Carboxypeptidase D Is Up-Regulated in RAW 264.7 Macrophages and Stimulates Nitric Oxide Synthesis by Cells in Arginine-Free Medium
- Departments of Pharmacology and Anesthesiology, University of Illinois College of Medicine at Chicago, Chicago, Illinois
Abstract
Membrane-bound carboxypeptidase D (CPD) is a B-type carboxypeptidase that specifically cleaves C-terminal Arg or Lys from peptides and proteins. RAW 264.7 cells contained significant membrane-bound CPD activity as shown by activity assays and immunoprecipitation. To determine whether CPD can increase nitric oxide (NO) synthesis by releasing precursor Arg, cells were activated in Arg-free medium with 50 U/ml interferon-γ (IFN-γ) and 0.1 μg/ml lipopolysaccharide (LPS) to up-regulate inducible NO synthase. Addition of the specific carboxypeptidase substrate, 200 μM furylacryloyl-Ala-Arg, stimulated NO production by 6-fold and this effect was blocked 83% by a specific inhibitor,dl-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA). MGTA did not inhibit NO synthesis stimulated by added free Arg. Lys, an inhibitor of Arg transport, also blocked the effect of the carboxypeptidase substrate. In cells stimulated with IFN-γ and LPS in Arg-free medium, CPD activity increased 2- to 3-fold between 8 and 16 h after treatment, but did not change in cells stimulated in medium containing 0.4 mM Arg. The NO synthase inhibitorN-monomethyl-l-arginine blocked the inhibitory Arg effect and the NO donorS-nitroso-acetylpenicillamine mimicked it, indicating that high levels of NO block the up-regulation of CPD. Immunohistochemical staining and Western analysis revealed an increase in CPD protein, and Northern analysis showed increased CPD mRNA upon stimulation of cells in Arg-free medium. CPD was localized both on the plasma membrane and in the Golgi. These data suggest that CPD expression is enhanced during inflammatory processes and may stimulate NO production by cleaving Arg from peptide substrates.
Footnotes
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Send reprint requests to: Randal A. Skidgel, Ph.D., Department of Pharmacology (m/c 868), University of Illinois College of Medicine, 835 S. Wolcott, Chicago, IL 60612. E-mail: rskidgel{at}uic.edu
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These studies were supported by National Heart Lung and Blood Institute Grant HL60678 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK41431.
- Abbreviations:
- NOS
- nitric-oxide synthase
- NO
- nitric oxide
- iNOS
- inducible nitric-oxide synthase
- LPS
- bacterial lipopolysaccharide
- IFN-γ
- interferon-γ
- CP
- carboxypeptidase
- DMEM
- Dulbecco's modified Eagle's medium
- MGTA
- dl-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid
- dansyl-Ala-Arg
- 5-dimethylaminonaphthalene-1-sulfonyl-L-alanyl-l-arginine
- Fa-Ala-Arg
- furylacryloyl-l-alanyl-l-arginine
- BSA
- bovine serum album
- SSC
- standard saline citrate
- PBS
- phosphate-buffered saline
- l-NMA, N-monomethyl-l-arginine
- NF-κB, nuclear factor-κB
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- Received August 31, 2000.
- Accepted February 26, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
