Amino Acids

, Volume 40, Issue 2, pp 517–526 | Cite as

Oxidative stress increases SNAT1 expression and stimulates cysteine uptake in freshly isolated rat cardiomyocytes

Original Article
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Abstract

Intracellular cysteine availability is an important rate-limiting factor governing glutathione synthesis in the heart. This is also dependent on the magnitude and rate of cysteine uptake into cardiomyocytes, which has been little studied. This study investigated the hypothesis that changes to cysteine transporter expression and activity during oxidative stress influence cardiomyocyte glutathione levels. The uptake of 0–3 mM l-[35S]cysteine into ventricular cardiomyocytes isolated from adult male Wistar rats was measured using oil filtration. Cysteine transporter expression was investigated by conventional and real-time quantitative reverse-transcription polymerase chain reaction and Western blotting. Glutathione levels were measured enzymatically. Oxidative stress was induced via 0–6 h incubation with 0.05 mM H2O2. Cysteine uptake was greatest in sodium-containing media and was inhibited by glutamine, 2-(methylamino)-isobutyric acid (αMeAIB), serine or alanine. The K m and V max of the αMeAIB insensitive and sensitive portions were 0.133 ± 0.01 mM and 468.11 ± 9.04 pmol/μl cell vol/min, and 0.557 ± 0.096 mM and 279.87 ± 16.06 pmol/μl cell vol/min, respectively. Cardiomyocytes expressed ASCT2, SNAT1 and SNAT2 but not ASCT1. Oxidative stress significantly enhanced cysteine uptake, which was attenuated by αMeAIB. This was accompanied by significantly enhanced SNAT1 expression, whilst SNAT2 and ASCT2 were unaffected. Incubation with cysteine significantly reduced the oxidative-stress-induced decline in cardiomyocyte glutathione as compared to cells incubated without cysteine or cells incubated with cysteine and αMeAIB. In conclusion, under control conditions SNAT transporters aid in the delivery of cysteine for cardiomyocyte GSH synthesis, whilst oxidative stress increases cardiomyocyte cysteine uptake and stimulates cardiomyocyte SNAT1 expression.

Keywords

SNAT1 Oxidative stress Isolated cardiomyocytes Glutathione 
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Notes

Acknowledgments

The authors would like to thank Dr. John McGivan for his constructive input into this work. This work was funded by a grant from the British Heart Foundation (PG/05030) and by the NIHR Bristol BRU in Cardiovascular Medicine.

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Bristol Heart InstituteUniversity of Bristol, Bristol Royal InfirmaryBristolUK
  2. 2.School of Science and TechnologyMcClymont Building, University of New EnglandArmidaleAustralia

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