Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR
Autor(a) principal: | |
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Data de Publicação: | 2005 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10316/8127 https://doi.org/10.1002/jnr.20308 |
Resumo: | We report a novel (13C, 2H) nuclear magnetic resonance (NMR) procedure to investigate lactate recycling through the monocarboxylate transporter of the plasma membrane of cells in culture. C6 glioma cells were incubated with [3-13C]lactate in Krebs-Henseleit Buffer containing 50% 2H2O (vol/vol) for up to 30 hr. 13C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at sim20.85 parts per million (ppm; -0.119 mumol/mg protein/hr) derived from the methyl carbon of [3-13C]lactate; and (2) an exponentially increasing shifted singlet at sim20.74 ppm (0.227 mumol/ mg protein/hr) from the methyl carbon of [3-13C, 2-2H]lactate. The shifted singlet appears because during its transit through the cytosol, [3-13C]lactate generates [3-13C, 2-2H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3-13C, 2-2H]lactate is shifted -0.11 ppm with respect to that of [3-13C]lactate, making it possible to distinguish between both molecules by 13C NMR. During incubations with 2.5 mM [1-13C]glucose and 3.98 mM [U-13C3]lactate or with 2.5 mM [1-13C]glucose and 3.93 mM [2-13C]pyruvate, C2-deuterated lactate was produced only from [1-13C]glucose or [U-13C3]lactate, revealing that this deuteration process is redox sensitive. When [1-13C]glucose and [U-13C3]lactate were used as substrates, no significant [3-13C]lactate production from [1-13C]glucose was detected, suggesting that glycolytic lactate production may be stopped under the high lactate concentrations prevailing under mild hypoxic or ischemic episodes or during cerebral activation. © 2004 Wiley-Liss, Inc. |
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Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMRWe report a novel (13C, 2H) nuclear magnetic resonance (NMR) procedure to investigate lactate recycling through the monocarboxylate transporter of the plasma membrane of cells in culture. C6 glioma cells were incubated with [3-13C]lactate in Krebs-Henseleit Buffer containing 50% 2H2O (vol/vol) for up to 30 hr. 13C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at sim20.85 parts per million (ppm; -0.119 mumol/mg protein/hr) derived from the methyl carbon of [3-13C]lactate; and (2) an exponentially increasing shifted singlet at sim20.74 ppm (0.227 mumol/ mg protein/hr) from the methyl carbon of [3-13C, 2-2H]lactate. The shifted singlet appears because during its transit through the cytosol, [3-13C]lactate generates [3-13C, 2-2H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3-13C, 2-2H]lactate is shifted -0.11 ppm with respect to that of [3-13C]lactate, making it possible to distinguish between both molecules by 13C NMR. During incubations with 2.5 mM [1-13C]glucose and 3.98 mM [U-13C3]lactate or with 2.5 mM [1-13C]glucose and 3.93 mM [2-13C]pyruvate, C2-deuterated lactate was produced only from [1-13C]glucose or [U-13C3]lactate, revealing that this deuteration process is redox sensitive. When [1-13C]glucose and [U-13C3]lactate were used as substrates, no significant [3-13C]lactate production from [1-13C]glucose was detected, suggesting that glycolytic lactate production may be stopped under the high lactate concentrations prevailing under mild hypoxic or ischemic episodes or during cerebral activation. © 2004 Wiley-Liss, Inc.2005info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10316/8127http://hdl.handle.net/10316/8127https://doi.org/10.1002/jnr.20308engJournal of Neuroscience Research. 79:1-2 (2005) 119-127Rodrigues, Tiago B.Gray, Heather L.Benito, MarinaGarrido, SusanaSierra, AlejandraGeraldes, Carlos F.Ballesteros, PalomaCerdán, Sebastiáninfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2022-05-25T03:18:47Zoai:estudogeral.uc.pt:10316/8127Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T20:55:44.413815Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR |
title |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR |
spellingShingle |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR Rodrigues, Tiago B. |
title_short |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR |
title_full |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR |
title_fullStr |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR |
title_full_unstemmed |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR |
title_sort |
Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR |
author |
Rodrigues, Tiago B. |
author_facet |
Rodrigues, Tiago B. Gray, Heather L. Benito, Marina Garrido, Susana Sierra, Alejandra Geraldes, Carlos F. Ballesteros, Paloma Cerdán, Sebastián |
author_role |
author |
author2 |
Gray, Heather L. Benito, Marina Garrido, Susana Sierra, Alejandra Geraldes, Carlos F. Ballesteros, Paloma Cerdán, Sebastián |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Rodrigues, Tiago B. Gray, Heather L. Benito, Marina Garrido, Susana Sierra, Alejandra Geraldes, Carlos F. Ballesteros, Paloma Cerdán, Sebastián |
description |
We report a novel (13C, 2H) nuclear magnetic resonance (NMR) procedure to investigate lactate recycling through the monocarboxylate transporter of the plasma membrane of cells in culture. C6 glioma cells were incubated with [3-13C]lactate in Krebs-Henseleit Buffer containing 50% 2H2O (vol/vol) for up to 30 hr. 13C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at sim20.85 parts per million (ppm; -0.119 mumol/mg protein/hr) derived from the methyl carbon of [3-13C]lactate; and (2) an exponentially increasing shifted singlet at sim20.74 ppm (0.227 mumol/ mg protein/hr) from the methyl carbon of [3-13C, 2-2H]lactate. The shifted singlet appears because during its transit through the cytosol, [3-13C]lactate generates [3-13C, 2-2H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3-13C, 2-2H]lactate is shifted -0.11 ppm with respect to that of [3-13C]lactate, making it possible to distinguish between both molecules by 13C NMR. During incubations with 2.5 mM [1-13C]glucose and 3.98 mM [U-13C3]lactate or with 2.5 mM [1-13C]glucose and 3.93 mM [2-13C]pyruvate, C2-deuterated lactate was produced only from [1-13C]glucose or [U-13C3]lactate, revealing that this deuteration process is redox sensitive. When [1-13C]glucose and [U-13C3]lactate were used as substrates, no significant [3-13C]lactate production from [1-13C]glucose was detected, suggesting that glycolytic lactate production may be stopped under the high lactate concentrations prevailing under mild hypoxic or ischemic episodes or during cerebral activation. © 2004 Wiley-Liss, Inc. |
publishDate |
2005 |
dc.date.none.fl_str_mv |
2005 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10316/8127 http://hdl.handle.net/10316/8127 https://doi.org/10.1002/jnr.20308 |
url |
http://hdl.handle.net/10316/8127 https://doi.org/10.1002/jnr.20308 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Journal of Neuroscience Research. 79:1-2 (2005) 119-127 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799133843766116352 |