Victor H. Morgenroth

Dopaminergic neurons—Alteration in the kinetic properties of tyrosine hydroxylase after cessation of impulse flow

Abstract Studies in vitro conducted on striatal tyrosine hydroxylase have demonstrated that addition of calcium chelators, such as EGTA, results in an alteration in the kinetic properties of tyrosine hydroxylase which can be completely reversed by addition of calcium to the incubation medium. Inhibition of impulse flow in the nigro-neostriatal pathway by either pharmacological or mechanical techniques causes the isolated striatal tyrosine hydroxylase to behave kinetically like the enzyme treated in vitro with EGTA. Tyrosine hydroxylase, isolated from the striatum of rats in which impulse flow has been interrupted in the dopamine pathways, has an increased affinity for both substrate and pterin cofactor and a reduced affinity for the end-product inhibitor, dopamine. The latter change is most dramatic; the K i of the enzyme for dopamine. increases more than 700-fold. These alterations in the kinetic properties of tyrosine hydroxylase can also be completely reversed by addition of calcium to the incubation medium. In rats with a pharmacological blockade of impulse flow induced by administration of γ-hydroxybutyrate, subsequent administration of dopamine receptor stimulants causes the isolated tyrosine hydroxylase to have properties similar to those observed for the enzyme prepared from untreated rats. Administration of dopamine receptor blockers prevents the dopamine receptor stimulants from altering the kinetic changes in tyrosine hydroxylase induced by a cessation of impulse flow. These data are discussed in terms of the possible role presynaptic receptors and calcium fluxes may play in the short-term regulation of tyrosine hydroxylase.

Noradrenergic neurons: allosteric activation of hippocampal tyrosine hydroxylase by stimulation of the locus coeruleus.

Robert H. Roth, Phyllis M. Salzman and Victor H. Morgenroth, III Departments of Pharmacology and Psychiatry Yale University School of Medicine New Haven, Connecticut 06510 Communicated by: Parkhurst A. Shore (Received B July 1974; accepted 23 July 1974) In peripheral noradrenergic neurons an increase in impulse flow results in an acceleration of norepinephrine biosynthesis. This increase in transmitter synthesis is due to an increase in the activity of the rate limiting enzyme tyrosine hydroxylase. Until recently this increase in tyrosine hydroxylase activity was thought to arise as a result of the removal of endproduct inhibition subsequent to the depletion of a small pool of norepinephrine which has access to tyrosine hydroxylase (1). Recent experiments in our laboratory, however, have suggested that the post-stimulation increase in tyrosine hydroxylase activity observed in sympathetic nerve endings after electrical stimulation of noradrenergic nerves occurs as a result of an allosteric activation of tyrosine hydroxylase (2). This allosteric activation appears to be mediated by an increase in affinity of the enzyme for both substrate and pteridine cofactor and a decreased affinity of the enzyme for the endproduct inhibitor norepinephrine, It is generally assumed that noradrenergic neurons in the central nervous system behave in a fashion quite similar to the postganglionic sympathetic noradrenergic neurons in the periphery. In fact, it has recently been demonstrated that stimulation of central noradrenergic neurons results in an increase in the turnover of norepinephrine as well as an increase in the accumu-

Activation of tyrosine hydroxylase in rat striatal slices by K+-depolarization— Effect of ethanol

