Browsing by Author "ROTH, RH"

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    ACTIVATION OF TYROSINE-HYDROXYLASE IN RAT STRIATAL SLICES BY K+-DEPOLARIZATION - EFFECT OF ETHANOL
    (1976) BUSTOS, G; ROTH, RH; MORGENROTH, VH
    Slices from rat corpus striatum were incubated for 10 min at 37.degree. C in freshly oxygenated Krebs-Ringer phosphate (KRP) media or KRP-high K+ (55 mM) media both in the presence and absence of ethanol (0.2-0.8%, wt/vol). 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 Km of the enzyme for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine HCl (DMPH4) was decreased from 0.82 to 0.09 mM and the Ki of the enzyme for dopamine (DA) was increased from 0.13 to 3.52 mM. Ethanol (0.2-0.8%, wt/vol) added directly to the KRP-high K+ media markedly blocked the K+-induced activation of tyrosine hydroxylase and 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 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 appeared 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. The blocking effect of ethanol upon the kinetic activation of tyrosine hydroxylase after K+-depolarization seemed to offer a likely explanation for the inhibitory effect of ethanol on K+-induced increase in DA synthesis reported by Gysling et al.
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    CHANGES IN EXTRACELLULAR LEVELS OF GLUTAMATE AND ASPARTATE IN RAT SUBSTANTIA-NIGRA INDUCED BY DOPAMINE-RECEPTOR LIGANDS - IN-VIVO MICRODIALYSIS STUDIES
    (1995) ABARCA, J; GYSLING, K; ROTH, RH; BUSTOS, G
    The microdialysis technique was utilized to study the local effects of D-1 and D-2 family type dopamine (DA) receptor (R) ligands on the in vivo release of endogenous glutamate (GLU) and aspartate (ASP) from rat substantia nigra (SN). Addition to the dialysis perfusion solution of either D-1-R and D-2-R agonists, such as SKF-38393 (50 and 100 mu M) and Quinpirole (5 and 10 mu M), resulted in dose-dependent increases in extracellular concentrations of GLU and ASP, respectively. The SKF-38393 and Quinpirole-induced effects were reduced by SCH-23390 (0.5 mu M), a D-1-R antagonist, and by Spiperone (1.0 mu M), a D-2-R antagonist, respectively. However, SCH-23390 and Spiperone did increase GLU and ASP extracellular concentrations. Local infusion with Tetrodotoxin (TTX) (1.0 mu M), a blocker of voltage-dependent Na+ channels, increased basal extracellular levels of GLU. In addition, co-infusion of TTX and SKF-38393 evoked increases in extracellular GLU levels higher than those observed after SKF-38393 alone. Finally, chemical lesions of nigral DA cells with 6-OH-DA. increased the basal extracellular levels of GLU. It is proposed that the release of GLU and ASP from SN may be regulated by D-1- and D-2- receptors present in this basal ganglia structure. In addition, part of the D-1 receptors present in SN might be located presynaptically on GLU-containing nerve endings.
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    TYROSINE-HYDROXYLASE ACTIVATION AND TRANSMITTER RELEASE FROM CENTRAL NORADRENERGIC NEURONS BY ELECTRICAL-FIELD STIMULATION
    (1978) BUSTOS, G; MORGENROTH, VH; HANCKE, JL; ROTH, RH
    Electrical 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). An in vitro system was developed to further study this interesting phenomenon. Rat hippocampal slices were stimulated in an electrical field using specially built chambers which permitted a continuous 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 [voltage], 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. K 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 labeled norepinephrine from hippocampal slices. Electrical field stimulation for 1.0 min produced a marked increase in the release of label. Absence of Ca 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 hypothesis that depolarization of central noradrenergic neurons results in an increase in transmitter synthesis mediated in part via a kinetic activation of tyrosine hydroxylase is supported. 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.