RESUMEN
PURPOSE: An active renin-angiotensin system has been found in the retina of rats and humans. Angiotensin-converting enzyme 2 (ACE2) is a recently discovered enzymatic homologue of Angiotensin-converting enzyme (ACE) that may be an important new component of the renin-angiotensin system (RAS). This study assesses the involvement of ACE2 in the normal and diabetic rodent retina and its modulation by ACE inhibition. METHODS: Sprague-Dawley rats were randomised into three groups, control, diabetes, and diabetes plus ramipril, with diabetes induced with the cell toxin streptozocin and the study run for 24 weeks. ACE2 and ACE gene levels were measured using quantitative real-time polymerase chain reaction (QRT-PCR), ACE2 protein expression was confirmed by Western blotting, and ACE and ACE2 catalytic activity were measured using specific activity assays in the rat retina. Localisation of ACE2 mRNA and protein were determined by in situ hybridisation and immunohistochemistry, respectively. RESULTS: ACE mRNA levels were decreased to approximately 50% in the diabetic retina, but ACE2 mRNA levels were not significantly changed. ACE but not ACE2 gene expression was influenced by ramipril treatment. Following immunostaining, both ACE2 and ACE protein were localised predominantly to the inner nuclear layer (INL) but also to photoreceptors. In the diabetic retina, ACE enzyme activity was decreased, whereas ACE2 enzyme activity was increased. CONCLUSIONS: This study has identified ACE2 gene and catalytically active protein in the rodent retina. In diabetes, the major changes were a decrease in ACE but an increase in ACE2 enzymatic activity. The ACE inhibitor ramipril did not reduce ACE2 enzymatic activity.
Asunto(s)
Carboxipeptidasas/metabolismo , Diabetes Mellitus Experimental/enzimología , Retinopatía Diabética/enzimología , Retina/enzimología , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Western Blotting , Carboxipeptidasas/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Hibridación in Situ , Masculino , Peptidil-Dipeptidasa A , ARN Mensajero/metabolismo , Ramipril/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mouse alpha- and gamma-nerve growth factor (NGF) are glandular kallikreins that form a non-covalent complex (7S NGF) with beta-NGF. gamma-NGF is an active arginine-specific esteropeptidase; the alpha-subunit is catalytically inactive and has a zymogen-like conformation. Site-directed mutagenesis of alpha-NGF to alter the N-terminus and three residues in loop 7, a region that contributes to the catalytic center, restored substantial catalytic activity against N-benzoyl arginine-p-nitroanilide as substrate in two derivatives although they were not as active as recombinant gamma-NGF. Seven of the 15 derivatives that remained more alpha-like were able to substitute for native alpha-NGF in reforming 7S complexes; the other eight derivatives that were more gamma-like showed greatly reduced ability to do so. However, the most gamma-like alpha-NGF derivative could not substitute for native gamma-NGF in 7S complex formation. These findings suggest that the alpha-NGF backbone can be corrected to a functional enzyme by the addition of a normal N-terminal structure and two catalytic site substitutions and that the 7S complex requires one kallikrein subunit in the zymogen form and one in an active conformation.