Journal
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
Volume 290, Issue 4, Pages H1713-H1720Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpheart.00826.2005
Keywords
coronary artery; endothelial cells; smoothelin
Funding
- NHLBI NIH HHS [R01 HL027339, HL-027339, R01 HL094447] Funding Source: Medline
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Mice have been used widely in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)- knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1mg/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 alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was similar to 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.
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