Yutaka Koyama

Involvement of the long-chain fatty acid receptor GPR40 as a novel pain regulatory system.

G-protein receptor (GPR) 40 is known to be activated by docosahexaenoic acid (DHA). However, reports studying the role and functions (including pain regulation) of GPR40 in the brain are lacking. We investigated the involvement of GPR40 in the brain on DHA-induced antinociceptive effects. Expression of GPR40 protein was observed in the olfactory bulb, striatum, hippocampus, midbrain, hypothalamus, medulla oblongata, cerebellum and cerebral cortex in the brain as well as the spinal cord, whereas GPR120 protein expression in these areas was not observed. Intracerebroventricular (i.c.v.), but not intrathecal (i.t.) injection of DHA (25 and 50μg/mouse) and GW9508 (a GPR40- and GPR120-selective agonist; 0.1 and 1.0μg/mouse) significantly reduced formalin-induced pain behavior. These effects were inhibited by pretreatment with the μ opioid receptor antagonist β-funaltrexamine (β-FNA), naltrindole (δ opioid receptor antagonist) and anti-β-endorphin antiserum. The κ opioid receptor antagonist norbinaltorphimine (nor-BNI) did not affect the antinociception of DHA or GW9508. Furthermore, the immunoreactivity of β-endorphin in the hypothalamus increased at 10 and 20min after i.c.v. injection of DHA and GW9508. These findings suggest that DHA-induced antinociception via β-endorphin release may be mediated (at least in part) through GPR40 signaling in the supraspinal area, and may provide valuable information on a novel therapeutic approach for pain control.

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.

Hypothalamic GPR40 Signaling Activated by Free Long Chain Fatty Acids Suppresses CFA-Induced Inflammatory Chronic Pain

GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freunds adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty acids might have an important role in this pain control system.

Endothelins stimulate the production of stromelysin-1 in cultured rat astrocytes.

The effects of endothelins (ETs) on the production of stromelysins, a sub-family of matrix metalloproteinases, were examined in cultured astrocytes. The treatment of cultured rat astrocytes with ET-1 increased stromelysin-1 mRNA levels, while stromelysin-2 and -3 mRNAs were not affected. Immunocytochemical observations showed that cultured astrocytes produced stromelysin-1 protein. ET-1 and Ala(1,3,11,15)-ET-1, an ET(B) receptor selective agonist, stimulated the release of stromelysin-1 from cultured astrocytes. Accompanying the increase in protein release, the peptidase activity of stromelysin-1 in the medium was also increased by ET-1. The effects of ET-1 on astrocytic stromelysin-1 expression were inhibited by PD98059, staurosporine, and Ca(2+) chelation, but not by SB203580 or pyrrolidine dithiocarbamate. These results show that activation of astrocytic ET receptors stimulates the production of stromelysin-1, suggesting a role for ETs in stromelysin production in brain pathologies.

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 Ala1,3,11,15‐ET‐1 (100 nM), an ETB 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 ETB 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 VEGF165 (100 ng/mL), an isopeptide of VEGF‐A, stimulated bromodeoxyuridine (BrdU) incorporation into cultured astrocytes. Treatment with ET‐1 and VEGF165 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 VEGF165 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.

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

AbstractBackgroundChemokines are involved in many pathological responses of the brain.nAstrocytes produce various chemokines in brain disorders, but little isnknown about the factors that regulate astrocytic chemokine production.nEndothelins (ETs) have been shown to regulate astrocytic functions throughnETB receptors. In this study, the effects of ETs on chemokinenproduction were examined in rat cerebral cultured astrocytes.MethodsAstrocytes were prepared from the cerebra of one- to two-day-old Wistar ratsnand cultured in serum-containing medium. After serum-starvation for 48nhours, astrocytes were treated with ETs. Total RNA was extracted using annacid-phenol method and expression of chemokine mRNAs was determined bynquantitative RT-PCR. The release of chemokines was measured by ELISA.ResultsTreatment of cultured astrocytes with ET-1 and Ala1,3,11,15-ET-1,nan ETB agonist, increased mRNA levels of CCL2/MCP1 andnCXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased innthe presence of ET-1 and Ala1,3,11,15-ET-1. The effect of ET-1 onnchemokine mRNA expression was inhibited by BQ788, an ETBnantagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes,nbut decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression bynET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50,nmithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression bynET-1 was inhibited by cycloheximide, Ca2+ chelation andnstaurosporine.ConclusionThese findings suggest that ETs are one of the factors regulating astrocyticnchemokine production. Astrocyte-derived chemokines are involved innpathophysiological responses of neurons and microglia. Therefore, thenET-induced alterations of astrocytic chemokine production are ofnpathophysiological significance in damaged brains.

