RESUMO
This study's rationale was that the expression and activity of aspartate transporters in hypertrophied hearts might be different from normal hearts, which could affect the use of aspartate in myocardial protection of hypertrophied hearts. mRNA expression of system X(ag)(-) transporters in hearts from normal (Wistar Kyoto) and hypertrophied (spontaneously hypertensive rat) rats was investigated by RT-PCR. EAAT3 protein expression in isolated cells and vesicles from normal and hypertrophied hearts was investigated by Western blotting. The same vesicles were also used to measure aspartate uptake. The effects of 0.5 mmol l(-1) aspartate supplementation on cardiac performance during ischaemia-reperfusion were investigated in isolated and perfused hearts. Both normal and hypertrophied hearts expressed EAAT1 and EAAT3 mRNA. EAAT3 protein expression was significantly greater in cells and vesicles from hypertrophied hearts compared to normal hearts. The velocity (V(max)) of aspartate uptake was faster at 24.4 +/- 2.2 pmol mg(-1) s(-1) in vesicles from hypertrophied hearts compared to 8.2 +/- 0.8 pmol mg(-1) s(-1) (P < 0.001, t test, n= 6, means +/-s.e.m.) in normal heart vesicles. The affinity (K(m)) was similar for both preparations. When recoveries were matched, 0.5 mmol l(-1) aspartate addition reduced reperfusion injury and increased functional recovery of hypertrophied hearts but not normal hearts. This was associated with a greater preservation of ATP, glutamate and glutamine and less lactate production during ischaemia in aspartate-treated hypertrophied hearts compared to all other experimental groups. These results suggest that increased aspartate transporter expression and activity in hypertrophy helps facilitate aspartate entry into hypertrophied cardiomyocytes, which in turn leads to improved myocardial protection.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Cardiomegalia/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Simportadores/metabolismo , Trifosfato de Adenosina/metabolismo , Alanina/sangue , Alanina/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacocinética , Western Blotting , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Transportador 1 de Aminoácido Excitatório , Transportador 3 de Aminoácido Excitatório , Glutamato Desidrogenase/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática , Ácido Glutâmico/sangue , Ácido Glutâmico/metabolismo , Glutamina/sangue , Glutamina/metabolismo , Coração/efeitos dos fármacos , Coração/fisiopatologia , Frequência Cardíaca/fisiologia , Isquemia/fisiopatologia , Cinética , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Masculino , Miocárdio/química , Miocárdio/metabolismo , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Reperfusão , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcolema/química , Sarcolema/metabolismo , Simportadores/genética , Pressão Ventricular/fisiologiaRESUMO
Glutamine is taken up into the rat hepatoma cell line H4-IIE-C3 by a Na+-dependent transport system which is specific for glutamine, alanine, serine, cysteine and asparagine and does not tolerate substitution of Na+ by Li+. Glutamine transport was relatively weakly inhibited by a 50-fold excess of leucine and was not inhibited by phenylalanine or N -methyl aminoisobutyrate. These general properties are characteristic of the recently identified ASCT/B0 family of transporters. Using a reverse transcriptase PCR-based homology cloning approach, we have characterized a cDNA for a novel member of this transporter family (H4-ASCT2) from H4-IIE-C3 cells. The cDNA encodes a 551-amino acid protein which exhibits similarities of between 75 and 85% with ASCT/B0 transporters previously cloned from other sources. When expressed in Xenopus oocytes, this transporter catalyses Na+-dependent glutamine uptake with characteristics very similar to those of glutamine uptake into the H4-IIE-C3 cells. This newly characterized transporter possesses a number of amino acid sequence differences from ASCT2 clones recently isolated from rat astroglial cells and from normal rat liver. In particular, the loop region between transmembrane helices 3 and 4 from H4-ASCT2 shares less than 60% sequence similarity with ASCT2 from rat liver; furthermore, there are some 25 single amino acid substitutions elsewhere in the H4-ASCT2 sequence compared with that from rat liver. Thus enhanced glutamine uptake in rat hepatoma cells is mediated by the expression of a novel ASCT/B0 transporter isoform rather than by increased expression of the ASCT2 mRNA found in normal rat liver.