF-A) is known to induce BBB dysfunction. Therefore maintaining BBB integrity by regulating astrocyte function is a potentially effective strategy for treating brain edema. In this review, we focus on the endothelin ETB receptor and its role in regulation of astrocyte functions. In mice, brain damage was induced by fluid percussion injury (FPI), and the resulting BBB disruption and brain edema were observed in the mouse cerebrum. BQ788, a selective ETB receptor antagonist, attenuated the FPI-induced BBB disruption and brain edema. Levels of brain VEGF-A increased after FPI, mainly in reactive astrocytes. BQ788 suppressed the FPI-induced increase in VEGF-A expression in reactive astrocytes. Moreover, intraventricular administration of VEGF neutralizing antibody also attenuated FPI-induced BBB disruption and brain edema. Claudin-5 is an endothelial tight junction protein essential for normal BBB structure and function. Levels of claudin-5 protein were reduced by FPI. Furthermore, VEGF neutralizing antibody blocked FPI-induced decrease in claudin-5. These results suggest that the ETB receptor antagonist BQ788 protects against brain edema by inhibiting VEGF-A-mediated decrease in claudin-5.

Improvement of cold injury-induced mouse brain edema by endothelin ETB antagonists is accompanied by decreases in matrixmetalloproteinase 9 and vascular endothelial growth factor-A.

Brain edema is a potentially fatal pathological state that often occurs after brain injuries such as ischemia and trauma. However, therapeutic agents that fundamentally treat brain edema have not yet been established. We previously found that endothelin ETB receptor antagonists attenuate the formation and maintenance of vasogenic brain edema after cold injury in mice. In this study, the effects of ETB antagonists on matrixmetalloproteinase (MMP)9 and vascular endothelial growth factor (VEGF)-A expression were examined in the cold injury model. Cold injury was performed in the left brain of male ddY mice (5-6 weeks old) for the induction of vasogenic edema. Expression of MMP9 and VEGF-A mRNA in the mouse cerebrum was increased by cold injury. Immunohistochemical observations showed that the MMP9 and VEGF-A were mainly produced in reactive astrocytes in the damaged cerebrum. Intracerebroventricular administration of BQ788 (10 µg) or IRL-2500 (10 µg) (selective ETB antagonists) attenuated brain edema and disruption of the blood-brain barrier after cold injury. BQ788 and IRL-2500 reversed the cold injury-induced increases in MMP9 and VEGF-A expression. The induction of reactive astrocytes producing MMP9 and VEGF-A in the damaged cerebrum was attenuated by BQ788 and IRL-2500. These results suggest that attenuations of astrocytic MMP9 and VEGF-A expression by ETB antagonists may be involved in the amelioration of vasogenic brain edema.

Pathogenesis of brain edema and investigation into anti-edema drugs.

Brain edema is a potentially fatal pathological state that occurs after brain injuries such as stroke and head trauma. In the edematous brain, excess accumulation of extracellular fluid results in elevation of intracranial pressure, leading to impaired nerve function. Despite the seriousness of brain edema, only symptomatic treatments to remove edema fluid are currently available. Thus, the development of novel anti-edema drugs is required. The pathogenesis of brain edema is classified as vasogenic or cytotoxic edema. Vasogenic edema is defined as extracellular accumulation of fluid resulting from disruption of the blood-brain barrier (BBB) and extravasations of serum proteins, while cytotoxic edema is characterized by cell swelling caused by intracellular accumulation of fluid. Various experimental animal models are often used to investigate mechanisms underlying brain edema. Many soluble factors and functional molecules have been confirmed to induce BBB disruption or cell swelling and drugs targeted to these factors are expected to have anti-edema effects. In this review, we discuss the mechanisms and involvement of factors that induce brain edema formation, and the possibility of anti-edema drugs targeting them.

Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1.

BACKGROUND: Chemokines are involved in many pathological responses of the brain. Astrocytes produce various chemokines in brain disorders, but little is known about the factors that regulate astrocytic chemokine production. Endothelins (ETs) have been shown to regulate astrocytic functions through ETB receptors. In this study, the effects of ETs on chemokine production were examined in rat cerebral cultured astrocytes. METHODS: Astrocytes were prepared from the cerebra of one- to two-day-old Wistar rats and cultured in serum-containing medium. After serum-starvation for 48 hours, astrocytes were treated with ETs. Total RNA was extracted using an acid-phenol method and expression of chemokine mRNAs was determined by quantitative RT-PCR. The release of chemokines was measured by ELISA. RESULTS: Treatment of cultured astrocytes with ET-1 and Ala(1,3,11,15)-ET-1, an ET(B) agonist, increased mRNA levels of CCL2/MCP1 and CXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased in the presence of ET-1 and Ala(1,3,11,15)-ET-1. The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ET(B) antagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression by ET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50, mithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca(2+) chelation and staurosporine. CONCLUSION: These findings suggest that ETs are one of the factors regulating astrocytic chemokine production. Astrocyte-derived chemokines are involved in pathophysiological responses of neurons and microglia. Therefore, the ET-induced alterations of astrocytic chemokine production are of pathophysiological significance in damaged brains.

Endothelin-1 stimulates cyclin D1 expression in rat cultured astrocytes via activation of Sp1.

