Insulin and IGF-I binding in isolated trout cardiomyocytes.
Insulin and IGF-I receptor binding were characterized in cardiac muscle cells isolated from the brown trout, Salmo trutta fario. Cardiomyocyte suspensions obtained by perfusion of ventricles with collagenase showed a high degree of viability as judged by trypan blue exclusion, LDH leakage, and morphology. Specific insulin binding was 2.88 +/- 0.28%/10 mg cells after overnight incubation at 4 degrees. Scatchard analysis indicated the presence of high affinity insulin binding sites with an apparent dissociation constant (Kd) of 0.285 +/- 0.043 nM and a binding site density of 1. 61 +/- 0.19 x 10(8)/mg cells. Specificity of insulin binding was determined by displacing labeled insulin with increasing concentrations of IGF-I, and the Kd value obtained was 4.77 +/- 2.82 nM, 17-fold higher than Kd values for displacement of insulin tracer by nonlabeled insulin. The percentage of IGF-I specific binding (6.70 +/- 1.42%/10 mg cells), affinity (Kd = 0.163 +/- 0.023 nM), and binding site density (4.00 +/- 1.13 x 10(8)/mg cells) were higher than those of insulin. Displacement curves of labeled IGF-I with nonlabeled insulin (Kd = 33.6 +/- 9.9 nM), indicated a high specificity of the IGF-I binding site. High concentrations of cold insulin and IGF-I were able to decrease markedly the specific binding to their own receptor. Incubation with cold IGF-I also induced a diminution in insulin binding in agreement with the lower specificity of the insulin receptor. These data suggest that insulin and IGF-I are able to down-regulate their own receptor number in cardiac muscle cells. The present results demonstrate that the isolated cardiac myocyte preparation from brown trout is a useful model for studying insulin and IGF-I binding in fish heart tissue.
Moon
T W
TW
Department of Biology, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada.
Castejón
C
C
Baños
N
N
Maestro
M A
MA
Plisetskaya
E M
EM
Gutiérrez
J
J
Navarro
I
I
D000818:Animals; D001667:Binding, Competitive; D002404:Catheterization; D002470:Cell Survival; D002478:Cells, Cultured; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000032:analysis / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008857:Microscopy, Interference; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D011869:Radioligand Assay / Q000379:methods; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013552:Swine; D014337:Trout
D007328:Insulin; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin