Ca2+- and GTP[γS]-induced translocation of the glucose transporter, GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method

✍ Scribed by Sabine Lehmann-Klose; Beatrice Beinbrech; J. Casper Rüegg; Gabriele Pfitzer; John Cuppoletti; Manfred Gratzl

• Publisher: Springer
• Year: 1995
• Tongue: English
• Weight: 932 KB
• Volume: 430
• Category: Article
• ISSN: 0031-6768
• DOI: 10.1007/bf00373907

✦ Synopsis

In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca 2+, as suggested by previous studies. To investigate the role of Ca 2+, we permeabilized neonatal rat myocytes with c~-toxin and incubated them for 1 h either at a pCa (i.e. -log10 [Ca2+]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5'-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipita~ion method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58-80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 gM Ca 2+ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca 2+ independent, i.e. it was identical in the absence and presence of Ca 2+ (10 gM). Guanosine 5'-O-(3-thiotriphosphate) (GTP[TS]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5fold by 200 gM GTP[TS] in the virtual absence of Ca 2 § (pCa 8) and even 3.5-fold at 10 gM free Ca 2 § We conclude that an increase in intracellular Ca 2+ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do