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

Bunyen Teng, Habib R. Ansari, Peter J. Oldenburg, J. Schnermann, S. Jamal Mustafa

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

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 (A 1WT) 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 α-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.

Original languageEnglish (US)
Pages (from-to)H1713-H1720
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume290
Issue number4
DOIs
StatePublished - Apr 1 2006

Keywords

  • Coronary artery
  • Endothelial cells
  • Smoothelin

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

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