Ca(2+)- and GTP[gamma S]-induced translocation of the glucose transporter, GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method.
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 Ca2+, as suggested by previous studies. To investigate the role of Ca2+, we permeabilized neonatal rat myocytes with alpha-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 immunoprecipitation 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 microM Ca2+ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca2+ independent, i.e. it was identical in the absence and presence of Ca2+ (10 microM). Guanosine 5'-O-(3-thio-triphosphate) (GTP[gamma S]), 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 2.5-fold by 200 microM GTP[gamma S] in the virtual absence of Ca2+ (pCa 8) and even 3.5-fold at 10 microM free Ca2+. We conclude that an increase in intracellular Ca2+ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[gamma S] in the absence of Ca2+, but that the effects of Ca2+ and GTP[gamma S] may be additive.
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D051275:Glucose Transporter Type 4; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects
D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002118:Calcium