SynopsisAtrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP-NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP-NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20 % of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP , whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP .

Background: Natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. Results: Data show that histone deacetylase inhibitors (HDACi) enhance NPRA expression by blocking HDACs and interacting with Sp1, histone acetyltransferase, and acetylated histones. Conclusion: Results suggest that epigenetic mechanisms regulate NPRA gene transcription. Significance: Findings will help to identify molecular targets regulating NPRA expression and function in vivo.

Along with its natriuretic, diuretic, and vasodilatory properties, atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) exhibit an inhibitory effect on cell growth and proliferation. However, the signaling pathways mediating this inhibition are not well understood. The objective of this study was to determine the effect of ANP-NPRA system on mitogen-activated protein kinases (MAPKs) and the downstream proliferative transcription factors involving activating protein-1 (AP-1) and cAMP-response element binding protein (CREB) in agonist-stimulated mouse mesangial cells (MMCs). We found that ANP inhibited vascular endothelial growth factor (VEGF)-stimulated phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38), to a greater extent in NPRA-transfected cells (50–60%) relative to vector-transfected cells (25–30%). The analyses of the phosphorylated transcription factors revealed that ANP inhibited VEGF-stimulated activation of CREB, and the AP-1 subunits (c-jun and c-fos). Gel shift assays demonstrated that ANP inhibited VEGF-stimulated AP-1 and CREB DNA-binding ability by 67 % and 62 %, respectively. The addition of the protein kinase G (PKG) inhibitor, KT-5823, restored the VEGF-stimulated activation of MAPKs, AP-1, and CREB, demonstrating the integral role of cGMP/PKG signaling in NPRA-mediated effects. Our results delineate the under lying mechanisms through which ANP-NPRA system exerts an inhibitory effect on MAPKs and down-stream effector molecules, AP-1 and CREB, critical for cell growth and proliferation.

Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

Atrial natriuretic peptide (ANP) is a member of the natriuretic peptide hormone family that maintains cardiovascular homeostasis and exerts antiproliferative effects in a variety of cell types. ANP mediates its effects by binding guanylyl cyclase‐A/natriuretic peptide receptor‐A (GC‐A/NPRA) and subsequently activating cGMP‐dependent protein kinase (PKG), but the signaling pathways regulated by this system have not yet been elucidated. The objective of this study was to determine the effects of ANP‐NPRA system on mitogen‐activated protein kinases (MAPKs) and proliferative transcription factors AP‐1 and CREB in murine Leydig tumor (MA‐10) cells. Measurement of phorphorylated protein levels by Western blot demonstrated that vascular endothelial growth factor (VEGF) treatment caused a 3‐ to 4‐fold increase in phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38). ANP treatment inhibited VEGF‐stimulated phosphorylation of MAPKs by 50–65%. VEGF treatment caused a 3‐ to 5‐fold increase in c‐jun, c‐fos, and CREB protein levels, an effect which was significantly inhibited by ANP treatment. ANP treatment inhibited VEGF‐stimulated AP‐1 and CREB transcriptional activity by 60–70% in a PKG‐dependent manner. Thus, the present study delineates that ANP/NPRA signaling can exert an inhibitory effect on the proliferative cellular mechanisms that play a critical role in angiogenic response in disease states.