A scrutiny of the biochemical pathways from Ang II to Ang-(3-4) in renal basolateral membranes

In a previous paper we demonstrated that Ang-(3-4) counteracts inhibition of the Ca(2+)-ATPase by Ang II in the basolateral membranes of kidney proximal tubules cells (BLM). We have now investigated the enzymatic routs by which Ang II is converted to Ang-(3-4). Membrane-bound angiotensin converting enzyme, aminopeptidases and neprilysin were identified using fluorescent substrates. HPLC showed that Plummer's inhibitor but not Z-pro-prolinal blocks Ang if metabolism, suggesting that carboxypeptidase N catalyzes the conversion Ang II -> Ang-(1-7). Different combinations of bestatin, thiorphan, Plummer's inhibitor, Ang II and Ang-(1-5), and use of short proteolysis times, indicate that Ang-(1-7)-> Ang-(1-5)-> Ang-(1-4)-> Ang-(3-4) is a major route. When Ang III was combined with the same inhibitors, the following pathway was demonstrated: Ang III -> Ang IV -> Ang-(3-4). Ca(2+)-ATPase assays with different Ang II concentrations and different peptidase inhibitors confirm the existence of these pathways in BLM and show that a prolylcarboxypeptidase may be an alternative catalyst for converting Ang II to Ang-(1-7). Overall, we demonstrated that BLM have all the peptidase machinery required to produce Ang-(3-4) in the vicinity of the Ca(2+)-ATPase, enabling a local RAS axis to effect rapid modulation of active Ca(2+) fluxes. (C) 2009 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Jose Bonifacio FoundationUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, BR-21941 Rio de Janeiro, BrazilInst Nacl Ciencia & Tecnol Biol Estrutural & Bioi, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Ciencias Biomed, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilWeb of Scienc