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ADENOSINE‐METABOLISING ENZYMES OF SKELETAL MUSCLE VASCULAR ENDOTHELIAL CELLS
Author(s) -
Le Gengyun,
Ballard Heather J
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a483
Subject(s) - adenosine , collagenase , differential centrifugation , adenosine kinase , adenosine monophosphate , 5' nucleotidase , adenosine deaminase , biochemistry , cytosol , nucleotidase , chemistry , microbiology and biotechnology , biology , enzyme
Adenosine is formed by vascular endothelial cells during systemic hypoxia: it is not known whether this adenosine is formed intracellularly by cytosolic‐5′‐nucleotidase (5′N) or extracellularly by ecto‐5′N. We isolated vascular endothelial cells and assayed the adenosine‐metabolising enzymes from them. Rat hindlimb muscles were perfused for 30 min with phosphate‐buffered‐saline and collagenase. The femoral vessels and soleus muscle were removed, minced and digested with collagenase for 45 min at 37°C. Intact cells were separated from cellular debris by differential centrifugation. Endothelial cells were isolated by immunomagnetic binding using CD31 + DynaBeads. The properties of the adenosine‐metabolising enzymes were assayed in a homogenate of this preparation: cytosolic and ecto‐5′N were separated by differential centrifugation. Recovery was 4.9±0.5 × 10 5 cells/ml and 90% of the cells were estimated to be endothelial cells following 12 days of primary culture. Under phase contrast microscopy, the typical cobblestone morphology of endothelial cells was seen. Vmax (nmol/min/mg) of the cytosolic enzymes were: adenosine deaminase, 1.3±0.1; adenosine kinase, 2.4±0.8; 5′N, 136.1±25.6. Ecto‐5′N had a Vmax of 40.6±5.1 nmol/min/mg. These data suggest that most adenosine formation by endothelial cells occurs intracellularly. Funded by the University of Hong Kong/HK Research Grants Council