Effect of the reducing environment on the accumulation of elastin and collagen in cultured smoothmuscle cells

Abstract

We show here that cultured neonatal-rabbit aortic smooth-muscle cells produce and accumulate significant amounts of insoluble elastin. When grown in the presence of ascorbic acid, the amount of insoluble elastin in these cultures decreases, whereas the accumulation of collagen increases. These changes have been attributed to increased hydroxylation of proline in elastin. The function of ascorbic acid in proline hydroxylase, do not demonstrate altered elastin accumulation. These studies are consistent with the molecular iron in the enzyme complex. This study shows that both ascorbic and isoascorbic acids act similarly to modify the accumulation of elastin and collagen in culture. On the other hand, cultures grown in the presence of dithiothreitol, a reducing agent previously shown to act as a cofactor for prolyl hydroxylase, does not demonstrate altered elastin accumulation. These studies are consistent with the suggestion that there is a specific role for ascorbic acid in this cellular system that cannot be replaced by other reducing cofactors. INTRODUCTIONElastin is an insoluble polymeric protein that is responsible for the elastic properties of vertebrate tissues. The insoluble elastin fibre is assembled from soluble precursor molecules termed tropoelastin. Though tropoelastin undergoes few post-translational modifications, one of the best characterized is the hydroxylation of 3-8 %/O of its prolyl residues (Davidson, 1987). The role, if any, of hydroxyproline in tropoelastin is unclear. This is in contrast with hydroxyproline's function in stabilizing the triple-helical confirmation of collagen molecules (Berg & Prockop, 1973;Uitto & Prockop, 1974). L-Ascorbic acid is a cofactor for the enzyme prolyl hydroxylase (EC 1.14.11.2), which catalyses the posttranslational hydroxylation of proline in nascent collagen and elastin molecules (Cardinale & Udenfriend, 1974). In addition, molecular oxygen, ferrous iron and 2-oxoglutarate are necessary cofactors for the normal functioning of proWyl hydroxylase (Adams & Frank, 1980). The biochemical activity of ascorbic acid, a 2-oxolactone, is generally considered to reside in the electroactivity of the keto-enol nature of the C-2 and C-3 atoms. D-Ascorbic acid (isoascorbic acid) is an epimer of Lascorbic acid that differs only by the transposition of the -OH and -H groups at C-5. Since the electroactivity of both molecules is conferred by the keto-enol portion of the ring, both epimers are essentially equivalent in terms of their oxidation-reduction behaviour (Lewin, 1976).The role of ascorbic acid in the mechanism of proline hydroxylation is not completely understood, and is still under investigation (Kiv-irikko & Myllyla, 1987 Udenfriend, 1965;Hutton et al., 1967;Rhoads et al., 1967;Rhoads & Udenfriend, 1970). Later studies with much more highly purified enzyme showed that ascorbic acid was quite specific, though some activity was demonstrated when DTT or L-cysteine was the reductive cofactor (Myllyla et al., 1978). Other investigations have suggested that ascorbic acid...