Metabolism of vasoactive peptides by vascular endothelium and smooth muscle aminopeptidase M
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F E Palmieri1, H H Bausback, P E Ward
1Department of Pharmacology, New York Medical College, Valhalla 10595.
Abstract:
The cellular localization of vascular plasma membrane aminopeptidase M (AmM; EC3.4.11.2) was examined in cultured porcine aorta endothelium and smooth muscle cells. AmM was 14-fold higher on smooth muscle (117 +/- 16 units/mg) than on endothelium (8.4 +/- 0.2). Proportional to its cellular distribution, AmM hydrolyzed the N-terminus of kallidin to produce bradykinin, and degraded des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and Met5-enkephalin. In contrast, bradykinin, angiotensin II and substance P were resistant to AmM-mediated hydrolysis. Peptide metabolism was optimal at pH 7.0 and was inhibited by o-phenanthroline, bestatin (Ki = 2.2 +/- 0.1 microM) and amastatin (Ki = 25 +/- 5 nM). Des(Asp1)angiotensin I and angiotensin III had the highest affinity (lowest Km) for AmM (Km = 2.2 +/- 0.5 and 2.0 +/- 0.4 microM respectively), followed by hepta(5-11)substance P (53.9 +/- 1.7 microM) and Met5-enkephalin (75.7 +/- 3.5 microM). In contrast, maximal velocities of hydrolysis were higher for Met5-enkephalin (313 +/- 2 nmol/min/mg) than for hepta(5-11)substance P (109 +/- 18 nmol/min/mg) or angiotensin III (26.5 +/- 1.0 nmol/min/mg). As expected for hydrolysis by a common enzyme, AmM-mediated enkephalin degradation was inhibited competitively by angiotensin III (Ki = 0.34 +/- 0.04 microM), hepta(5-11)substance P (43.7 +/- 6.3 microM) and kallidin (62 microM). These data suggest that vascular AmM may modulate vasoactive peptide levels in vivo, particularly within the microenvironment of endothelial and smooth muscle cell surface receptors.
Insights
Vascular plasma membrane aminopeptidase M (AmM) is more abundant in smooth muscle cells than endothelial cells. This enzyme degrades vasoactive peptides like angiotensin and enkephalin, suggesting a role in regulating blood pressure.
Area of Science:
- Biochemistry
- Cell Biology
- Vascular Biology
Background:
- Vascular plasma membrane aminopeptidase M (AmM) is an enzyme found in blood vessels.
- Its specific localization and function within different vascular cell types are not fully understood.
Purpose of the Study:
- To investigate the cellular localization of AmM in porcine aorta endothelium and smooth muscle cells.
- To characterize the peptide substrates and kinetic properties of AmM in these cells.
Main Methods:
- Cultured porcine aorta endothelial and smooth muscle cells were used.
- Cellular AmM activity was measured.
- Hydrolysis of various vasoactive peptides by AmM was analyzed.
- Enzyme kinetics, including affinity (Km) and maximal velocity (Vmax), were determined.
- Inhibition studies with specific inhibitors and competing peptides were performed.
Main Results:
- AmM was significantly more abundant in smooth muscle cells (117 units/mg) compared to endothelial cells (8.4 units/mg).
- AmM effectively hydrolyzed kallidin, des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P, and Met5-enkephalin, but not bradykinin, angiotensin II, or substance P.
- The enzyme exhibited highest affinity for des(Asp1)angiotensin I and angiotensin III.
- Hydrolysis rates varied, with Met5-enkephalin showing the highest maximal velocity.
Conclusions:
- Vascular AmM is differentially localized in the aorta, with higher expression in smooth muscle cells.
- AmM plays a role in the metabolism of multiple vasoactive peptides.
- These findings suggest AmM may regulate local concentrations of vasoactive peptides, influencing vascular function.