Complete response to vasopressin requires epithelial organization in A6 cells in culture

Abstract

Arginine vasopressin (AVP) stimulates adenylate cyclase activity in A6 epithelia grown on filters but not in A6 epithelia grown on plastic culture dishes. When A6 cells are subcultured from culture dishes and seeded at high density on filters, stimulation of adenylate cyclase by AVP is not evident for approximately 3 days. Peak stimulation by AVP occurs between 5 and 10 days. The time course for development of responsiveness to AVP corresponds to the development of an ordered epithelium with polarized cells, tight junctions, and a high transepithelial resistance. A similar correlation is seen when mature filter-grown epithelia that respond to AVP are dissociated by chelation of calcium and subcultured as single cells onto another filter. Stimulation of adenylate cyclase is not evident until an epithelium with significant electrical resistance is reformed. Filters seeded at 1.6 X 10(6) cells/cm2 require 1-2 days to show AVP sensitivity and electrical resistance; filters seeded at 0.4 X 10(6) cells/cm2 require 4 days. Experiments to test the function of the subunits of adenylate cyclase with forskolin (catalytic subunit) and with cholera toxin (Ns, guanine nucleotide-binding subunit) indicate that they are functional during the entire time and that it is the receptor for AVP that is not functional until an ordered epithelium has been formed.