C Type Natriuretic Peptide Receptor Activation Inhibits Sodium Channel Activity in Human Aortic Endothelial Cells by Activating the Diacylglycerol-Protein Kinase C Pathway

Figure 1. Western blot analysis of EnNaC alpha in human aortic endothelial cells. (A). Western blot of EnNaC alpha from cell lysates of 4 independents experiments in which hAoECs were treated with either vehicle (veh) or cANF for 8 h before harvesting the cells for protein. (B). Densitometric analysis of the immunoreactive bands indicated by arrows and normalized to the Actin band in the Western blot in (A). A Student’s t test was performed to compare the two groups. n = 4 samples in each of the two groups. * represents a p-value < 0.05. *** represents a p-value < 0.001.

Figure 2. The NPRC agonist cANF inhibits EnNaC activity in hAoEC. Representative single-channel recordings (A), I-V curve of EnNaC (B), and summary plot of EnNaC activity (NPo) (C). A Student t-test was used to compare the control (Ctrl) and cANF experimental groups. n = 30 patches for the control group and n = 34 patches for the cANF group. * represents a p-value < 0.05.

Figure 3. EnNaC alpha subunit protein expression in hAoEC transfected with NPRC specific siRNA compared to cells transfected with non-targeting siRNA. (A). Western blot for EnNaC alpha protein after hAoECs were transfected with NPRC specific siRNA or non-targeting (NT) siRNA and then treated with or without cANF before harvesting the cells for protein. (B). Densitometric analysis of the 150 kDa, 75 kDa, and 60 kDa immunoreactive bands in (A). Each group includes hAoEC cell lysates from three separate siRNA experiments that were loaded on the same gel and probed with EnNaC alpha antibody. A one-way ANOVA was used for statistical analysis of the experimental groups with a Holm–Sidak post hoc test. n = 3 samples for each of the three groups. * represents a p-value < 0.05.

Figure 4. Quantification of diacylglycerols in hAoECs treated with cANF or vehicle. (A). DAG(18:1/20:2), (B). DAG(18:2/20:4), (C). DAG(18:0/18:1), (D). DAG(16:1/18:3) were among some diacylglycerols quantified in hAoECs treated with vehicle (Veh) or cANF for 8 h. A Student’s t-test was performed to make comparisons between the two groups. n = 3 samples in the vehicle and cANF groups. * represents a p-value < 0.05. ** represents a p-value < 0.01.

Figure 5. Protein Kinase C activity in hAoEC treated with cANF or vehicle. n = 3 independent cANF versus vehicle (VEH) control treatment experiments. POS CTRL represents Positive Control. A Student’s t-test was performed to make comparisons between the two groups. n = 3 samples for each of the two groups. ** Represents a p-value < 0.01.

Figure 6. EnNaC alpha protein expression after inhibition of PKC.(A). Western blot for EnNaC alpha protein after hAoECs were treated with or without the PKC inhibitor GF109203X and with or without the NPRC agonist cANF for 3 h. (B). Densitometric analysis of the 150 kDa, 75 kDa, and 60 kDa immunoreactive bands in (A). A Student’s t-test was performed to make comparisons between the two groups. n = 3 samples for each of the three groups. * Represents a p-value < 0.05. ** Represents a p-value < 0.01.

Figure 7. Schematic of the proposed model for the inhibition of EnNaC following NPRC activation. NPRC is activated by ligand binding. The NPRC agonist cANF is shown to selectively bind to NPRC. NPRC ligand biding results in the activation of phospholipase C (PLC) leading to the hydrolysis of PIP2. Hydrolysis of PIP2 produces the hydrophobic molecule diacylglycerol (DAG) which is retained in the plasma membrane and activates protein kinase C (PKC). PKC is known to inhibit the activity of ion channels including epithelial sodium channels (ENaC) and presumably endothelial sodium channels (EnNaC). Schematic was created in part using the BioRender software.