Abstract
Purpose: :
To evaluate the distribution of angiotensin II (Ang II) and Ang–(1–7) in retina from mice with homozygous disruption of angiotensin–converting enzyme (ACE –/–) and (ACE+/+).
Methods: :
ACE null C57BL/6 mice were generated by gene targeting. Genotype was confirmed by Southern analysis. Mice were sacrificed by carbon dioxide asphyxiation. The eyes were enucleated and fixed in PBS– paraformaldehyde. Localization of Ang II and Ang–(1–7) was determined by paraffin sections using a commercial antibody for Ang II (Phoenix H–200–12) and an in–house antibody for Ang–(1–7) (CCF–Core 1). Since both primary antisera were polyclonal rabbit antisera, Ang II and Ang–(1–7) were probed on separate sections and resolved with anti–rabbit Alexa 488 secondary antiserum. Specificity of the immunostaining was verified by substituting the primary antibodies with equivalent dilution of the non–immune IgG. Slides were mounted in anti–fade medium containing DAP1. A confocal laser–scanning microscope was used for immunoflorescence analysis. Immunoreactivity was quantified using Image Pro.
Results: :
Retinal tissue in both ACE+/+ and ACE–/– mice were histologically normal and Ang II and Ang–(1–7) immunoreactivity was detected in the retinal Müller cells extending from the endfeet throughout the entire cell. The two peptides appear to be co–localized. In the ACE–/– mice both peptides are upregulated, Ang II by 1.7 fold and Ang–(1–7) by 2.4 fold. Increase in Ang II may be due to the stimulation of alternative pathways for the synthesis of Ang II. ACE gene deletion leads to the accumulation of Ang I, the precursor of Ang II. Ang I is converted to Ang–(1–9) and Ang–(1–7). Ang–(1–9) is also converted to Ang–(1–7). In addition, Ang II synthesized by the alternative pathways may also be converted to Ang–(1–7). Furthermore, the lack of ACE inhibits the metabolism of Ang–(1–7) to Ang–(1–5). Thus, Ang–(1–7) is enhanced by multiple mechanisms.
Conclusions: :
Ang II and Ang–(1–7) in retinal Müller cells may be synthesized by the ACE pathway and alternate pathway(s) or may be totally independent of ACE.
Keywords: genetics • Muller cells • retina