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Conference Paper: Interaction of caveolin-1, cholesterol and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells
Title | Interaction of caveolin-1, cholesterol and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells |
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Authors | |
Issue Date | 2009 |
Publisher | Sage Publications Ltd. |
Citation | The 24th International Symposium on Cerebral Blood Flow and Metabolism & the 9th International Conference on Quantification of Brain Function with PET, Chicago, IL., 29 June-3 July 2009. In Journal of Cerebral Blood Flow and Metabolism, 2009, v. 29 n. 1 suppl., p. S459, abstract no. 709 How to Cite? |
Abstract | Objectives: 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 Identifier | http://hdl.handle.net/10722/63740 |
ISSN | 2023 Impact Factor: 4.9 2023 SCImago Journal Rankings: 1.937 |
DC Field | Value | Language |
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dc.contributor.author | Shen, J | en_HK |
dc.date.accessioned | 2010-07-13T04:31:00Z | - |
dc.date.available | 2010-07-13T04:31:00Z | - |
dc.date.issued | 2009 | en_HK |
dc.identifier.citation | The 24th International Symposium on Cerebral Blood Flow and Metabolism & the 9th International Conference on Quantification of Brain Function with PET, Chicago, IL., 29 June-3 July 2009. In Journal of Cerebral Blood Flow and Metabolism, 2009, v. 29 n. 1 suppl., p. S459, abstract no. 709 | en_HK |
dc.identifier.issn | 0271-678X | - |
dc.identifier.uri | http://hdl.handle.net/10722/63740 | - |
dc.description.abstract | Objectives: 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.language | eng | en_HK |
dc.publisher | Sage Publications Ltd. | - |
dc.relation.ispartof | Journal of Cerebral Blood Flow & Metabolism | - |
dc.title | Interaction of caveolin-1, cholesterol and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells | en_HK |
dc.type | Conference_Paper | en_HK |
dc.identifier.email | Shen, J: shenjg@hkucc.hku.hk | en_HK |
dc.identifier.authority | Shen, J=rp00487 | en_HK |
dc.identifier.doi | 10.1038/jcbfm.2009.157 | - |
dc.identifier.hkuros | 158835 | en_HK |
dc.identifier.volume | 29 | - |
dc.identifier.issue | 1 suppl. | - |
dc.identifier.spage | S459, abstract no. 709 | - |
dc.identifier.epage | S459, abstract no. 709 | - |
dc.identifier.issnl | 0271-678X | - |