The activation of supraspinal GPR40/FFA1 receptor signalling regulates the descending pain control system

The ω‐3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid‐induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons.

Signaling molecules regulating phenotypic conversions of astrocytes and glial scar formation in damaged nerve tissues

Phenotypic conversion of astrocytes from resting to reactive (i.e., astrocytic activation) occurs in numerous brain disorders. Astrocytic activation in severely damaged brain regions often leads to glial scar formation. Because astrocytic activation and glial scar largely affect the vulnerability and tissue repair of damaged brain, numerous studies have been made to clarify mechanisms regulating the astrocytic phenotype. The phenotypic conversion is accompanied by the increased expression of intermediate filament proteins and the induction of hypertrophy in reactive astrocytes. Severe brain damage results in proliferation and migration of reactive astrocytes, which lead to glial scar formations at the injured areas. Gliogenesis from neural progenitors in the adult brain is also involved in astrocytic activation and glial scar formation. Recent studies have shown that increased expression of connexin 43, aquaporin 4, matrix metalloproteinase 9, and integrins alter the function of astrocytes. The transcription factors: STAT3, OLIG2, SMAD, NF-κB, and Sp1 have been suggested to play regulatory roles in astrocytic activation and glial scar formation. In this review, I discuss the roles of these key molecules regulating the pathophysiological functions of reactive astrocytes.

Functional alterations of astrocytes in mental disorders: pharmacological significance as a drug target

Astrocytes play an essential role in supporting brain functions in physiological and pathological states. Modulation of their pathophysiological responses have beneficial actions on nerve tissue injured by brain insults and neurodegenerative diseases, therefore astrocytes are recognized as promising targets for neuroprotective drugs. Recent investigations have identified several astrocytic mechanisms for modulating synaptic transmission and neural plasticity. These include altered expression of transporters for neurotransmitters, release of gliotransmitters and neurotrophic factors, and intercellular communication through gap junctions. Investigation of patients with mental disorders shows morphological and functional alterations in astrocytes. According to these observations, manipulation of astrocytic function by gene mutation and pharmacological tools reproduce mental disorder-like behavior in experimental animals. Some drugs clinically used for mental disorders affect astrocyte function. As experimental evidence shows their role in the pathogenesis of mental disorders, astrocytes have gained much attention as drug targets for mental disorders. In this paper, I review functional alterations of astrocytes in several mental disorders including schizophrenia, mood disorder, drug dependence, and neurodevelopmental disorders. The pharmacological significance of astrocytes in mental disorders is also discussed.

Decreases in rat brain aquaporin-4 expression following intracerebroventricular administration of an endothelin ETB receptor agonist

Aquaporins (AQPs) comprise a family of water channel proteins, some of which are expressed in brain. Expressions of brain AQPs are altered after brain insults, such as ischemia and head trauma. However, little is known about the regulation of brain AQP expression. Endothelins (ETs), vasoconstrictor peptides, regulate several pathophysiological responses of damaged nerve tissues via ET(B) receptors. To show possible roles of ET(B) receptors in the regulation of brain AQP expression, the effects of intracerebroventricular administration of an ET(B) agonist were examined in rat brain. In the cerebrum, the copy numbers of AQP4 mRNAs were highest among AQP1, 3, 4, 5 and 9. Continuous administration of 500 pmol/day Ala(1,3,11,15)-ET-1, an ET(B) selective agonist, into rat brain for 7 days decreased the level of AQP4 mRNA in the cerebrum, but had no effect on AQP1, 3, 5 and 9 mRNA levels. The level of AQP4 protein in the cerebrum decreased by the administration of Ala(1,3,11,15)-ET-1. Immunohistochemical observations of Ala(1,3,11,15)-ET-1-infused rats showed that GFAP-positive astrocytes, but not neurons, activated microglia or brain capillary endothelial cells, had immunoreactivity for AQP4. These findings indicate that activation of brain ET(B) receptors causes a decrease in AQP4 expression, suggesting that ET down-regulates brain AQP4 via ET(B) receptors.