Activation of CREB by ethanol and acetaldehyde in NG108-15 cells
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Abstracts / Neuroscience Research 58S (2007) S1–S244 S195 P3-b41 Expression analysis of VGLUT during the postnatal maturation of rat pineal gland S. Yoshida, A. Ina, J. Konno, K. Ogasawara, T. Wu, F. Shutoh, H. Nogami, S. Hisano Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan In the rat pineal gland, pinealocytes contain synaptic-like microvesicles which have vesicular glutamate transporter (VGLUT) 1 and 2. Recently, we reported early postnatal VGLUT1 expression in the gland whose mat- uration completes in the first 3 weeks after birth. Despite inhibition of melatonin synthesis by exogenous glutamate in adult pinealocytes in vitro, the role of glutamate in the developing gland is still unclear. To address this issue, we studied ontogeny of rat pineal VGLUT with in situ hybridization and immunohistochemistry. VGLUT1-staining was always seen only in pinealocytes stained for synaptopysin (a pinealocyte marker) at all ages examined. In contrast, VGLUT2-staining was seen in both pinealocytes and synaptophysin-unstained cells until the second post- natal week and later confined to pinealocytes, with the dominant type of pineal VGLUT postnatally shifting from VGLUT2 to VGLUT1. Our data suggest that pineal maturation processes are regulated under the age- dependent and VGLUT type-dependent exocytotic release of glutamate. P3-b42 Warm temperature-activated Ca 2+ response in cul- tured fibroblasts, neuronal cells and keratinocytes Noriko Mochizuki-Oda 1 , Tomoyuki Kusuno 1 , Haruhito Yamashita 1 , Yasuharu Takamori 2 , Hisao Yamada 2 , Hironari Yamada 1 1 Synchrotron Light Life Science Center, Ritsumeikan University, Kusatsu, Japan; 2 Department Anatomy and Cell Science, Moriguchi, Japan Among six TRP channels, TRPV3 and TRPV4 are identified as ‘warmth’ receptors, as their thresholds are around 33–35 ◦ C. We investigated the effects of warm temperature on the intracellular Ca 2+ in cultured fibrob- lasts (NIH3T3), neuronal cells (NG108-15) and keratinocytes (M308). The ‘warmth’ stimulus was applied by increasing the temperature of the bath solution from 33 to 38 ◦ C. M308 cells expressing TRPV3 and TRPV4 showed rapid increase of cellular Ca 2+ in normal bath solution contain- ing Ca 2+ . Without the extra-cellular Ca 2+ , slow and reduced response was observed, suggesting contribution of Ca 2+ mobilization from intracellular stores. NIH3T3 cells also responded to ‘warmth’ in the normal solution, but not in Ca 2+ -free solution. This suggests that they have only influx of Ca 2+ . On the other hand, NG108-15 cells showed both Ca 2+ influx and mobilization. The variety of the mechanisms of ‘warmth’ sensing will be discussed. Research funds: KAKENHI (17657052) P3-b43 Effect of oxidized galectin-1 on mRNA expression in macrophage Haruhisa Kawasaki 1 , Yukie Kogawa 2 , Kenichi Sasaguri 2 , Sadao Sato 2 , Hiroko Inoue 3 , Hidenori Horie 4 1 Department of Biology, Keio University, Yokohama, Japan; 2 Department of Craniofacial Growth and Development Dentistry, Kanagawa Dental College, Kanagawa, Japan; 3 School of Science and Engineering, Waseda University, Tokyo, Japan; 4 Brain and Oral Science Center, Kanagawa Dental College, Kanagawa, Japan Oxidized galectin-1 (GAL-1/Ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. But, the mechanism of GAL-1/Ox, how to regulate macrophage, remains to be clarified. Here we demonstrated effects of GAL-/Ox on mRNA expression of four injury related molecules, IL-1, IL-6, LIF, and iNOS in cultured rat peritoneal macrophages under 10 ng/ml LPS treated condition. RT-PCR analysis revealed that IL-6 as well as iNOS mRNA expressions were sig- nificantly up-regulated by GAL-1/Ox, but the other mRNA did not show significant differences