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INNOVATIVE METHODOLOGY

Isolation and characterization of coronary endothelial and smooth muscle cells from A₁ adenosine receptor-knockout mice

¹Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and ²National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Submitted 3 August 2005 ; accepted in final form 10 November 2005

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A₁ receptor (A₁AR)-knockout (A₁KO) mice and their wild-type (A₁WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle -actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A₁WT and A₁KO mice. Furthermore, the A₁AR was characterized by Western blot analysis using an A₁AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.

coronary artery; endothelial cells; smoothelin