Differences between human and goose erythrocytes in response to phorbol esters and expression of phorbol ester receptors.
Phorbol esters inhibited the uptake of a fluorescent glucose analogue in goose but not in human erythrocytes. Specific phorbol-12,13-dibutyrate (PDB) binding sites were identified in both goose and human erythrocytes. In the absence of Ca2+ and phospholipid, PDB binding in whole cell lysates was similar to that in intact cells, but addition of Ca2+ (0.5 mM) and phosphatidyl serine (96 micrograms/ml) caused a 4-fold increase in the binding detected in lysates. Nonlinear least-squares analysis of the PDB binding isotherm revealed that the data for lysates from both goose and human cells were best fit by a two-site model, with goose erythrocytes having approximately 3 times as many sites per class of receptors. Subcellular fractionation of human lysates indicated that the high (Kd = 3.6 +/- 2.2 nM) and low (Kd = 20 +/- 5 nM) affinity sites could be accounted for by the contributions from cytosol and crude membrane, respectively. Separation of the high and low affinity sites was not achieved in goose lysates. PDB binding to intact goose erythrocytes exhibited the lower affinity (Kd approximately 30 nM) and was enhanced approximately 2-fold by incubation at 37 degrees C relative to incubation at 4 degrees C. This was due to an increased Bmax, with no change in Kd of the whole cell binding. Human erythrocytes did not demonstrate this temperature-enhanced binding of PDB to intact cells. These data are consistent with a temperature-induced translocation of PDB receptors from cytosol to membrane in goose erythrocytes. The failure of human erythrocytes to respond to PDB is not due to an absence of PDB receptors but may be related to the diminished number of receptors or to the lack of a temperature-induced increase in whole cell receptor number.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005777:Geese; D005944:Glucosamine / Q000031:analogs & derivatives*; D006801:Humans; D010069:Oxadiazoles / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D011493:Protein Kinase C; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000032:analysis*; D013045:Species Specificity; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010069:Oxadiazoles; D010703:Phorbol Esters; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; C045400:6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoglucose; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D009327:4-Chloro-7-nitrobenzofurazan; D005944:Glucosamine; D013755:Tetradecanoylphorbol Acetate