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Conference Paper: Interaction of caveolin-1, cholesterol and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

TitleInteraction of caveolin-1, cholesterol and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells
Authors
Issue Date2009
PublisherSage Publications Ltd.
Citation
The XXIVth International Symposium on Cerebral Blood Flow and Metabolism & The IXth International Conference on Quantification of Brain Function with PET, Chicago, IL, 29 June - 3 July 2009. In Journal of Cerebral Blood Flow & Metabolism, 2009, v. 1 n. suppl, p. S459 Abstract no. 709 How to Cite?
AbstractObjectives: Interaction between nitric oxide (NO) and caveolins appears to be a potential cellular signal pathway in ischemic brain injury. To elucidate the interaction of NO and caveolins in hypoxic neural cells, we investigated the expressions of caveolin-1 and caveolin-2 and NO production in human SK-N-MC neuroblastoma cells exposed to different periods of hypoxia. Methods: Human neuroblastoma SK-N-MC cells were grown as a monolayer in minimum essential medium (MEM, Sigma) supplemented with fetal bovine serum (10%, v/v), glutamine (10 mg/ml) and antibiotics (penicillin and streptomycin, 10 mg/ml). For hypoxic treatment, confluent monolayers in multiple-well plates were placed in a modular incubator chamber. The cells were consistently incubated with 2% O2 plus 5% CO2 balanced with N2 gas for 8, 15, 24 and 36 hrs at 37°C respectively. To understand the interactions of caveolin-1, cholesterol and nitric oxide synthases in the hypoxic SK-N-MC cells, the cells were treated with different reagents to change the contents of NO and cholesterol. Results: The expression of caveolin-1 mRNA and protein was transiently upregulated by 15 hrs of hypoxia but downregulated by 24 hrs or longer exposure to hypoxia, whereas the expression of caveolin-2 mRNA down-regulated by hypoxia. In normoxic SK-N-MC cells, both S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) and 3-morpholinosydnonimine (SIN-1, peroxynitrite donor) up-regulated the expression of caveolin-1. Moreover, in the SK-N-MC cells exposed to 15 hrs of hypoxia, pretreatments of NG-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), 1400W (a inducible NOS inhibitor) or FeTMPyP (a peroxynitrite decomposition catalyst) attenuated the increase of caveolin-1 expression, but there were no obvious changes in the expression of caveolin-1 by incubation of 7-nitroindazole (7-NI, a selective neural NOS inhibitor) and l-N5-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor). Meanwhile, hypoxia treatment induced intracellular cholesterol accumulation. Pretreatments of methyl-β-cyclodextrin (MCD) and methyl-β-cyclodextrin-cholesterol (MCD-CHOL) and oleic acid inhibited and enhanced the expression of caveolin-1 respectively. Those results suggest that the upregulation of caveolin-1 could be associated with iNOS-induced reactive nitrogen species production and lipid accumulation in the hypoxic neural cells. Furthermore, we compared the expression of eNOS, iNOS, and nNOS and NO production in wild-type and caveolin-1 over-expressed SK-N-MC cells after 15 hrs of hypoxia treatment. Exposure to 15 hrs of hypoxia inhibited eNOS expression but induced iNOS expression and NO production in the wild-type cells. However, over-expression of caveolin-1 prevented the loss of eNOS but downregulated the expression of iNOS and inhibited NO production. Conclusion: The results suggest that augmentation of caveolin-1 in response to hypoxia stimulation could be a physiological regulating mechanism to inhibit iNOS-induced NO production. Overall, the complex interactions of reactive nitrogen species and caveolin-1 could be an important signal pathway in the modulation of NO production in hypoxic neural cells. Acknowledgement: This work was supported by Hong Kong RGC GRF grant No. 7495/04 M and No. 7748/08 M.
Persistent Identifierhttp://hdl.handle.net/10722/63740
ISSN
2015 Impact Factor: 4.929
2015 SCImago Journal Rankings: 3.004

 

