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In order to understand angiotensin metabolism in the canine brain, we determined the molecular forms of angiotensin peptides present in the hypothalamus of the dog and carried out measurements of the metabolism of 125l-angiotensin I in homogenates of that tissue. Angiotensin peptides were extracted from canine hypothalamic tissue and quantified by specific radioimmunoassays combined with high-performance liquid chromatography. The major angiotensin peptides detected were angiotensin-(2-7) (391.2 ± 16.8 pg/g tissue) and angiotensin-(3-7) (864.8 ± 128.1 pg/g). Angiotensin II immunoreactivity was mainly composed of angiotensin-(3—8) (117.5 ± 64 pg/g) and trace amounts of angiotensin II and angiotensin III. Angiotensin I immunoreactivity was composed of angiotensin I (52.3 ± 5.8 pg/g). In separate experiments, addition of 125l-angiotensin I into supernatants (18000g for 2min) of canine hypothalamic homogenates resulted in the accumulation of 125l-angiotensin- (1-7) as the major peptide product (14% of the total 125l-radioactivity) at 2 min. Incubation of the homogenate supernatants with enalaprilat (I (j.mol/1), phosphoramidon (10 nmol/l), or ethylenediamine tetraacetic acid (1 mmol/l) did not inhibit the production of 125l-angiotensin-(1-7). In contrast, addition of Z-Pro-Prolinal (1 |imol/l), a specific inhibitor of prolyl endopeptidase, prevented the generation of 125l-angiotensin-(1-7) from 125!-angiotensin I by 47.0 ± 8.0% (n=6). The predominance of angiotensin-(2-7) and angiotensin-(3-7) in the canine hypothalamus suggests the importance of the proline (Pro)7-phenylalanine (Phe)8 cleavage. These findings suggest that prolyl endopeptidase is a major component of the enzymatic pathways that participate in angiotensin metabolism in canine hypothalamus.