Enzymatic pathways of the brain renin-angiotensin system: unsolved problems and continuing challenges

Abstract

The brain renin-angiotensin system continues to be enigmatic more than 40 years after the brain was first recognized to be a site of action of angiotensin II. This review focuses on the enzymatic pathways for the formation and degradation of the growing number of active angiotensins in the brain. A brief description and nomenclature of the peptidases involved in the processing of angiotensin peptides in the brain is given. Of primary interest is the array of enzymes that degrade radiolabeled angiotensins in receptor binding assays. This poses major challenges to studies of brain angiotensin receptors and it is debatable whether an accurate determination of brain angiotensin receptor binding kinetics has yet been made. The quandary facing the investigator of brain angiotensin receptors is the need to protect the radioligand from metabolic alteration while maintaining the characteristics of the receptors in situ. It is the tenet of this review that we have yet to fully understand the binding characteristics of brain angiotensin receptors and the extent of their distribution in the brain because of our inability to fully protect the angiotensins from metabolic alteration until equilibrium binding conditions can be attained.

Fig. 1

Pathways of formation of angiotensin peptides in the brain. Abbreviations used: ACE — angiotensin converting enzyme; ACE2 — human homolog of angiotensin converting enzyme; APA — aminopeptidase A; A-LAP — adipocyte derived leucine-aminopeptidase; L-RAP — leukocyte-derived arginine aminopeptidase; NEP — neutral endopeptidase; TOP — thimet endopeptidase; Pro-EP — prolyl-endopeptidase; Pro-CP — prolyl-carboxypeptidase; APX — aminopeptidase X; DAP — aspartyl aminopeptidase; P-LAP/IRAP — placental leucine-aminopeptidase/insulin-regulated aminopeptidase; APN/APM — aminopeptidase N/M; APB — aminopeptidase B; DPP I — dipeptidyl peptidase I; DPP III — dipeptidyl peptidase III. Numbering of amino acid residues in all fragments is based on the numbering in angiotensinogen. Larger sized arrows indicate the “classical” metabolic pathways for angiotensin peptides.

Fig. 2

Time course of specific binding of ¹²⁵I–Ang III to a rat brain membrane preparation. Rat brain membranes (50 mg initial wet weight/ml) were prepared as described previously . The incubation medium contained standard assay buffer 150 mM NaCl, 5 mM EDTA, 0.1 mM bacitracin, and 50 mM NaPO₄, pH 7.1–2, plus the following peptidase inhibitors: o-phenanthroline (1 mM), puromycin (3 mM) phenylmethylsulfonyl fluoride (1 mM). A total of 2.5 mg initial wet weight of brain membranes was present in 100 μl for this assay which was carried out at 21–24 °C. Nonspecific binding was determined in the presence of 3 μM Ang II and subtracted from total binding to derive specific binding.

Fig. 3

Metabolic fate of ¹²⁵I–Ang II bound to a rat brain membrane preparation. Rat brain membranes were prepared as described previously . The incubation medium contained 150 mM NaCl, 5 mM EDTA, 0.1 mM bacitracin, and 50 mM NaPO₄, pH 7.1–2. A total of 12.5 mg initial wet weight of brain membranes was present in 500 μl for this assay, which was carried out for 60 min at 21–24 °C. After one hour incubation the membrane suspension was centrifuged and the supernatant discarded. The pellet was resuspended in HPLC mobile phase: 21% acetonitrile: 79% triethylamine phosphate (83 mM phosphate, pH 3.0), periodically vortexed during 20 min and recentrifuged. The supernatant was fitered through a 0.22 µm filter, applied to Sep-Pak® C18 (Waters Inc.) column and eluted with 21% acetonitrile: 79% triethylamine phosphate (83 mM phosphate, pH 3.0). The eluate was run on a reverse-phase (C₁₈) column with a mobile phase of either 13% acetonitrile: 87% triethylamine phosphate (83 mM phosphate, pH 3.0), to allow better resolution of smaller fragments, or 21% acetonitrile: 79% triethylamine phosphate (83 mM phosphate, pH 3.0) at a flow rate of 1.2 ml/min. Radiolabeled Ang II and fragments were identified based on the elution times of radioiodinated standards of Ang II and its fragments under the same HPLC conditions. 15 s fractions of the column eluate were collected and counted in a gamma counter.