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Neutral thiol-activated peptidases present in the pH 5-soluble fraction of rabbit brain (separated by step-elution chromatography on diethylaminoethyl cellulose) were screened for the hydrolysis of bradykinin. Lys-bradykinin, Met-Lys-bradykinin, angiotensin I, angiotensin II, substance P, luteinizing hormone-releasing hormone (LH-RH), and neurotensin by bioassay. The column effluent was monitored for bradykinin inactivation and arylamidase activity and combined in six pools on the basis of bradykinin inactivation. The pools were characterized by determining the peptide fragments and amino acids released from bradykinin with an amino acid analyzer. Pools 1 through 3 contained 80% of the kininase activity and essentially all of the endopeptidase A and B activity, whereas pools 4 through 6 accounted for 98% of the recovered arylamidase activity. Bradykinin, angiotensin I, angiotensin II, and substance P were inactivated by all the pools, whereas LH-RH and neurotensin were inactivated by pools 3 and 4, and pools 3, 4, and 5, respectively. These data show that rabbit brain contains peptidases having some selectivity for the inactivation of neuropeptides. Endopeptidase B purified from pool 3 is inhibited by bradykinin-potentiating peptide 9a (BPP9a, SQ 20881) (< Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), a competitive inhibitor of the hydrolysis of bradykinin (Km = 3.5 X 10(-5) M, Ki = 3 X 10(-6) M) which also completely inhibits the inactivation of LH-RH.

Bradykinin is a potent vasodilator peptide; however, its half-life in vivo is very short because of various plasma and tissue peptidases that hydrotyze bradykinin to inactive fragments. We studied the role of kininase II (angiotensin converting enzyme) and neutral endopeptidase 24.11 (enkephalinase) in the catabolism of bradykinin in vascular tissue by determining the effect of inhibitors of kininase II (captopril) and of endopeptidase 24.11 (phosphoramidon) on the action of bradykinin on rat isolated mesenteric arteries. Because bradykinin may induce prostaglandin formation and release, we also studied the effect of a cyclooxygenase inhibitor, indomethacin, on the action of bradykinin. The mesenteric bed was isolated from rats (250-300 g) with rats under ether anesthesia and was perfused with Krebs' solution (4 ml/min) containing phenylephrine (0.5-1.0 /tg/ml) to produce a mean perfusion pressure of 120-130 mm Hg. Bradykinin (2.5-40.0 ng), injected as a bolus, produced a dose-dependent decrease in perfusion pressure. In the presence of indomethacin (1.0 fig/ml), the amplitude of the vasodilator responses to bradykinin was not significantly affected, although the duration of the responses was increased approximately two to four times. In the presence of captopril (1.0 Mg/ml), bradykinin elicited either a vasodilator or a biphasic effect The vasodilator effect was greatly potentiated by captopril, whereas the duration of the response was unchanged when compared with control experiments. When present, the pressor responses were also dose related. In the presence of indomethacin plus captopril, bradykinin produced only a fall in perfusion pressure that lasted five to six times longer than without any treatment, Phosphoramidon (1-200 ng/ml) did not affect the responses to bradykinin. We conclude that bradykinin induces vasodilation of rat isolated mesenteric arteries followed by a vasoconstrictor effect due to prostaglandin release. The contribution of neutral endopeptidase 24.11 to bradykinin inactivation seems to be negligible, whereas kininase II plays an important role in the metabolism of bradykinin in vascular tissue. KEYWORDS • kinins • prostaglandins • angiotensin converting enzyme inhibitors • angiotensin converting enzymes • peptide peptidohydrolases by guest on March 22, 2015 http://hyper.ahajournals.org/ Downloaded from M C Salgado, H Caldo and M C Rodrigues Effect of bradykinin on isolated mesenteric arteries of the rat.

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The distribution and properties of neutral peptidases acting on the peptide hormone bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) were determined in several rabbit tissues. The supernatant and particulate fractions prepared from tissue homogenates (25000g for 60min) were studied. Bradykinin inactivation (kininase activity) was measured by bioassay with the isolated guinea-pig ileum. The sites of peptide-bond cleavage were determined in the amino acid analyser, which permits detection and measurement of amino acids and peptides derived from bradykinin. The results indicate that kininases are present in a wide range of concentrations in different tissues, kidney and lung having the most activity. Kininases present in different tissues were distinguished on the basis of sensitivity to the effects of EDTA, dithiothreitol and ZnCl2 and by the site of peptide-bond hydrolysis in bradykinin.

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