ANALYSIS OF NATRIURETIC PEPTIDE RECEPTOR A INTERNALIZATION BY RIBONUCLEIC ACID INTERFERENCE.

Abstract

Guanylyl cyclase/natriuretic peptide receptor A (GC-A/NPRA) is considered the principal receptor for both atrial and brain natriuretic peptides (ANP and BNP). Binding of these hormones to NPRA leads to the generation of the intracellular second messenger cGMP, which, in turn, maintains blood pressure and cardiovascular homeostasis. The objective of the present study was to determine whether ANP-NPRA complexes are internalized to release the bound ANP from the receptor in the intracellular compartments. In these studies, we used human embronic kidney-293 (HEK-293) cells stably transfected with murine NPRA cDNA. Cells were cultured in Dulbecco9s Modified Eagle9s Medium (DMEM) supplemented with 10% bovine fetal serum and maintained at 37°C under an atmosphere of 5% CO 2 /95% O 2 . Receptor internalization studies were performed using 125 I-ANP in intact HEK-293 cells. All hormonal treatments and binding assays were performed in DMEM serum-free medium containing 0.1% bovine serum albumin. We used vector-based artificial microRNA for expression of short-hairpin RNA (shRNA) to more potently silence the target gene expression of NPRA. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses were applied to confirm the specificity and the efficiency of the constructs. The results showed that the expression of shRNA accounted for more than 80% down-regulation of NPRA. Furthermore, the results revealed that the internalized 125 I-ANP radioactivities were drastically reduced by almost 80% in cells depleted with NPRA compared with wild-type control cells. The results demonstrate that NPRA undergoes internalization as part of its normal trafficking and metabolism. The findings of this study are important toward the understanding of the signaling mechanisms and the functional role of NPRA to maintain the cardiovascular homeostasis.