Biosynthesis of fibronectin by rabbit aorta

• Published in: The Journal of biological chemistry Vol. 266; no. 26; pp. 17686 - 17694
• Main Authors: TAKASAKI, I, CHOBANIAN, A. V, BRECHER, P
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-09-1991
• Subjects: Analytical, structural and metabolic biochemistry; Animals; aorta; Aorta - metabolism; Biological and medical sciences; Cells, Cultured; Fibronectins - biosynthesis; Fundamental and applied biological sciences. Psychology; Glycoproteins; In Vitro Techniques; Male; Muscle, Smooth, Vascular - metabolism; Proteins; Rabbits; Cell culture; Vertebrata; Mammalia; Blotting assay; Rabbit; Aorta; Glycoproteins; Biosynthesis; Lagomorpha; Fibronectin; Endothelium
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)47426-7

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both
cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo
in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta
using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated
with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation
was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most
abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied,
fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed
that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1
h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized
fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble
and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly
synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation
caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent
with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased
following a 24-h preincubation of the aortic rings, and concomitant increases in steady state mRNA for fibronectin were found.
These in vitro studies documented the utility of aortic rings for the general purpose of studying protein synthesis in vascular
cells and provide new information on the characteristics of fibronectin biosynthesis by aortic tissue.