Tetrahydrobiopterin Turnover in Cultured Rat Sympathetic Neurons: Developmental Profile, Pharmacologic Sensitivity, and Relationship to Norepinephrine Synthesis

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Abstract
We have examined the turnover of 5,6,7,8-tetrahydrobiopterin (BH4) and the effect of decreasing BH4 levels on in situ tyrosine hydroxylase (TH) activity and norepinephrine (NE) content in a homogeneous population of NE-containing neurons derived from the superior cervical ganglion (SCG) of the neonatal rat and maintained in tissue culture. Initial studies indicated that the level of BH4 within SCG cultures increased fourfold between 5 and 37 days in vitro (DIV). This increase in BH4 levels was determined to result from an increase in the rate of BH4 biosynthesis without a change in the rate of degradation. Regardless of culture age, the BH4 content of SCG neurons was observed to turn over with a half-life of ∼2.5 h. BH4 synthesis by SCG neurons was found to be five times more sensitive to inhibition by 2,4-diamino-6-hydroxypyrimidine (DAHP) and 25 times less sensitive to inhibition by N-acetylserotonin than was previously reported for CNS neurons in culture. Under basal conditions, the rates of in situ TH activity and BH4 biosynthesis were similar. In response to inhibition of BH4 biosynthesis by DAHP and a 90–95% decrease in BH4 levels, in situ TH activity declined by 75%. NE levels declined by 30% following a 24-h period of inhibition of BH4 synthesis. After 2 days of BH4 synthesis inhibition, the level of NE was decreased by 47%. On treatment days 3 and 4, the decline in NE content plateaued at 24% of control levels. In contrast, treatment of cultures for 24 h with the direct-acting inhibitor of TH, α-methyl-p-tyrosine, produced an 84% decline in NE content that was maintained over the 4-day treatment period, indicating that the slow decline in NE content following inhibition of BH4 synthesis was not the result of the slow turnover rate of NE. These results demonstrate that despite an almost complete loss of BH4, sympathetic neurons were able to maintain neurotransmitter content, albeit at reduced levels, by retaining a level of TH activity above the value that might have been predicted based on the reduced level of BH4.

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