Figure 1.

NPR-A undergoes agonist-dependent (homologous) desensitization. (A) NPR-A desensitization is biphasic. 293 cells stably expressing NPR-A were treated with 1 μM ANP for increasing periods, and then crude membranes were prepared and assayed for guanylyl cyclase activity in the presence of the activators indicated or fractionated by SDS-PAGE, blotted to Immobilon membrane, and probed with a NPR-A–specific antiserum. The antigen–antibody complex was quantitated by incubating with an ¹²⁵I-coupled donkey anti-rabbit–directed secondary antibody. Control equals no ANP incubation. (B) Long-term declines in ANP-dependent activity are explained by decreases in NPR-A protein. The ratio of ANP/ATP/Mg²⁺-dependent over Mn²⁺ and Triton X-100-dependent guanylyl cyclase activities from A (ordinate) was plotted versus time of ANP incubation (abscissa). The vertical bars contained within each symbol represent the range of values obtained from two separated samples, which were determined in duplicate. For some points the range is smaller than the diameter of the symbol and is not visible.