Y-1 adrenal cortical
tumor cells in culture, which contain substantial amounts of
tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-
tetrahydropterin] (BH4) and
GTP cyclohydrolase (
GTP-CH), were used to study the regulation of BH4 biosynthesis by
ACTH and cAMP.
ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and
GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of
ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by
ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by
ACTH at higher cell densities was due to an increase in the specific activity of
GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of
steroids, since exogenous
steroids added to low density cultures also resulted in an increase in the specific activity of the
enzyme. Addition of
steroids had no effect on
ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP,
8-bromo-cAMP, and
forskolin all produced time- and dose-dependent increases in BH4 levels,
GTP-CH activity, and steroidogenesis. Maximum increases in
GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1
cAMP-dependent protein kinase with an altered regulatory subunit,
ACTH was unable to increase BH4 levels or
GTP-CH activity at a concentration that produced maximal stimulation of BH4 and
steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.