Abstract
Previous reports show that bradykinin B2 receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.
Footnotes
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This research was supported by grants from the University of Buenos Aires (Grant M-003). F.G.P. and W.N. are research fellows of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). A.E.E. is member of CONICET.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.113381.
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ABBREVIATIONS: BK, bradykinin; HUA, human umbilical artery; ACE, angiotensin-converting enzyme; NEP, neural endopeptidase; CRC, concentration-response curve; 5-HT, 5-hydroxytryptamine, serotonin; APM, aminopeptidase M; ECE, endothelin-converting enzyme; LPS, lipopolysaccharide; IL, interleukin.
- Received September 3, 2006.
- Accepted November 1, 2006.
- The American Society for Pharmacology and Experimental Therapeutics