Induction of tyrosine hydroxylase by nerve growth factor and by elevated K+ concentrations in cultures of dissociated sympathetic neurons

Tyrosine hydroxylase (TH) is induced by nerve growth factor (NGF) as well as by elevated K’ concentrations in cultures of sympathetic neurons dissociated from superior cervical ganglia of newborn rats. The two inductions were found to be mediated by different mechanisms. Cultures of sympathetic neurons required the continuous presence of 10 rig/ml of 2.5 S NGF for maximal survival. Increasing the NGF concentration in cultures grown at 10 rig/ml for 5 days to 100 to 1,000 rig/ml for an additional 2 to 3 days resulted in an elevation of the specific TH activity by 50 to 100%. Immunotitration demonstrated that the elevated TH activity was due to an increased number of enzyme molecules. The fact that the NGF-mediated TH induction was blocked by cycloheximide but not by a-amanitin suggests a regulation at the post-transcriptional level. Both dibutyryl CAMP and cholera toxin failed to elevate the activity of TH, indicating that CAMP does not act as a second messenger. The same is true for Ca’+, since the NGF-mediated TH induction was neither reduced by an inhibitor of transmembrane Ca2+ flux (D-600) nor by an inhibitor of cahnodulin (trifluoperazine) . Exposing cultures grown at 1,000 ng of NGF/ml for 1 to 3 days to medium with elevated K’ concentrations (55 mM) resulted in a further increase of TH activity by about 100%. Stimulation was maximal when normal medium was replaced by medium with elevated K’ concentrations 7 days after plating; no inductioin was observed when cultures were switched to elevated K’ concentrations later than 14 days thereafter. As for NGF, the K+-mediated TH increase was due to an augmented number of enzyme molecules (immunotitration). In contrast to the NGF-mediated induction, the K+-mediated induction appeared to be regulated at the transcriptional level, since it was markedly reduced by a-amanitin. The extent of TH induction by increasing concentrations of K+ paralleled the reduction in membrane potential as assessed by the uptake of methyltriphenylphosphonium. Reduction of the Ca2’ concentrations in the medium had no influence on TH induction. However, it was inhibited by D-600, Mg2+, and diphenylhydantoin, substances interfering with transmembrane Ca2+ fluxes. Moreover, TH induction was blocked by trifluoperazine and felodipine, drugs interfering selectively with cahnodulin-mediated processes. In electron micrographs of cells grown at elevated K+ concentrations, the incidence of mitochondria containing calcium precipitates was reduced by 40%, suggesting that the K+-induced depolarization interfered with the mitochondrial sequestration of Ca’+. Nerve growth factor (NGF) is a protein which plays a central role in the development and maintenance of function of sympathetic and sensory neurons of the peripheral nervous system. One of the most characteristic ‘Supported by a fellowship from the Alexander von HumboldtStiftung, Bonn, Federal Republic of Germany. Present address: Preclinical Research, Sandoz AG, CH-4002, Basel, Switzerland. * To whom correspondence should be addressed at Department of Neurochemistry, Max Planck Institute for Psychiatry, D-8033 Martinsried, Federal Republic of Germany. 155 effects of NGF on sympathetic neurons is the selective induction of tyrosine hydroxylase (TH) and dopamine ,L&hydroxylase (DBH), enzymes necessary for the synthesis of norepinephrine (for review, see Thoenen and Barde, 1980). After the administration of NGF to newborn or adult rats, the activity of these enzymes is elevated in sympathetic ganglia and in the adrenal medulla (Thoenen et al., 1971; Otten et al., 1977). NGF selectively induces TH and DBH, whereas dopa decarboxylase, the third enzyme involved in norepinephrine synthesis, increases only to the extent of the NGF-mediated hyper-