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

Abstract

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 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). 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 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 Ca(2+)-ATPase, enabling a local RAS axis to effect rapid modulation of active Ca(2+) fluxes.

Exposure of luminal membranes of LLC-PK1cells to ANG II induces dimerization of AT1/AT2receptors to activate SERCA and to promote Ca2+mobilization

“…Whereas basolateral ANG II increases cytosolic Ca 2ϩ by inhibition of PMCA, the positively self-sustained luminal stimulus on SERCA provides a refined mechanism for further Ca 2ϩ control. It is noteworthy that, despite their polarity, the ANG II effect on Ca 2ϩ homeostasis in proximal tubule cells is biphasic concerning the agonist concentration (5,6) (Fig. 1); however, the processes involved seem to be different.…”

“…The activities of ACE, ACE2, neprilysin (NEP), and amino peptidases (AP) were measured using fluorescent substrates containing the fluorogenic group 4-methylcoumaryl-7-amide (MCA) or ortho-amino benzoic acid (Abz) and the fluorescent suppressor groups dinitrophenyl (Dnp) or 2,4-dinitrophenyl-ethylendiamino (EDDnp), in the absence or presence of the inhibitors lisinopril (for ACE), thiorphan (for NEP), DX600 (for ACE2), and bestatin (for AP), as previously described (6,41). Briefly, hydrolysis of substrates was monitored by spectrofluorimetry (Hitachi F-4500, Tokyo, Japan).…”

“…A possible mechanism for ANG II-mediated SERCA activation is the internalization of the peptide (36,47) or the formation of small ANG II-derived activating peptides (5,6). To test the hypothesis that ANG II is internalized or partially metabolized, LLC-PK 1 cells were incubated with 8 M ANG II for different times and the resulting supernatants were analyzed by HPLC (Fig.…”

“…These pumps control intracellular Ca 2ϩ concentration by regulating intracellular Ca 2ϩ stocks, fine-tuning cytosolic Ca 2ϩ activity in many cell types (12). Therefore, renal SERCA and PMCA are excellent targets for hormones, including ANG II and angiotensin-derived peptides (6). We found that picomolar ANG II inhibits PMCA activity via a PLC/protein kinase C (PKC) pathway triggered by the peptide binding to AT 1 R/AT 2 R heterodimers (4,5).…”

“…We found that picomolar ANG II inhibits PMCA activity via a PLC/protein kinase C (PKC) pathway triggered by the peptide binding to AT 1 R/AT 2 R heterodimers (4,5). Micromolar ANG II is metabolized by peptidases associated with the basolateral membrane, generating Ang-(3-4) that reactivates PMCA (5,6).…”