Tetrahydrobiopterin Synthesis Rate and Turnover Time in Neuronal Cultures from Embryonic Rat Mesencephalon and Hypothalamus

Abstract

6-(R)-(L-erythro-1',2'-Dihydroxypropyl)-2-amino- 4-hydroxy-5,6,7,8-tetrahydropteridine (tetrahydrobiopterin, BH4) synthesis rate and turnover time were estimated in cultures derived from the embryonic rat mesencephalon (MES) and hypothalamus (HYP) by following the decline in BH4 levels after blockade of BH4 biosynthesis by N-acetylserotonin (NAS) or 2,4-diamino-6-hydroxypyrimidine (DAHP). BH4 content of both culture systems decreased by 75% following an 8-h incubation with maximally effective concentrations of NAS (200 microM) or DAHP (10 mM). Parameters describing BH4 metabolism were calculated from steady-state levels of BH4 and first-order rate constants determined by a nonlinear regression analysis of the exponential BH4 decline. These parameters were confirmed using an alternative procedure that examined the first-order rate of recovery of BH4 following termination of BH4 synthesis inhibition. Steady-state levels of BH4 in HYP cultures (70.3 +/- 9.4 pg/culture) were significantly greater than that for MES (46.5 +/- 2.8 pg/culture). The average fractional rate constants of BH4 loss for MES (0.153 +/- 0.015/h) and HYP (0.159 +/- 0.014/h) were equivalent. The calculated rate of BH4 synthesis was significantly greater for HYP (11.29 +/- 2.13 pg/culture/h) than for MES (7.11 +/- 0.85 pg/culture/h), owing to the greater steady-state concentration of BH4. BH4 turnover time for MES (6.68 +/- 0.67 h) and HYP (6.40 +/- 0.62 h) and half-life for MES (4.63 +/- 0.46 h) and HYP (4.44 +/- 0.43 h) did not differ. The turnover of the cofactor is thus rapid enough that alterations in its synthesis or degradation could acutely modify the rate of monoamine biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)