Effect of a new aminopeptidase P inhibitor, apstatin, on bradykinin degradation in the rat lung.
Bradykinin (Bk), a potent vasoactive and cardioprotective peptide hormone, is almost completely inactivated during a single circulation through the rat lung. It has been hypothesized that membrane-bound aminopeptidase P, which can hydrolyze the Arg1-Pro2 bond of Bk, and angiotensin-converting enzyme (ACE) act in concert to degrade Bk in the pulmonary circulation. To test this hypothesis, an inhibitor of aminopeptidase P was designed and synthesized. N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-prolyl-L-prolyl-L - alaninamide (apstatin) inhibited purified rat lung membrane-bound aminopeptidase P with a Ki value of 2.6 microM (linear mixed-type inhibition, alpha = 5.1, beta = 0). Apstatin did not inhibit ACE or other known Bk-degrading enzymes. Apstatin and an ACE inhibitor, ramiprilat, were tested for their ability to inhibit Bk degradation in the isolated perfused rat lung. [2,3-Proly-3,4-3H(N)]-bradykinin ([3H]-Bk) was perfused through the isolated lung in the presence or absence of inhibitors. The perfusate was then subjected to HPLC to identify and quantitate radiolabeled fragments. In the absence of inhibitors, no intact [3H]-Bk was found in the perfusate. In the presence of ramiprilat (0.5 microM), only 22% +/- 6% of the radioactivity in the perfusate was intact [3H]-Bk, and the remaining radioactivity indicated cleavage of the Arg1-Pro2 bond. When apstatin (40 microM) was perfused along with ramiprilat, degradation of [3H]-Bk was almost completely blocked (92% +/- 4% intact [3H]-Bk in the perfusate). The results indicate that the Bk-degrading activity in the rat pulmonary vascular bed can be fully accounted for by aminopeptidase P (30%) and ACE (70%).
Prechel
M M
MM
Department of Molecular and Cellular Biochemistry, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois, USA.
Orawski
A T
AT
Maggiora
L L
LL
Simmons
W H
WH
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D001920:Bradykinin / Q000378:metabolism*; D002417:Cattle; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006868:Hydrolysis; D066298:In Vitro Techniques; D008168:Lung / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D010455:Peptides; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; C097277:apstatin; D001920:Bradykinin