Presynaptic Dopamine Autoreceptors Control Tyrosine Hydroxylase Activation in Depolarized Striatal Dopaminergic Terminals

The possible control of tyrosine hydroxylase (TH) activity by dopaminergic receptor-dependent mechanisms was investigated using rat striatal slices or synaptosomes incubated in the presence of various 3,4-dihydroxyphenylethylamine (dopamine or DA) agonists and antagonists. Under "normal" conditions (4.8 mM K+ in the incubating medium), the DA agonists apomorphine, 6,7-dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99), 7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT), Trans-(-)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-2H-pyrazolo-3,4- quinoline, and 3-(3-hydroxyphenyl)-N-n-propylpiperidine decreased TH activity in soluble extracts of incubated tissues. In the case of the catechol-containing drugs apomorphine and TL-99, this effect was partly due to a direct inhibition of the enzyme, but in all other cases it appeared to depend on the stimulation of presynaptic DA autoreceptors. No effect of DA antagonists was detected on TH activity under "normal" conditions. In contrast, when tissues were incubated in a K+ -enriched (60 mM) medium, (-)-sulpiride and other DA antagonists enhanced TH activation due to depolarization whereas DA agonists were ineffective. Because (-)-sulpiride also increased the enzyme activity in striatal slices exposed to drugs inducing release of DA, such as veratridine and d-amphetamine, it is concluded that the stimulating effect of the DA antagonist resulted in fact from the blockade of the negative control of TH normally triggered by endogenous DA acting on presynaptic autoreceptors. In contrast to TH activation due to K+ -induced depolarization, the activation evoked by tissue incubation with dibutyryl cyclic AMP was unaffected by the typical agonist 7-OH-DPAT or the antagonist (-)-sulpiride. This would suggest that TH control via presynaptic DA autoreceptors normally concerns possible modulations of the cyclic AMP-dependent phosphorylation of the enzyme.     

Further Studies on the Nature of Postsynaptic Dopamine Uptake and Metabolism in Rat Striatum: Sodium Dependency and Investigation of a Possible Role for Carrier-Mediated Uptake into Serotonin Neurons

The nature of postsynaptic sites involved in the uptake and metabolism of striatal 3,4-dihydroxyphenylethylamine (dopamine, DA) was investigated. The accumulation of [3H]DA (10(-7) M) into slices of rat striatum was found to be greatly dependent (greater than 99%) on the presence of sodium ion in the incubation medium. However, the formation of the [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) was only partially reduced in the absence of sodium (DOPAC, 27% of control; HVA, 47% of control). Inhibition of carrier-mediated DA neuronal uptake with nomifensine (10(-5) M) significantly decreased DA accumulation (18% of control) and [3H]DOPAC formation (62% of control), but enhanced [3H]HVA production (143% of control). Inhibition of the 5-hydroxytryptamine (5-HT, serotonin) neuronal uptake system with fluoxetine (10(-6) M) or selective 5-HT neuronal lesions with 5,7-dihydroxytryptamine (5,7-DHT) had no effect on [3H]DOPAC or [3H]HVA formed from [3H]DA in the presence or absence of nomifensine. These results demonstrate that the uptake and subsequent metabolism of striatal DA to DOPAC and HVA is only partially dependent on carrier-mediated uptake mechanism(s) requiring sodium ion. These data support our previous findings suggesting a significant role for synaptic glial cell deamination and O-methylation of striatal DA. Further, experiments with fluoxetine or 5,7-DHT suggest that 5-HT neurons do not significantly contribute in the synaptic uptake and metabolism of striatal DA.     

Drug Potencies on Partially Purified Brain D2 Dopamine Receptors

To examine the sensitivities of partially purified dopamine receptors to various dopaminergic agonists and antagonists, canine brain striatum dopamine receptors were enriched by isoelectric focusing. The digitonin-solubilized receptors were prelabelled with [3H]spiperone and focused for two time periods. After 5 h (incomplete focusing), radioactive peaks were detected at pH 6 and 9-11. Only the pH 6 peak revealed drug sensitivities expected of D2 receptors. Receptor recovery of the pH 6 peak was 79% with purification being sevenfold. After focusing overnight to equilibrium, the pH 6 peak further separated into peaks at pH 4.6 and 6.8. The receptor was identified only in the pH 4.6 fraction. The recovery of receptors in the pH 4.6 peak was low (10%), indicating little enrichment of the receptor. The rank order of binding of neuroleptics and dopamine agonists to the purified material was similar to that of the original preparation of soluble receptors. Dopamine did not bind to the purified pH 4.6 fraction unless the phosphate buffer (used during focusing) was replaced with Tris buffer. The absence of receptors in the pH 6.8 and pH 10 fractions, although both contained prelabeled [3H]spiperone, indicates the importance of testing agonists and antagonists on each fraction at each step in purification.     

