The effects of exogenous ANP and A71915 on cGMP production in E11.5 ventricular cells and assessment of ANP bioactivity. (A) Cellular cGMP levels in E11.5 ventricular cells treated with ANP (1 μg/ml) and or A71915 (1 µM) were determined using a competitive HTRF immunoassay. The baseline level of cGMP (Cont) was measured to be 28.1 ± 0.2 nM/ 100,000 cells. Addition of exogenous ANP resulted in a significant increase in cGMP. Co-treatment of cells with both ANP and A71915 resulted in lower levels cGMP compared to untreated cells. N = 3 independent experiments, performed in duplicate wells. Each bar represents mean ± SEM. *P < 0.005 Vs. Cont, #P < 0.005 Vs. Cont or ANP (1 μg/ml), One-way ANOVA with Tukey post hoc test. (B) Assessment of ANP bioactivity in the conditioned media of E11.5 ventricular cells: Conditioned media was collected at 1, 4 and 12-hour time points and transferred to HEK293 cells, and cGMP levels were measured. As a negative control (Cont), cGMP production was measured in HEK293 cells that were not treated with ANP. As a positive control, cGMP levels were measured in HEK293 cells treated directly with fresh ANP (1 μg/ml). Over 12 hours, conditioned media treatment maintained significantly higher levels of cGMP with a minimal decline in bioactivity. N = 4 independent experiments, performed in duplicate wells. Each bar represents mean ± SEM. *P < 0.005 Vs. Cont, #p < 0.05 Vs. ANP or 1 hr, One-way ANOVA with Tukey post hoc test.