Localization and characterization of binding sites for endothelin in Harder's gland in rabbits

The characterization and localization of binding sites for endothelin-1 (ET-1) labeled with iodine 125I were investigated in homogenized tissues and sections of Harder's glands of normal rabbits. The membrane of Harder's glands was harvested and incubated with 125I-ET-1 (0.25-1 nmol/L) in 20 +/- 4 mg of protein per 0.25 mL at 37 degrees C for 90 min in the presence of protease inhibitors. Specific labeling was assessed by coincubating unlabeled ET-1, ET-2, ET-3 and other unrelated cytokines. The tissue labeled with 125I-ET-1 was collected by filtration and counted in a gamma counter. For an in vitro autoradiography study, 15 microns cryostat sections were incubated with 125I-ET-1 (0.1 nmol/L). They were fixed, dipped in liquid emulsion and kept for 6 days before development. Membrane counting showed that the binding of 125I-ET-1 to Harder's gland was saturable. Scatchard data analysis revealed one class of binding with a dissociation constant (Kd) of 0.33 nmol/L and a maximal density of binding (Bmax) of 794 attomole/mg of protein. The binding was inhibited most by ET-1, followed by ET-2 and then ET-3 but not by unrelated peptides. Emulsion-dipped slides with sections showed specific high-density labeling mainly over structures identified from serial sections stained by hematoxylin-eosin as the walls of capillaries, arterioles, arteries, and veins of the glands. Less dense binding was found in both white and pink lobes of the gland. No binding was found in fat and connective tissues. The distribution of endothelin action sites in the glandular blood vessels and Harder's gland suggests that the peptide may have a role in the regulation of blood circulation and glandular secretion in the normal rabbit.