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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2009.tde-01072009-112943
Document
Author
Full name
Olivia Beloto da Silva
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Souza, Maria Oliveira de (President)
Gomes, Guiomar Nascimento
Machado, Ubiratan Fabres
Title in Portuguese
Efeito da glicose sobre recuperação do pHi em células HEK-293.
Keywords in Portuguese
Células epiteliais
Co-transportador Na+/glicose
Glicose
pH intracelular
Transporte através da membrana
Trocador Na+/H+
Abstract in Portuguese
Os estudos foram realizados em cultura de células HEK-293 (human embrionic kidney cells). Por microscopia de fluorescência, avaliou-se a velocidade de recuperação do pHi (dpHi/dt). Por Western blot, avaliou-se a expressão de SGLTs e NHEs e a translocação dos SGLTs foi avaliada por imunofluorescência. Resultados: No controle, a dpHi/dt foi de 0,169 ± 0,020 unid pH/min (n=6). A glicose modula dose e tempo dependentemente a dpHi/dt. O tratamento crônico aumentou esse parâmetro e somente Florizina (inibidor dos SGLTs), H-89 (inibidor da PKA) e BAPTA (quelante de Ca2+intracelular Ca2+i) reduziram esse efeito. O tratamento crônico induziu a internalização do SGLT1, manteve o SGLT2 no citosol e aumentou sua expressão. Conclusões: No tratamento crônico, a internalização do SGLT1 depende da PKA, independe de Ca2+i e a permanência do SGLT2 no citosol depende tanto da PKA quanto do Ca2+i. Assim, a distribuição celular do SGLT2 altera a atividade dos NHEs.
Title in English
Effect of glucose on pHi recovery in HEK-293 cells.
Keywords in English
Epithelial cells
Glucose
Intracellular pH
Membrane transport
Na+/Glucose cotransporter
Na+/H+ exchanger
Abstract in English
In this work we used human embryonic kidney (HEK-293 cells). The pHi recovery rate (dpHi/dt) was evaluated through fluorescence microscopy. The expression of SGLT´s and NHEs was analysed through Western blot and translocation of SGLTs was evaluated through Imunofluorescence. Results: In the control situation, the dpHi/dt was 0,169 ± 0,020 units pH/min (n=6). This parameter was modulated by glucose in a concentration and time dependent manner. Chronic treatment increased the dpHi/dt and this stimulatory effect was inhibited by Phlorizin (SGLTs inhibitor), H-89 (PKA inhibitor) and BAPTA (intracellular Ca2+ cheleator - Ca2+i). The chronic treatment induced internalization of SGLT1, increased the expression of SGLT2 and kept it in the cytosol. Conclusions: In chronic treatment, the internalization of SGLT1 involves a PKA-dependent and Ca2+i- independent mechanism. The maintenance of SGLT2 in the cytosol depends on PKA and Ca2+i. Thus, the cellular distribution of SGLT2 is associated with NHEs activity.
 
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Publishing Date
2009-09-01
 
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