calcimycin and bepafant

This report confirms that human umbilical vein endothelial cells activated by A23187 produce platelet-activating factor (PAF) (22.4 +/- 9.9 ng/10(6) cells/h; mean +/- S.E.). A proportion of the PAF produced (56%) was released by cells into the medium. The PAF released, however, was not detected without prior organic extraction, and the method of organic extraction was critical for detection. Extraction with 80% ethanol was not successful, but a modified methanol/chloroform extraction method was. These observations may explain some of the conflicting reports in the literature on release of PAF by activated endothelial cells. The requirements for organic extraction may reflect the nature of cell-released PAF's binding by albumin; it was observed that PAF added to identical media could be detected in a bioassay without the requirement for extraction. Such PAF was also readily degraded by PAF-acetylhydrolase added to media, while PAF released from cells was resistant to such degradation, suggesting that it was released in a "protected" configuration. Stimulation of cells was performed in media with albumin as the only extracellular macromolecule. Limited proteolytic digestion of the albumin with trypsin and pepsin showed that PAF released by cells was located exclusively between amino acids 240 and 386 (domain II), while no synthetic PAF added to media was located on this region. These results are identical to those described for the release of PAF by the early embryo. Albumin exposed to embryos had a higher thiol concentration (0.77 +/- 0.04 micromol of thiol/micromol of albumin; mean +/- S.E.) than control media to which an equivalent amount of synthetic PAF was added (0.59 +/- 0.02 micromol of thiol/micromol of albumin) (measured with Ellman's reagent). Furthermore, albumin from conditioned media was more susceptible to reduction by 10 mM dithiothreitol than control albumin, as assessed by its mobility on PAGE. The protected configuration of released PAF was caused by cell-dependent conformational changes to albumin involving cysteine-cysteine disulfide bonds. Partial reduction with dithiothreitol of albumin exposed to cells resulted in released PAF being able to be detected directly in a bioassay without the requirement for prior organic extraction.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Animals; Azepines; Biological Availability; Blastocyst; Calcimycin; Cattle; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Humans; Ionophores; Mice; Pepsin A; Peptide Mapping; Phospholipases A; Platelet Activating Factor; Platelet Aggregation Inhibitors; Protein Binding; Protein Conformation; Serum Albumin; Serum Albumin, Bovine; Sulfhydryl Reagents; Triazoles; Trypsin