DC FieldValueLanguage
dc.contributor.authorShen, Jen_HK
dc.date.accessioned2010-07-13T04:31:00Z-
dc.date.available2010-07-13T04:31:00Z-
dc.date.issued2009en_HK
dc.identifier.citationThe XXIVth International Symposium on Cerebral Blood Flow and Metabolism & The IXth International Conference on Quantification of Brain Function with PET, Chicago, IL, 29 June - 3 July 2009. In Journal of Cerebral Blood Flow & Metabolism, 2009, v. 1 n. suppl, p. S459 Abstract no. 709en_HK
dc.identifier.issn0271-678X-
dc.identifier.urihttp://hdl.handle.net/10722/63740-
dc.description.abstractObjectives: Interaction between nitric oxide (NO) and caveolins appears to be a potential cellular signal pathway in ischemic brain injury. To elucidate the interaction of NO and caveolins in hypoxic neural cells, we investigated the expressions of caveolin-1 and caveolin-2 and NO production in human SK-N-MC neuroblastoma cells exposed to different periods of hypoxia. Methods: Human neuroblastoma SK-N-MC cells were grown as a monolayer in minimum essential medium (MEM, Sigma) supplemented with fetal bovine serum (10%, v/v), glutamine (10 mg/ml) and antibiotics (penicillin and streptomycin, 10 mg/ml). For hypoxic treatment, confluent monolayers in multiple-well plates were placed in a modular incubator chamber. The cells were consistently incubated with 2% O2 plus 5% CO2 balanced with N2 gas for 8, 15, 24 and 36 hrs at 37°C respectively. To understand the interactions of caveolin-1, cholesterol and nitric oxide synthases in the hypoxic SK-N-MC cells, the cells were treated with different reagents to change the contents of NO and cholesterol. Results: The expression of caveolin-1 mRNA and protein was transiently upregulated by 15 hrs of hypoxia but downregulated by 24 hrs or longer exposure to hypoxia, whereas the expression of caveolin-2 mRNA down-regulated by hypoxia. In normoxic SK-N-MC cells, both S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) and 3-morpholinosydnonimine (SIN-1, peroxynitrite donor) up-regulated the expression of caveolin-1. Moreover, in the SK-N-MC cells exposed to 15 hrs of hypoxia, pretreatments of NG-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), 1400W (a inducible NOS inhibitor) or FeTMPyP (a peroxynitrite decomposition catalyst) attenuated the increase of caveolin-1 expression, but there were no obvious changes in the expression of caveolin-1 by incubation of 7-nitroindazole (7-NI, a selective neural NOS inhibitor) and l-N5-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor). Meanwhile, hypoxia treatment induced intracellular cholesterol accumulation. Pretreatments of methyl-β-cyclodextrin (MCD) and methyl-β-cyclodextrin-cholesterol (MCD-CHOL) and oleic acid inhibited and enhanced the expression of caveolin-1 respectively. Those results suggest that the upregulation of caveolin-1 could be associated with iNOS-induced reactive nitrogen species production and lipid accumulation in the hypoxic neural cells. Furthermore, we compared the expression of eNOS, iNOS, and nNOS and NO production in wild-type and caveolin-1 over-expressed SK-N-MC cells after 15 hrs of hypoxia treatment. Exposure to 15 hrs of hypoxia inhibited eNOS expression but induced iNOS expression and NO production in the wild-type cells. However, over-expression of caveolin-1 prevented the loss of eNOS but downregulated the expression of iNOS and inhibited NO production. Conclusion: The results suggest that augmentation of caveolin-1 in response to hypoxia stimulation could be a physiological regulating mechanism to inhibit iNOS-induced NO production. Overall, the complex interactions of reactive nitrogen species and caveolin-1 could be an important signal pathway in the modulation of NO production in hypoxic neural cells. Acknowledgement: This work was supported by Hong Kong RGC GRF grant No. 7495/04 M and No. 7748/08 M.-
dc.languageengen_HK
dc.publisherSage Publications Ltd.-
dc.relation.ispartofJournal of Cerebral Blood Flow & Metabolism-
dc.titleInteraction of caveolin-1, cholesterol and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cellsen_HK
dc.typeConference_Paperen_HK
dc.identifier.emailShen, J: shenjg@hkucc.hku.hken_HK
dc.identifier.authorityShen, J=rp00487en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1038/jcbfm.2009.157-
dc.identifier.hkuros158835en_HK

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