Erythrocytes L-arginine y+ transporter inhibition by N-ethylmaleimide in ice-bath.
Erythrocytes L: -arginine uptake is conveyed by y+ and y+L membrane transport systems. Pre-incubation with N-ethylmaleimide for 10 min at 37°C inhibits the y+ system. The aim of this study was to determine the ideal pre-incubation temperature in evaluating y+ and y+L systems. Cells were pre-incubated with or without N-ethylmaleimide for 10 min at 4°C and 37°C. L: -Arginine uptake was quantified by radioisotope and standard erythrocytes membrane flux methodology. Results demonstrate that erythrocytes L: -arginine content is depleted by pre-incubation at 37°C for 10 min, thus changing the V (max) measurement. The inhibitory effect of N-ethylmaleimide pre-incubation was temperature independent and already complete after 1 min of incubation. No significant difference in kinetic parameters was detected between cells pre-incubated at 37°C or 4°C, under zero-trans conditions. In conclusion, we suggest that measurement of erythrocytes L: -arginine uptake by y+ and y+L systems could be carried out without N-ethylmaleimide pre-incubation at 37°C.
Pinheiro da Costa
Bartira Ercília
BE
Programa de Pós-graduação em Medicina e Ciências da Saúde (Nefrologia), Faculdade de Medicina/Instituto de Pesquisas Biomédicas/Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, 2° Andar - Laboratório de Nefrologia, Instituto de Pesquisas Biomédicas/HSL, Porto Alegre, RS 90610-000, Brazil. bart@pucrs.br
de Almeida
Priscilla Barcellos
PB
Conceição
Ioná Rosine
IR
Antonello
Ivan Carlos Ferreira
IC
d'Avila
Domingos O
DO
Poli-de-Figueiredo
Carlos Eduardo
CE
D027182:Amino Acid Transport System y+ / Q000037:antagonists & inhibitors* / Q000378:metabolism; D027241:Amino Acid Transport System y+L / Q000037:antagonists & inhibitors / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D003584:Cytological Techniques / Q000379:methods*; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007053:Ice; D007700:Kinetics; D013696:Temperature
D027182:Amino Acid Transport System y+; D027241:Amino Acid Transport System y+L; D007053:Ice; D001120:Arginine; D005033:Ethylmaleimide