by the application of GAL-1/Ox. Increased IL-6 may promotes axonal regeneration in peripheral nerves, but it is not certain whether increased iNOS contributes to the regeneration. Research funds: KAKENHI (17500265) P3-b45 Essential role of activation of PKA and ERK by BDNF for AMPA receptor translocation to postsynaptic sites Hiroko Nakata 1,2 , Shun Nakamura 1 1 National Institute of Neuroscience, Tokyo, Japan; 2 Tokyo Institute of Psychiatry, Tokyo, Japan We have recently shown that brain-derived neurotrophic factor (BDNF) induced an AMPA-type glutamatergic receptor (AMPAR) subunit, GluR1 translocation to the postsynaptic density (PSD) of cultured cortical pyra- midal neurons. The Ca 2+ signaling through IP3R as well as store-operated cation channel TRPC was critical in this translocation. The present study investigates the signaling mechanism of GluR1 translocation after the calcium raise. Acute BDNF application to the cultured neurons increased protein phosphorylation of protein kinase A (PKA) substrates and extra- cellular signal-regulated kinase (ERK) within 5–10 min at most of the dendritic spines. A PLC inhibitor blocked this effect. Half the PSD- translocation of GluR1 was prevented by each inhibitor of adenylyl cyclase (AC), PKA, or MEK. An AC (or PKA) inhibitor together with MEK one completely blocked the translocation which may partially involve exocytic process. These findings suggest that AC/PKA and ERK work downstream of PLC activated by BDNF. Research funds: Health sciences research grant of nano-1 P3-b47 Activation of CREB by ethanol and acetaldehyde in NG108-15 cells Junko Morita, Hiroko Inoue Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan It has been shown that ethanol promotes cAMP signaling, leading to phosphorylation of cAMP response element binding protein (CREB) and induction of CRE-mediated gene transcription. Acetaldehyde has long been suggested to be involved in a number of ethanol’s pharmacological and behavioral effects, but the role of acetaldehyde in ethanol’s effects has not been cleared. In the present study, we investigated the effects of ethanol and acetaldehyde on the activation of CREB. When NG108-15 cells were incubated with ethanol or acetaldehyde, activation of CREB was observed. The effect of ethanol was attenuated in the presence of catalase inhibitor, and was enhanced in the presence of aldehyde dehydrogenase inhibitor. Therefore, acetaldehyde appeared to be partly responsible for ethanol-induced CREB activation in NG108-15 cells. P3-c0 1 Characterization of novel CK1-mediated phosphoryla- tion of SCS-binding Rho GTPases in vitro Fumitaka Kawakami, Kanzo Suzuki, Kenzo Ohtsuki Laboratory of Signal Biology, Graduate School of Kitasato University, Kanagawa, Japan Purpose: Recently, we found that casein kinase 1 (CK1) preferentially phosphorylates sulfatide and cholesterol-sulfate (SCS)-binding proteins (SCS-BPs), such as myelin basic protein and tau protein, containing novel phosphorylation motif [K/R-X-K/R-X-X-S/T] for CK1 in vitro. These findings suggest that CK1 may be responsible for the regulation of various SCS-BPs through their preferential phosphorylation at high level of SCS in brain. Therefore, the present study was carried out to identify novel SCS-BPs involved in the novel CK1-mediated signal transduction in brain. Results and Discussion: It was found that (i) RhoA (a small GTPase) was a SCS-binding protein; and (ii) RhoA was highly phosphorylated by CK1 (δ and γ ) in the presence of SCS in vitro. No effect of GTP(S and GDP on the CK1-mediated phosphorylation of RhoA GTPase was observed in vitro. These results suggest that the CK1-mediated phosphorylation of Rho family GTPases at the high level of SCS may be involved in the preferential modulation of the Rho-mediated signaling neuronal system in brain.
