on January 15, 2004
Published online before print January 15, 2004, doi: 10.1161/01.ATV.0000117176.71143.a1
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Submitted on July 11, 2003
Accepted on January 5, 2004
Regulation of Endopeptidases EC3.4.24.15 and EC3.4.24.16 in Vascular Endothelial Cells by Cyclic Strain: Role of Gi Protein Signaling
Eoin J. Cotter ;
From the Vascular Health Research Centre (E.J.C., N.V.O.S., P.M. Coen, Y.A.B., P.A.C., P.M. Cummins), Faculty of Science and Health, Dublin City University, Glasnevin, Dublin, Ireland; and the Department of Biochemistry & Molecular Biology (M.J.G.), FUHS/Chicago Medical School, North Chicago, IL.
* To whom correspondence should be addressed. E-mail: phil.cummins{at}dcu.ie.
Objective--Endopeptidase EC3.4.24.15 (EP24.15)- and EC3.4.24.16 (EP24.16)-specific peptide hydrolysis plays an important role in endothelium-mediated vasoregulation. Given the significant influence of hemodynamic forces on vascular homeostasis and pathology, we postulated that these related peptidases may be mechanosensitive. The objective of this study, therefore, was to investigate the putative role of cyclic strain in regulating the expression and enzymatic activity of EP24.15 and EP24.16 in bovine aortic endothelial cells (BAECs).
Methods and Results--BAECs were cultured under conditions of defined cyclic strain (0% to 10% stretch, 60 cycles/min, 0 to 24 hours). Strain significantly increased EP24.15 and EP24.16 soluble activity in a force- and time-dependent manner, with elevations of 2.3±0.4- and 1.9±0.3-fold for EP24.15 and EP24.16, respectively, after 24 hours at 10% strain. Pharmacological agents and dominant-negative G protein mutants used to selectively disrupt Gi
- and G
-mediated signaling pathways attenuated strain-dependent (24 hours, 5%) increases for both enzymes. Differences in the inhibitory profile for both enzymes were also noted, with EP24.15 displaying greater sensitivity to Gi2/3 inhibition and EP24.16 exhibiting greater sensitivity to Gi1/2 and G inhibition. Cyclic strain also increased levels of secreted EP24.15 and EP24.16 activity by 2.6±0.02- and 3.6±0.2-fold, respectively, in addition to mRNA levels for both enzymes (EP24.15 x42%, EP24.16 x56%).
Conclusions--Our findings suggest that cyclic strain putatively regulates both the mRNA expression and enzymatic function of EP24.15 and EP24.16 in BAECs via alternate Gi protein signaling pathways.
Key words: metallopeptidase • endothelial function • cyclic strain • G protein
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