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 Citations (Scopus)

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)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume290
Issue number4
DOIs
StatePublished - Apr 1 2006

Fingerprint

Adenosine A1 Receptors
Knockout Mice
Smooth Muscle Myocytes
Anti-Bacterial Agents
Thoracic Aorta
Endothelial Cells
Western Blotting
Staining and Labeling
Smooth Muscle Myosins
Trypsin Inhibitors
Myosin Heavy Chains
Collagenases
Soybeans
Smooth Muscle
Actins
Antibodies
Enzymes
Serum
Research

Keywords

  • Coronary artery
  • Endothelial cells
  • Smoothelin

ASJC Scopus subject areas

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

Cite this

Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice. / Teng, Bunyen; Ansari, Habib R.; Oldenburg, Peter J; Schnermann, J.; Mustafa, S. Jamal.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 290, No. 4, 01.04.2006.

Research output: Contribution to journalArticle

@article{a8a72ae8208241eb86a88a4569cb63cb,
title = "Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice",
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.",
keywords = "Coronary artery, Endothelial cells, Smoothelin",
author = "Bunyen Teng and Ansari, {Habib R.} and Oldenburg, {Peter J} and J. Schnermann and Mustafa, {S. Jamal}",
year = "2006",
month = "4",
day = "1",
doi = "10.1152/ajpheart.00826.2005",
language = "English (US)",
volume = "290",
journal = "American Journal of Physiology - Renal Physiology",
issn = "0363-6127",
publisher = "American Physiological Society",
number = "4",

}

TY - JOUR

T1 - Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

AU - Teng, Bunyen

AU - Ansari, Habib R.

AU - Oldenburg, Peter J

AU - Schnermann, J.

AU - Mustafa, S. Jamal

PY - 2006/4/1

Y1 - 2006/4/1

N2 - 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.

AB - 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.

KW - Coronary artery

KW - Endothelial cells

KW - Smoothelin

UR - http://www.scopus.com/inward/record.url?scp=33646096754&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33646096754&partnerID=8YFLogxK

U2 - 10.1152/ajpheart.00826.2005

DO - 10.1152/ajpheart.00826.2005

M3 - Article

VL - 290

JO - American Journal of Physiology - Renal Physiology

JF - American Journal of Physiology - Renal Physiology

SN - 0363-6127

IS - 4

ER -