Transcript of Activation of CREB by ethanol and acetaldehyde in NG108-15 cells
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Abstracts / Neuroscience
3-b41 Expression analysis of VGLUT during the postnatalaturation of rat pineal gland
. Yoshida, A. Ina, J. Konno, K. Ogasawara, T. Wu, F. Shutoh, H.ogami, S. Hisano
Graduate School of Comprehensive Human Sciences, University ofsukuba, Tsukuba, Japan
n the rat pineal gland, pinealocytes contain synaptic-like microvesicleshich have vesicular glutamate transporter (VGLUT) 1 and 2. Recently,e reported early postnatal VGLUT1 expression in the gland whose mat-ration completes in the first 3 weeks after birth. Despite inhibition ofelatonin synthesis by exogenous glutamate in adult pinealocytes in
itro, the role of glutamate in the developing gland is still unclear. Toddress this issue, we studied ontogeny of rat pineal VGLUT with in situybridization and immunohistochemistry. VGLUT1-staining was alwayseen only in pinealocytes stained for synaptopysin (a pinealocyte marker)t all ages examined. In contrast, VGLUT2-staining was seen in bothinealocytes and synaptophysin-unstained cells until the second post-atal week and later confined to pinealocytes, with the dominant typef pineal VGLUT postnatally shifting from VGLUT2 to VGLUT1. Our datauggest that pineal maturation processes are regulated under the age-ependent and VGLUT type-dependent exocytotic release of glutamate.
3-b42 Warm temperature-activated Ca2+ response in cul-ured fibroblasts, neuronal cells and keratinocytesoriko Mochizuki-Oda1, Tomoyuki Kusuno1, Haruhito Yamashita1,asuharu Takamori2, Hisao Yamada2, Hironari Yamada1
Synchrotron Light Life Science Center, Ritsumeikan University,usatsu, Japan; 2 Department Anatomy and Cell Science,origuchi, Japan
mong six TRP channels, TRPV3 and TRPV4 are identified as ‘warmth’eceptors, as their thresholds are around 33–35 ◦C. We investigated theffects of warm temperature on the intracellular Ca2+ in cultured fibrob-asts (NIH3T3), neuronal cells (NG108-15) and keratinocytes (M308).he ‘warmth’ stimulus was applied by increasing the temperature of theath solution from 33 to 38 ◦C. M308 cells expressing TRPV3 and TRPV4howed rapid increase of cellular Ca2+ in normal bath solution contain-ng Ca2+. Without the extra-cellular Ca2+, slow and reduced response wasbserved, suggesting contribution of Ca2+ mobilization from intracellulartores. NIH3T3 cells also responded to ‘warmth’ in the normal solution,ut not in Ca2+-free solution. This suggests that they have only influx ofa2+. On the other hand, NG108-15 cells showed both Ca2+ influx andobilization. The variety of the mechanisms of ‘warmth’ sensing will be
iscussed.
