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 II 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 prolyl-carboxypeptidase 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.