Premium
Small interfering RNA‐mediated functional silencing of natriuretic peptide receptor‐A in human embryonic kidney ‐293 cells
Author(s) -
Somanveen K,
Arise Kiran K,
Wu Edwin,
Pandey Kailash N
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a254-a
Subject(s) - internalization , atrial natriuretic peptide , hek 293 cells , receptor , microbiology and biotechnology , transfection , intracellular , messenger rna , small interfering rna , microrna , medicine , chemistry , endocrinology , biology , gene , biochemistry
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 intracellular second messenger cGMP, which maintains blood pressure and cardiovascular homeostasis. The objective of this study was to determine whether ANP‐NPRA complexes are internalized into intracellular compartments. Internalization studies were performed utilizing 125 I‐ANP in intact human embryonic kidney (HEK)‐293 cells stably transfected with murine NPRA cDNA. We used artificial microRNA to silence target gene expression of NPRA. Reverse transcription‐polymerase chain reaction and northern blot analyses applied to confirm the specificity and efficiency of the vector. The results showed that the expression of microRNA accounted for more than 80% down regulation of NPRA. In addition, internalized 125 I‐ANP radioactivity was reduced by almost 80% in cells depleted with NPRA, as compared with control cells. We conclude that NPRA undergoes internalization as part of its normal trafficking and metabolism. The findings of this study are important towards understanding the signal mechanisms and functional role of NPRA in maintaining the cardiovascular homeostasis. Supported by NIH Grant HL‐57531.