AJP - Cell Physiology, Vol 255, Issue 5 C622-C632, Copyright © 1988 by American Physiological Society

ARTICLES

Binding and internalization of a fluorescent vasopressin analogue by collecting duct cells

K. L. Kirk
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.

The kinetics of binding and internalization of a fluorescent vasopressin analogue [1-desamino-8-rhodamine lysine vasopressin (rhoda LVP)] by principal cells within the microperfused rabbit cortical collecting tubule were quantitatively assessed with computer-assisted video microscopy. At 25 degrees C, binding of rhoda LVP exhibited saturation kinetics with half-maximal binding at 2 nM and maximal binding at concentrations greater than 5 nM. Rhoda LVP binding could be prevented by the simultaneous addition of a 10-fold higher concentration of arginine vasopressin (AVP) or the V2-receptor agonist, 1-desamino-8-D-arginine vasopressin (desmopressin). No obvious internalization or rhoda LVP was detected at 25 degrees C, i.e., the rhoda LVP fluorescence remained localized to the basal pole of each principal cell for at least 100 min after rhoda LVP addition and could be largely reversed by the subsequent addition of AVP. Conversely, warming the cells to 38 degrees C after binding was initiated resulted in a rapid (less than 30 min) migration of the fluorescence into the cell interior and a loss of AVP-displaceable binding from the cell surface. These results document the utility of this noninvasive optical strategy for quantitatively monitoring hormone binding to intact collecting tubule cells and demonstrate that rhoda LVP binds reversibly and with high affinity to V2 receptors on principal cells in the collecting tubule. The internalization (and presumed inactivation) of the hormone-receptor complex at 38 degrees C may contribute to the desensitization of collecting tubule cells to vasopressin at physiological temperature.

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