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 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.
Axelband
Flavia
F
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Dias
Juliana
J
Miranda
Filipe
F
Ferrão
Fernanda M
FM
Barros
Nilana M
NM
Carmona
Adriana K
AK
Lara
Lucienne S
LS
Vieyra
Adalberto
A
D000804:Angiotensin II / Q000378:metabolism*; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001485:Basement Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007668:Kidney / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D001121:Lysine Carboxypeptidase / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D015244:Thiorphan / Q000494:pharmacology
D000806:Angiotensin-Converting Enzyme Inhibitors; D000804:Angiotensin II; D015244:Thiorphan; D007703:Peptidyl-Dipeptidase A; D001121:Lysine Carboxypeptidase; D007930:Leucine; C012211:ubenimex