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 Ca2+-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). Ca2+-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 Ca2+-ATPase, enabling a local RAS axis to effect rapid modulation of active Ca2+ fluxes. (C) 2009 Elsevier B.V. All rights reserved.