Figure 4.

Guanylyl cyclase measurements of tissues from Npr2-7A and Npr-7E mutants. A, Changes in ANP- and CNP-dependent guanylyl cyclase activities in brains from Npr2^wt/wt, Npr2^wt/7A, and Npr2^7A/7A mice. Crude membranes were prepared from flash-frozen brains, and 60–75 μg was assayed in duplicate for each condition; n = 5 or 6. Error bars indicate mean ± SEM. *p ≤ 0.05, statistical significance from wild-type samples. **p ≤ 0.01, statistical significance from wild-type samples. ***p ≤ 0.001. B, Changes in ANP- and CNP-dependent guanylyl cyclase activities in lungs from Npr2^wt/wt, Npr2^wt/7A, and Npr2^7A/7A mice. Crude membranes were prepared from flash-frozen lungs, and 10–17 μg was assayed in duplicate for each condition; n = 5 or 6. Error bars indicate mean ± SEM. *p ≤ 0.05, statistical significance from WT samples. **p ≤ 0.01, statistical significance from WT samples. C, CNP-stimulated guanylyl cyclase activity measured in brains from Npr2^wt/wt and Npr2^7E/7E mice does not differ. Guanylyl cyclase activity was measured in crude membranes from postnatal day 25 male and female Npr2^wt/wt and Npr2^7E/7E mice in the presence of excess MgCl₂, 0.1 mm GTP, 1 mm ATP, and the indicated concentrations of CNP or with 0.1 mm GTP and excess Mn²⁺ and 1% Triton X-100 (Mn/Triton). n = 6 mice for each genotype with 3 males and 3 females for each treatment. A, B, cGMP measurements were done by a highly sensitive radioimmunoassay without removal of cross-reacting GTP. C, cGMP was determined by an ELISA-based method after removal of GTP by sodium carbonate precipitation and purification of cGMP over an alumina column.