esearch funds: KAKENHI (17657052)
3-b43 Effect of oxidized galectin-1 on mRNA expression inacrophagearuhisa Kawasaki1, Yukie Kogawa2, Kenichi Sasaguri2, Sadaoato2, Hiroko Inoue3, Hidenori Horie4
Department of Biology, Keio University, Yokohama, Japan;Department of Craniofacial Growth and Development Dentistry,anagawa Dental College, Kanagawa, Japan; 3 School of Sciencend Engineering, Waseda University, Tokyo, Japan; 4 Brain andral Science Center, Kanagawa Dental College, Kanagawa, Japan
xidized galectin-1 (GAL-1/Ox) stimulates macrophages to promotexonal regeneration in peripheral nerves after nerve injury. But, theechanism of GAL-1/Ox, how to regulate macrophage, remains to be
larified. Here we demonstrated effects of GAL-/Ox on mRNA expressionf four injury related molecules, IL-1�, IL-6, LIF, and iNOS in cultured rateritoneal macrophages under 10 ng/ml LPS treated condition. RT-PCRnalysis revealed that IL-6 as well as iNOS mRNA expressions were sig-
ificantly up-regulated by GAL-1/Ox, but the other mRNA did not showignificant differences by the application of GAL-1/Ox. Increased IL-6 mayromotes axonal regeneration in peripheral nerves, but it is not certainhether increased iNOS contributes to the regeneration.esearch funds: KAKENHI (17500265)
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rch 58S (2007) S1–S244 S195
3-b45 Essential role of activation of PKA and ERK by BDNFor AMPA receptor translocation to postsynaptic sitesiroko Nakata1,2, Shun Nakamura1
National Institute of Neuroscience, Tokyo, Japan; 2 Tokyonstitute of Psychiatry, Tokyo, Japan
e have recently shown that brain-derived neurotrophic factor (BDNF)nduced an AMPA-type glutamatergic receptor (AMPAR) subunit, GluR1ranslocation to the postsynaptic density (PSD) of cultured cortical pyra-
idal neurons. The Ca2+ signaling through IP3R as well as store-operatedation channel TRPC was critical in this translocation. The present studynvestigates the signaling mechanism of GluR1 translocation after thealcium raise. Acute BDNF application to the cultured neurons increasedrotein phosphorylation of protein kinase A (PKA) substrates and extra-ellular signal-regulated kinase (ERK) within 5–10 min at most of theendritic spines. A PLC� inhibitor blocked this effect. Half the PSD-ranslocation of GluR1 was prevented by each inhibitor of adenylylyclase (AC), PKA, or MEK. An AC (or PKA) inhibitor together with MEKne completely blocked the translocation which may partially involvexocytic process. These findings suggest that AC/PKA and ERK workownstream of PLC� activated by BDNF.
esearch funds: Health sciences research grant of nano-1
3-b47 Activation of CREB by ethanol and acetaldehyde inG108-15 cellsunko Morita, Hiroko Inoue
Graduate School of Advanced Science and Engineering, Wasedaniversity, Tokyo, Japan
t has been shown that ethanol promotes cAMP signaling, leading tohosphorylation of cAMP response element binding protein (CREB) and
nduction of CRE-mediated gene transcription. Acetaldehyde has longeen suggested to be involved in a number of ethanol’s pharmacologicalnd behavioral effects, but the role of acetaldehyde in ethanol’s effectsas not been cleared. In the present study, we investigated the effects ofthanol and acetaldehyde on the activation of CREB. When NG108-15 cellsere incubated with ethanol or acetaldehyde, activation of CREB wasbserved. The effect of ethanol was attenuated in the presence of catalase
nhibitor, and was enhanced in the presence of aldehyde dehydrogenasenhibitor. Therefore, acetaldehyde appeared to be partly responsible forthanol-induced CREB activation in NG108-15 cells.
3-c0� 1 Characterization of novel CK1-mediated phosphoryla-ion of SCS-binding Rho GTPases in vitroumitaka Kawakami, Kanzo Suzuki, Kenzo Ohtsuki
Laboratory of Signal Biology, Graduate School of Kitasatoniversity, Kanagawa, Japan
urpose: Recently, we found that casein kinase 1 (CK1) preferentiallyhosphorylates sulfatide and cholesterol-sulfate (SCS)-binding proteinsSCS-BPs), such as myelin basic protein and tau protein, containing novelhosphorylation motif [K/R-X-K/R-X-X-S/T] for CK1 in vitro. These findingsuggest that CK1 may be responsible for the regulation of various SCS-BPshrough their preferential phosphorylation at high level of SCS in brain.herefore, the present study was carried out to identify novel SCS-BPs
nvolved in the novel CK1-mediated signal transduction in brain.esults and Discussion: It was found that (i) RhoA (a small GTPase) was
itro. These results suggest that the CK1-mediated phosphorylation of Rhoamily GTPases at the high level of SCS may be involved in the preferential
odulation of the Rho-mediated signaling neuronal system in brain.
