Use of 6-diazo-5-oxo-L-norleucine to study interaction between myocardial glycoconjugate secretion and endothelial activation in the early embryonic chick heart

Abstract

The glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), a glycoconjugate inhibitor, was used to probe the relationships between myocardial secretion of extracellular matrix and endothelial differentiation and formation of cushion mesenchyme (primordia of A V values). When DON was given to stage 12 chick embryos maintained in shell-less culture, the myocardial secretion gradient of glucose- and sulfate-labeled matrix was blocked. Concomitantly, the endothelium failed to complete activation but continued to divide and incorporate thymidine. By varying DON concentration, two distinct phases of endothelial differentiation were identified: the first (labile to 0.5 micrograms) involved hypertrophy, the second (labile to 0.25 micrograms) acquisition of migratory appendages with resultant mesenchyme formation. Glucosamine + DON (but not inosine, glucose, or glutamine) restored the matrical secretion gradient and to varying degrees both phases of endothelial activation. Endothelia totally suppressed from forming mesenchyme in situ acquired this capacity when explanted into three-dimensional collagen gel culture. The capacity was enhanced by glucosamine given in situ as an inhibitory override, dependent upon serum concentration, inhibited by heat-inactivated serum or by adding DON to the medium, but unaffected by hyaluronate. These results were compared to those obtained by co-culturing endothelium and myocardium and discussed in terms of the hypothesis that cushion mesenchyme formation results from an epithelial interaction mediated by glycoconjugates.