Endothelins (ETs), a family of vasoconstrictor peptides, are up-regulated in several pathological conditions in the brain, and induce astrocytic proliferation. We previously observed that ET-1 increased the expression of cyclin D1 protein. Thus, we confirmed the intracellular up-regulation of cyclin D1 by ET-1 in rat cultured astrocytes. Real-time PCR analysis indicated that ET-1 (100 nM) and Ala(1,3,11,15)-ET-1 (100 nM), a selective agonist of the ETB receptor, induced a time-dependent and transient increase in cyclin D1 mRNA. The effect of ET-1 was diminished by an ETB antagonist (1 µM BQ788) or inhibitors of Sp1 (500 nM mithramycin), ERK (50 µM PD98059), p38 (20 µM SB203580) and JNK (1 µM SP600125), but not inhibitors of NF-κB (10 µM SN50 and 100 µM pyrrolidine dithiocarbamate). The binding assay for Sp1 indicated that ET-1 increased the binding activity of Sp1 to consensus sequences, and two oligonucleotides of the cyclin D1 promoter including the Sp1-binding sites diminished the effect of ET-1. Western blot analysis showed that ET-1 induced time-dependent and transient phosphorylation of Sp1 on Thr453 and Thr739 via the ETB receptor. ET-1-induced phosphorylation of Sp1 was attenuated by PD98059 and SP600125. Additionally, ET-1 increased the incorporation of bromodeoxyuridine (BrdU) in cultured astrocytes and the number of BrdU-positive cells decreased in the presence of PD98059, SP600125 and mithramycin. These results suggest that ET-1 increases the expression of cyclin D1 via activation of Sp1 and induces astrocytic proliferation.

Endothelins reciprocally regulate VEGF-A and angiopoietin-1 production in cultured rat astrocytes: implications on astrocytic proliferation.

Vascular endothelial growth factors (VEGFs) and angiopoietins (ANGs) are involved in pathophysiological responses in damaged nerve tissues. Astrocytes produce VEGFs and ANGs upon brain ischemia and traumatic injury. To clarify the extracellular signals regulating VEGF and ANG production, effects of endothelins (ETs), a family of endothelium-derived peptides, were examined in cultured rat astrocytes. ET-1 (100 nM) and Ala(1,3,11,15)-ET-1 (100 nM), an ET(B) receptor agonist, increased VEGF-A mRNA levels in cultured astrocytes, while ANG-1 mRNA was decreased by ETs. ET-1 did not affect astrocytic VEGF-B, placental growth factor (PLGF), and ANG-2 mRNA levels. The effects of ET-1 on VEGF-A and ANG-1 mRNAs were inhibited by BQ788, an ET(B) antagonist. Release of VEGF-A proteins from cultured astrocytes was increased by ET-1. In contrast, ET-1 reduced release of astrocytic ANG-1. Exogenous ET-1 (100 nM) and VEGF(165) (100 ng/mL), an isopeptide of VEGF-A, stimulated bromodeoxyuridine (BrdU) incorporation into cultured astrocytes. Treatment with ET-1 and VEGF(165) increased the numbers of cyclin D1-positive astrocytes. Exogenous ANG-1 (250 ng/mL) did not stimulate the BrdU incorporation. Increases in BrdU incorporation by ET-1 and VEGF(165) were not affected by ANG-1. In 60-70% confluent cultures, SU4312 (10 µM), a VEGF receptor tyrosine kinase inhibitor, partially reduced the effects of ET-1 on BrdU incorporation and cyclin D1 expression. ET-induced BrdU incorporation and cyclin D1 expression were reduced by a neutralizing antibody against VEGF-A. Our findings suggest that ET-1 is a factor regulating astrocytic VEGF-A and ANG-1, and that increased VEGF-A production potentiates ET-induced astrocytic proliferation by an autocrine mechanism.

Depolarizing stimuli cause persistent and selective loss of orexin in rat hypothalamic slice culture.

A hypothalamic neuropeptide orexin (hypocretin) is a critical regulator of physiological processes including sleep/wakefulness and feeding. Using organotypic slice culture of rat hypothalamus, we found that exposure to elevated extracellular concentration of K(+) (+10-30 mM) for 24-72h led to a substantial decrease in the number of neurons immunoreactive for orexin and a co-existing neuropeptide dynorphin-A. In contrast, the same treatment affected neither the number of melanin-concentrating hormone-immunoreactive neurons nor the number of total neurons. A substantial decrease of orexin-immunoreactive neurons was also induced by 72h treatment with 1-10 microM veratridine, a Na(+) channel activator. The effect of elevated K(+) was only partially reversible, and that of veratridine was virtually irreversible, although the decrease in orexin immunoreactivity was not associated with signs of cell damage assessed by propidium iodide uptake and Hoechst 33342 nuclear staining. In addition, the level of preproorexin mRNA did not decrease during treatment with elevated K(+) or veratridine. After treatment with elevated K(+) and veratridine, c-Fos immunoreactivity appeared in orexin-immunoreactive neurons but not in melanin-concentrating hormone-immunoreactive neurons, suggesting selective excitation of orexin neurons. However, the amount of orexin released extracellularly was paradoxically decreased by treatment with elevated K(+) and veratridine. Overall, these characteristics of orexin neurons may be taken into consideration to understand the behaviors of these neurons under physiological and pathophysiological conditions.