PYRIDINE-NUCLEOTIDE LEVELS UNDER CONDITIONS OF 5-ALPHA-DIHYDROTESTOSTERONE-STIMULATED 17-BETA-ESTRADIOL FORMATION FROM ESTRONE AND PATHWAY OF NICOTINAMIDE ADENINE-DINUCLEOTIDE BIOSYNTHESIS IN PLACENTAL VILLI INVITRO

It has been reported that testosterone, 5-alpha-dihydrotestosterone and 20-alpha-dihydroprogesterone, substrates for pyridine nucleotide-dependent hydroxysteroid oxidoreductases, stimulate the conversion of estrone to 17-beta-estradiol (E2) by placental villi in vitro. Their enzyme-catalyzed oxidation generates either NADH or NADPH. If the concentration of either reduced nucleotide were rate determining in the conversion of estrone to E2, then increases in NADH or NADPH levels as the result of steroid oxidation could stimulate E2 formation. In this investigation, enzymatic assays were used to quantitate NAD, NADP, NADH, and NADPH in villous tissue from term placenta under conditions where E2 formation was stimulated by 5-alpha-dihydroxytestosterone. On the basis of concurrent observations that NAD levels varied initially and decreased in tissue samples in culture over a 24-h period, the ability of villi to incorporate [C-14]nicotinic acid or [C-14]nicotinamide into NAD was also examined. No changes were detected in the ratios of oxidized to reduced [C-14]nicotinamide nucleotides under stimulatory conditions. NAD was formed only from nicotinic acid. The data suggest that NAD and NADP reduction, if it is the basis for stimulation, is tightly coupled to reoxidation. It would also appear that media used widely for villous tissue and cell culture may not be optimal for pyridine nucleotide synthesis.