Abstract Slices from rat corpus striatuum were incubated for 10 min at 37° in freshlyy oxygenated Krebs-Ringer phosphate (KRP) media or KRP-high K + (55mM) media both in the presence and absence of ethanol (0.2 to 0.8%, w/v). Thereafter, the slices were homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 105,000 g supernatant fraction. The presence of K + (55 mM) in the incubation media increased about 3-fold the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, when compared to that found in striatal slices incubated in normal KRP media. Incubation of striatal slices in a KRP-high K + media also produced changes in the kinetic properties of tyrosine hydroxylase. The K m of the enzyme for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine HCl (DMPH 4 ) was decreased from 0.82 to 0.09 mM and the K i of the enzyme for dopamine (DA) was increased from 0.13 to 3.52 mM. Ethanol (0.2 to 0.8%, w/v) added directly to the KRP-high K + media markedly blocked the K + -induced activation of tyrosine hydroxylase as well as the kinetic alterations in the enzyme observed after K + -depolarization of the striatal slices. In contrast, the presence of ethanol did not modify the activity and kinetic characteristics of tyrosine hydroxylase isolated from slices incubated in normal KRP media. The results reported in this work suggest that the increase in DA synthesis observed in striatal slices after K + -depolarization might be mediated in part via an allosteric activation of tyrosine hydroxylase. This activation appears to be mediated by an increase in the affinity of the enzyme for the pterin cofactor and a decreased affinity for the end-product inhibitor DA. Also, the blocking effect of ethanol upon the kinetic activation of tyrosine hydroxylase after K + -depolarization seems to offer a likely explanation for the inhibitory effect of ethanol on K + -induced increase in DA synthesis reported recently by Gysling et al. (Biochem. Pharmac. 25 , 157 (1976)).

Tyrosine hydroxylase activation and transmitter release from central noradrenergic neurons by electrical field stimulation

SummaryElectrical stimulation of the noradrenergic neurons in the locus coeruleus of the rat results in a marked increase in the tyrosine hydroxylase activity of the hippocampus on the stimulated side (Roth et al., 1975). We have developed an in vitro system to further study this interesting phenomenon. Rat hippocampal slices were stimulated in an electrical field using specially built chambers which permitted a continous superfusion with Krebs Ringer Phosphate solution while stimulation was taking place. The slices were then homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 104000 g supernate fraction. Electrical field stimulation (60 V, 4 ms, 5–20 Hz, 5 min) induced a stimulus-dependent increase in tyrosine hydroxylase activity. The increase in tyrosine hydroxylase activity was directly dependent on the number of pulses of stimulation applied. Potassium depolarization of hippocampal slices also resulted in a marked increase in the activity of the enzyme. Electrical field stimulation appears to activate tyrosine hydroxylase by causing an increase in its affinity for both substrate and pteridine cofactor and by decreasing its affinity for the endproduct inhibitor, norepinephrine. No change in Vmax was observed. The superfusion system was also used to study spontaneous and electrically evoked release of labelled norepinephrine from hippocampal slices. Electrical field stimulation for 1.0 min produced a marked increase in the release of label. Absence of calcium from the superfusion solution almost completely abolished the electrically evoked release of exogenously taken up 3H-norepinephrine. The release of 3H-norepinephrine was also found to be dependent on the number of pulses of stimulation used. The results reported support the hypothesis that depolarization of central noradrenergic neurons results in an increase in transmitter synthesis mediated in part via a kinetic activation of tyrosine hydroxylase. The concomitant use of superfusion and electrical field stimulation of hippocampal slices provides a simple system to study neurotransmitter synthesis and release in central noradrenergic neurons.

Sympathetic neurotransmitter metabolism in Hirschsprung's disease

Tyrosine hydroxylase activity was measured in high speed supernatants obtained from full thickness segments of aganglionic and ganglionic colon of three children with Hirschsprungs disease. Tyrosine hydroxylase activity expressed as pmole DOPA/mg protein/min was 0.93 +/- 0.16 in ganglionic and 2.67 +/- 0.21 in aganglionic colon. Tyrosine hydroxylase activity in ganglionic colon rose to 2.29 +/- 0.11 following calcium stimulation (100 muM) but could not be further increased in aganglionic colon. Addition of norepinephrine (2 X 10(-4) M) to tissue homogenates inhibited tyrosine hydroxylase activity in ganglionic colon by 57 +/- 8% but only by 14 +/- 3% in aganglionic colon, suggesting that the enzyme present in aganglionic colon is insensitive to feedback inhibition by endogenous norepinephrine. The elevation of tyrosine hydroxylase activity in aganglionic colon and its insensitivity to calcium stimulation and norepinephrine inhibition is further evidence of sympathetic overactivity in the aganglionic colon and suggests a basic enzymatic abnormality in the pathogenesis of Hirschsprungs disease.