Cell Specificity of Vasopressin Binding in Renal Collecting Duct: Computer-Enhanced Imaging of a Fluorescent Hormone Analog

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 16; pp. 6000 - 6004
• Main Authors: Kirk, Kevin L., Buku, Angeliki, Eggena, Patrick
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.08.1987; National Acad Sciences
• Subjects: Animals; Biological and medical sciences; Cell lines; Cell membranes; Cell physiology; Cells; Digitized images; Epithelial cells; Female; Fluorescence; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Hormonal regulation; Image Processing, Computer-Assisted; Kidney Tubules - metabolism; Kidney Tubules, Collecting - metabolism; Lectins; Lypressin - analogs & derivatives; Lypressin - metabolism; Molecular and cellular biology; Peanuts; Pixels; Rabbits; Rhodamines - metabolism; Time Factors; Vasopressins - metabolism; Xanthenes - metabolism; Renal tubule; Fixation; Antidiuretic hormone; Investigation method; Image processing; Digital processing; Digital image; Localization; Mechanism of action; Cell; Fluorescence microscopy; Vasopressin
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.16.6000

A noninvasive microscopic method was used to assess the cell specificity of vasopressin binding within the heterogeneous collecting duct. The binding of a fluorescent vasopressin analog (1-desamino-8-rhodamine-L-lysine vasopressin) to cells of the microperfused rabbit cortical collecting tubule was visualized and quantitated with image-intensified video microscopy and digital image processing. Binding to the basolateral membranes of a subpopulation of cells could be detected within 1-2 min of addition of the fluorescent analog (10 nM) to the peritubular bath. Binding could be prevented or reversed by the addition of a 10-fold excess of the native hormone, which indicates that the fluorescent analog binds specifically to vasopressin receptors. The time course of binding paralleled and slightly preceded hyperpolarization of the lumen-negative transepithelial voltage, an electrical response that is also elicited by the native hormone. Double-label experiments in which the intercalated cell population was stained with fluorescein-labeled peanut lectin revealed that binding of the vasopressin analog was localized to the remaining cell type, the principal cell. Our results support the following conclusions. First, the principal cell constitutes the primary target cell for vasopressin in the rabbit cortical collecting tubule, although the intercalated cell may possess a limited number of receptors at a density below the detection limit of this optical approach. Second, computer-enhanced video microscopy is a powerful, noninvasive method for assessing the kinetics and spatial pattern of hormone binding. We have used computer-enhanced imaging techniques to directly visualize and quantitate the binding of a fluorescent vasopressin analog to individual epithelial cells. This noninvasive optical approach has allowed the generation of spatial and temporal ``maps'' of hormone binding, maps that were used to correlate directly (in individual experiments) the kinetics, magnitude, and spatial pattern of binding with the physiologic effects of the hormone analog.