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Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase
- Source :
- Journal of Molecular and Cellular Cardiology. 51:855-861
- Publication Year :
- 2011
- Publisher :
- Elsevier BV, 2011.
-
Abstract
- In certain cases of endothelial dysfunction l-arginine becomes rate-limiting for NO synthesis in spite of sufficiently high plasma concentrations of the amino acid. To better understand this phenomenon, we investigated routes of substrate supply to endothelial nitric oxide synthase (eNOS). Our previous data with human umbilical vein (HUVEC) and EA.hy.926 endothelial cells demonstrated that eNOS can obtain its substrate from the conversion of l-citrulline to l-arginine and from protein breakdown. In the present study, we determined the quantitative contribution of proteasomal and lysosomal protein degradation and investigated to what extent extracellular peptides and l-citrulline can provide substrate to eNOS. The RFL-6 reporter cell assay was used to measure eNOS activity in human EA.hy926 endothelial cells. Individual proteasome and lysosome inhibition reduced eNOS activity in EA.hy926 cells only slightly. However, the combined inhibition had a pronounced reducing effect. eNOS activity was fully restored by supplementing either l-citrulline or l-arginine-containing dipeptides. Histidine prevented the restoration of eNOS activity by the dipeptide, suggesting that a transporter accepting both, peptides and histidine, mediates the uptake of the extracellular peptide. In fact, the peptide and histidine transporter PHT1 was expressed in EA.hy926 cells and HUVECs (qRT/PCR). Our study thus demonstrates that l-citrulline and l-arginine-containing peptides derived from either intracellular protein breakdown or from the extracellular space seem to be good substrate sources for eNOS.
- Subjects :
- Proteasome Endopeptidase Complex
Nitric Oxide Synthase Type III
Arginine
Endothelium
Leupeptins
Peptide
Nitric Oxide
Umbilical vein
Cell Line
Genes, Reporter
Enos
Lysosome
Human Umbilical Vein Endothelial Cells
medicine
Extracellular
Humans
Histidine
Protease Inhibitors
Molecular Biology
Chromatography, High Pressure Liquid
chemistry.chemical_classification
biology
Membrane Transport Proteins
Biological Transport
Chloroquine
Dipeptides
Atherosclerosis
biology.organism_classification
medicine.anatomical_structure
Biochemistry
chemistry
Proteolysis
Citrulline
Endothelium, Vascular
Lysosomes
Cardiology and Cardiovascular Medicine
Oligopeptides
Subjects
Details
- ISSN :
- 00222828
- Volume :
- 51
- Database :
- OpenAIRE
- Journal :
- Journal of Molecular and Cellular Cardiology
- Accession number :
- edsair.doi.dedup.....9f83e835d90d235a70e3f656181b7ac0