Regulation of Platelet-activating Factor Receptors in Rat Kupffer Cells

Abstract

Ligand binding studies indicate that 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) down-regulates its own receptors on the plasma membrane of isolated rat Kupffer cells but has no significant effect on the binding affinity of the receptor for AGEPC. Exposure of isolated rat Kupffer cells to 10(-8) and 10(-6) M AGEPC resulted in a rapid, time-dependent reduction in the number of cell surface AGEPC receptors to a new steady state concentration (54.1 +/- 5.0% and 38.6 +/- 5.4% of control, respectively). During the observation period (6 h), the half-time of surface AGEPC receptors was about 60 and 45 min in the presence of 10(-8) and 10(-6) M AGEPC, respectively. Both the rate of loss and the maximal loss of the receptors were dependent upon the AGEPC concentration. With receptor synthesis inhibited by cycloheximide in the absence of AGEPC, the half-time of the surface AGEPC receptor was about 4 h, suggesting that AGEPC receptors are not recycled and that the loss of AGEPC receptors from the plasma membrane is accelerated by AGEPC binding. When incubated with Kupffer cells at 37 degrees C for 3 h, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (1.0 microM), an inactive metabolite of AGEPC, did not cause the loss of AGEPC receptors. Under the same conditions, AGEPC antagonists such as BN52021 (2 x 10(-5) M) or U66985 (2 x 10(-5) M) alone had no effect (97.0 +/- 3.9% of control for BN52021) or only a relatively slight effect (78.4 +/- 1.8% for U66985) on the number of surface AGEPC receptors. However, AGEPC antagonists inhibited the AGEPC-induced down-regulation of AGEPC receptors in a concentration-dependent manner, suggesting that the AGEPC-induced down-regulation of AGEPC receptors is a receptor-mediated process. The AGEPC-mediated decrease in receptor number on rat Kupffer cells is reversible. Upon removing AGEPC from the culture medium, about 67% of the lost receptors were replaced within 2 h. Cycloheximide, an inhibitor of protein synthesis, prevented the restoration of the AGEPC receptors. Similar results were obtained when Kupffer cells were incubated with Pronase followed by removing Pronase and reincubating the cells with or without cycloheximide. These observations suggest that the restored AGEPC receptor is newly synthesized rather than recycled. The present study demonstrates that under non-stimulatory (i.e. in the absence of AGEPC) conditions AGEPC receptors are lost from the plasma membrane and are reformed in the cells continuously.(ABSTRACT TRUNCATED AT 400 WORDS)