Conclusion
We conclude that the NRK-52E cells express an intracellular RAS localized to the nucleus and may be an appropriate cell model to elucidate the functional relevance of this system.
Methods
RAS components were visualized by immunofluorescent staining in intact cells and protein expression in isolated nuclei.
Results
An antibody to the angiotensin I (Ang I) sequence of AGT (AI-AGT) revealed only cytosolic staining, while an antibody to an internal sequence of AGT (Int-AGT) revealed primarily nuclear staining. Immunoblots of nuclear and cytosolic fractions confirmed the differential cell staining of AGT. Immunostaining for renin was present on nuclei of intact cells. Nuclear renin activity averaged 0.77±0.05 nmol/mg protein/h that was reduced by aliskiren (0.13±0.01 nmol/mg/h, n=3, p<0.01); trypsin activation increased activity three-fold. Peptide staining localized angiotensin II (Ang II) and Ang-(1-7) to the nucleus and peptide content averaged 59±2 and 57±22 fmol/mg (n=4), respectively. Peptide metabolism in isolated nuclei revealed the processing of Ang I to Ang-(1-7) by thimet oligopeptidase.