INSULIN-LIKE GROWTH FACTOR-I ENHANCES TYROSINE-HYDROXYLASE ACTIVATION IN BOVINE CHROMAFFIN CELLS

Previous studies have shown that insulin-like growth factor-I (IGF-I) enhances secretagogue-stimulated Ca2+ uptake and catecholamine release in bovine chromaffin cells. This report describes the effect of IGF-I on the activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2), the major regulatory enzyme in the pathway of catecholamine biosynthesis. Tyrosine hydroxylase activity was assayed by measuring 3,4-dihydroxyphenylalanine (Dopa) accumulation in the presence of brocresine, an inhibitor of Dopa decarboxylase. Chromaffin cells cultured in serum-free medium produced approximately 40% less Dopa when stimulated by 55 mM K+ than did cells that had been cultured in the presence of serum. Incubation of cells for 3 days in serum-free medium containing 10 nM IGF-I restored high K+-stimulated Dopa accumulation to a level comparable to that seen in cells cultured continuously in serum-containing medium. In eight experiments, IGF-I increased high K+-stimulated Dopa accumulation (expressed as picomoles per minute per milligram of protein) by 96 +/- 13%. IGF-I increased the protein content of chromaffin cells by approximately 30%; consequently, its effect on tyrosine hydroxylase activity was even greater when Dopa synthesis was expressed as picomoles per minute per 10(7) cells. IGF-I also enhanced the rate of Dopa accumulation in cells stimulated by dimethylphenyl-piperazinium, 8-bromo-cyclic AMP, phorbol 12,13-dibutyrate, or Ba2+. The effect of IGF-I on high K+-stimulated tyrosine hydroxylase activity was measurable when enzyme activity was assayed in vitro, suggesting that this effect was due to a stable modification of the enzyme. Immunoblotting studies indicated that cells cultured in the presence of IGF-I had a small selective increase (36 +/- 12%) in the amount of tyrosine hydroxylase per unit of protein. The effect of IGF-I on tyrosine hydroxylase activity and catecholamine synthesis appears to be due to an integrated response of the cells to this agonist: IGF-I increases the amount of tyrosine hydroxylase in the cell and promotes the activation of this enzyme by a variety of agents.