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Endothelin, a potent vasoactive peptide originally isolated from the vascular endothelial cells, exerts glycogenolytic and vasoconstrictive actions in the perfused rat liver. In this paper we demonstrate high-affinity binding sites for endothelin-1 (ET-1) on rat hepatocytes. Upon incubation at 37 degrees C, association of ET-1 with hepatocytes occurred in a time-dependent manner, was maximal between 3 and 6 h, and subsequently declined; at this temperature ET-1 was rapidly internalized with the internalized ligand exceeding the surface-bound ligand at all time points. The rate of association of 125I-ET-1 with hepatocytes was much slower when the binding assay was performed at 4 degrees C; sequestration of ET-1 in hepatocytes was also substantially reduced at this temperature. ET-1 was extremely potent in stimulating phosphoinositide metabolism in hepatocytes, with significant activation of this signal transduction process occurring at ET-1 concentrations as low as 0.1 pM, with an EC50 of 1 pM. The effect of ET-1 was coupled via a pertussis toxin-sensitive G-protein. Cholera toxin did not affect ET-1-mediated phosphoinositide metabolism and neither toxin influenced the association of 125I-ET-1 with hepatocytes. PAGE of hepatocyte membranes following exposure of the cells to 125I-ET-1 and cross-linking revealed labelling of three major proteins with apparent molecular masses of 32, 49 and 72 kDa. 125I-ET-1 labelling of each of these proteins was inhibited by unlabelled ET-1, whereas unlabelled ET-3 inhibited the labelling of only the 32 and 49 kDa proteins. 125I-ET-3 labelled the 49 kDa protein and this labelling was inhibited by both unlabelled ET-1 and ET-3. Each of these receptors appears to be functional, since both ET-1 and ET-3 stimulated phosphoinositide metabolism in hepatocytes. Down-regulation of ET-1 association and desensitization of ET-1-induced phosphoinositide metabolism occurred upon incubation of hepatocytes with the homologous ligand. Following down-regulation, the ET-1 receptor was restored to the surface of the hepatocyte by prolonged incubation, although the ET-1-stimulated phosphoinositide response remained inhibited even after complete recovery of the ET-1 association capability. These results demonstrate the presence of multiple high-affinity receptors for ET-1 on hepatocytes and the direct action of this peptide on hepatic parenchymal cells via the phosphoinositide signal transduction pathway.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is one of the most potent mediators of vascular permeability. PAF levels change in the rabbit endometrium just prior to implantation, which suggests that PAF may be a key substance transducing preimplantation embryonic signals. To study whether PAF was present in the human endometrium, and if so, to determine the cellular origin and hormonal regulation of endometrial PAF, specimens were obtained from 14 women (aged 23-42 yr) undergoing elective hysterectomy during the luteal phase of the cycle (plasma progesterone levels greater than 2 ng/ml). No specimens were taken from women with malignant uterine pathology. Stromal cells and epithelial glandular cells were separated by collagenase and DNAse digestion, and then cultured to confluence in vitro in medium 199. Radioimmunoassays of prostaglandin F (PGF) and prolactin in the culture media were used to confirm cell type and viability. PGF release into the culture medium from stromal cells was low (control 1.52 +/- 0.20 ng/ml), and unchanged by hormone treatment. In contrast, release of PGF from unstimulated glandular cells was 6.05 +/- 0.52 ng/ml, and was significantly increased (p less than 0.05) by estradiol or progesterone plus estradiol, to 12.17 +/- 1.67, and 8.60 +/- 0.81, respectively. Progesterone alone was without effect. Prolactin was secreted by stromal cell cultures, increasing steadily from 24 to 120 h. The levels in the medium were increased by progesterone. PAF activity was assessed by rabbit platelet aggregation and serotonin-release bioassays after lipid extraction and separation by thin-layer chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

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