Visualization of binding sites for bovine parathyroid hormone (PTH 1-84) on cultured kidney cells with a biotinyl-b-PTH (1-84) antagonist.
Parathyroid hormone (PTH) receptors have been found in a subpopulation of kidney cells. In this report, we investigated the feasibility of techniques that apply a partial antagonist of PTH conjugated to biotin to localize receptors cytochemically on bovine kidney cortical cells in monolayer culture at the light microscopic level. Biotinylated bovine PTH (1-84) (biotinyl-PTH) was bound to the cultured cells for 1-30 min at 37 degrees C in the amounts of 10(-5) -10(-10) M. In a different set of experiments, the cells were also exposed to a solution containing 10(-6) M biotinylated PTH and an excess of unlabeled PTH, insulin, adrenocorticotropin, or calcitonin for 10 and 30 min at 37 degrees C to test the specificity of the binding. The cells were then fixed in 2.5% glutaraldehyde and stained with the avidin-biotin peroxidase complex (ABC) technique. Diffuse labeling was evident on 30% of the cells in 10 min with concentrations of biotinyl-PTH as low as 10(-8) M. The stain was diffuse, but more intense after 1-10 min in higher concentrations (10(-6) M). If a 15-1500-fold excess of unlabeled PTH was added to the biotinyl-PTH, no staining was observed. The other peptides (insulin, ACTH or calcitonin) had no effect on binding. Longer times in biotinyl-PTH (10(-6) M for 10-30 min) resulted in intense patches of label on the cells resembling caps (in addition to the pale diffuse label). The percentage of labeled cells in the monolayer (30%) did not change with time. These studies show that a partial antagonist of PTH can be used as a cytochemical probe for specific PTH receptors in a subpopulation of cultured cortical kidney cells.
D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D006651:Histocytochemistry / Q000379:methods; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism; D008857:Microscopy, Interference; D008858:Microscopy, Phase-Contrast; D010281:Parathyroid Hormone / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D013997:Time Factors
D010281:Parathyroid Hormone; C047977:biotinyl-parathyroid hormone; D001710:Biotin