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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2018.tde-26022018-145459
Document
Author
Full name
Érika Yumi Taniguchi
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2017
Supervisor
Committee
Cassola, Antonio Carlos (President)
Moreira, Thiago dos Santos
Oliveira, Eduardo Rebelato Lopes de
Onuchic, Luiz Fernando
Title in Portuguese
pH intracelular nos neurônios dos gânglios das raízes dorsais.
Keywords in Portuguese
Microscopia de fluorescência
Neurônio sensorial
pH intracelular
Rato wistar
Trocador Na+/H+
Abstract in Portuguese
O objetivo do trabalho foi determinar o pHi, a capacidade tamponante do citosol na ausência de CO2/HCO3-i) de neurônios dos gânglios das raízes dorsais e investigar a expressão de trocadores Na+/H+ e sua função na regulação do pHi. O pHi foi estimado com o indicador fluorescente BCECF. A operação do trocador era quantificada na recuperação da acidose intracelular induzida experimentalmente. Na ausência do tampão CO2/HCO3- a taxa de alcalinização (k) deve-se, por hipótese, ao transporte de H+ pelo trocador. A hipótese foi confirmada pela ação de agentes farmacológicos, e.g., amiloride. Em soluções tamponadas por CO2/HCO3- as células tem pHi de 7,24 e, em soluções tamponadas com HEPES, 7,04. A βi foi de 8,17 mM/pH. As células se recuperam da acidose com k médio de 0,0138 s-1. O efeito inibitório do amiloride em concentração de 1 mM deve-se ao fato dos fenótipos celulares expressarem diferentes isoformas do trocador. Segundo RT-PCR, todas as 5 isoformas do trocador são expressas e a quantidade de RNAm, avaliada por qPCR, é maior para a NHE1, seguida de NHE5.
Title in English
Intracellular pH in neurons of dorsal root ganglia.
Keywords in English
Fluorescence microscopy
Intracellular pH
Na+/H+ exchanger
Sensory neuron
Wistar rat
Abstract in English
The objective here was to determine intracellular pH (pHi), cytosolic buffering power in CO2/HCO3- free medium (βi) of neurons from dorsal root ganglia and to investigate the functional expression of the Na+/H+ exchangers in the regulation of pHi. pHi was estimated with fluorescence indicator BCECF. Exchanger operation was quantified during recovery from intracellular acidification induced experimentally. In CO2/HCO3- free medium the alkalinization rate (k) is due, hypothetically, H+ extrusion by the exchanger. This assumption was confirmed by action of pharmacologic agents, e.g., amiloride. In medium buffered with CO2/HCO3- cells have pHi of 7.24 and, in medium buffered with HEPES, 7.04. βi calculated was 8.17 mM/pH. Cells recovery from acidosis with mean k of 0.0138 s-1. Inhibitory effect of amiloride in 1 mM concentration is due to cellular phenotypes expressing different Na+/H+ exchanger isoforms. According to RT-PCR, all the five exchanger isoforms are expressed and mRNA quantity, evaluated by qPCR, is greater to NHE1, followed by NHE5.
 
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Release Date
2020-02-26
Publishing Date
2018-02-26
 
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