Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes |
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Authors: | Vetter Roland Kott Monika Schulze Wolfgang Rupp Heinz |
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Institution: | (1) Max Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany;(2) Institute of Clinical Pharmacology and Toxicology, Free University of Berlin, Berlin, Germany;(3) Molecular Cardiology Laboratory, Philipps University Marburg, Marburg |
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Abstract: | This study investigates sarcoplasmic reticulum (SR) calcium-(Ca2+) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca2+ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca2+ uptake of +33%. Specific Ca2+ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca2+ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca2+ re-uptake function of the SR under defined culture conditions. |
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Keywords: | neonatal rat cardiomyocyte culture sarcoplasmic reticulum phospholamban calcium ATPase calcium transport thyroid hormone |
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