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Differences between Human and Goose Erythrocytes in Response to Phorbol Esters and Expression of Phorbol Ester Receptors¹

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908

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 µg/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 (K_d = 3.6 ± 2.2 nM) and low (K_d = 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 (K_d 30 nM) and was enhanced 2-fold by incubation at 37°C relative to incubation at 4°C. This was due to an increased B_max, with no change in K_d 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.

¹ Supported by NIH Grant GM31184.

² Initially supported by NIH Training Grant HL07284. Present address: Division of Pharmacology, M-013H, Dept. of Medicine, University of California, San Diego, San Diego, CA 92093. To whom requests for reprints should be addressed.

³ Recipient of American Cancer Society Grant FRA-306.

Received 10/22/86. Revised 6/ 1/87. Accepted 6/23/87.