Influence of δ-Opioid Receptors on Production of Labeled Methionine5-Enkephalin in Murine Neuroblastoma Cells

Synthesis of methionine5-enkephalin by intact cells of murine neuroblastoma clone N1E-115 has been demonstrated both immunocytochemically and biochemically. In addition, N1E-115 cells possess homogeneous enkephalin (delta) receptors which inhibit prostaglandin E1-induced intracellular cyclic AMP formation. An assay was developed for measuring de novo synthesis of methionine5-enkephalin by pulsing cells in culture with radioactive methionine and isolating this pentapeptide to radiochemical purity by a procedure that included immunoaffinity chromatography specific for oxidized methionine5-enkephalin. This assay indicated that production of radiolabeled-methionine5-enkephalin was increased upon lengthy exposure of intact N1E-115 cells in the late logarithmic phase of growth to a nonproteolyzable analog of methionine5-enkephalin. This increase in synthesis of intracellular methionine5-enkephalin relative to control cells was prevented by prior incubation of the clone with naloxone, indicating that the response was mediated by the delta receptor.     

Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1-Methyl-4-Phenylpyridinium Ion

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.     

Dopamine Sulfoconjugation in the Rat Brain: Regulation by Monoamine Oxidase

An increase of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain such as is found following 3,4-dihydroxyphenylalanine (L-DOPA) administration or an intraventricular injection of free dopamine did not result in DA sulfate formation, despite the presence of phenolsulfotransferase activity in various regions of the brain and the high affinity of DA for this enzyme. However, when rats were pretreated with pargyline, a monoamine oxidase inhibitor, the same treatment with L-DOPA or free DA led to active synthesis of DA sulfate. The increase in DA sulfate was significantly correlated with the degree of monoamine oxidase inhibition and directly proportional to free DA concentrations in the hypothalamus (r = 0.86), striatum (r = 0.54), and brainstem (r = 0.89). The highest ratio of DA sulfate to free DA was found in the hypothalamus, suggesting that sulfoconjugation is most active in this region. Prior treatment of rats with 6-hydroxydopamine did not decrease DA sulfate concentrations, indicating that sulfoconjugation occurs most likely in extraneuronal tissues not destroyed by the neurotoxin. The results are compatible with the notion that phenolsulfotransferase may be highly compartmentalized and that inhibition of monoamine oxidase allows the newly generated free DA to become accessible to the sulfoconjugating enzyme, resulting in increase in DA sulfation.     

Association of Serotonin, Dopamine, or Noradrenaline with an Actin-Like Component in Pheochromocytoma (PC12) Cells

A rat pheochromocytoma (PC12) cell line was used to examine the possibility that 5-hydroxytryptamine (serotonin), 3,4-dihydroxyphenylethylamine (dopamine), or noradrenaline may be associated with cytoplasmic actin, as was suggested by previous in vitro binding studies on an actin-like protein from rat brain synaptosomes. When PC12 cells were incubated with [3H]serotonin. [3H]dopamine, or [3H]noradrenaline for 30 min at 37 degrees C, approximately 2-4% of the radioactivity present in the cells was found to be associated with a high-molecular-weight (actin-like) component in supernatant fractions. Evidence relating this monoamine binding component to actin filaments includes: (a) its strong absorption by myosin filaments at low ionic strength: (b) a decrease in its affinity for myosin in the presence of 1 mM ATP, which lowers the affinity of authentic actin for myosin: (c) displacement of bound [3H]serotonin from it by DNase I, which binds strongly to actin and which inhibits [3H]serotonin binding to actin in vitro; (d) an increase in its binding of each monoamine (by 25-40%) after PC12 cells were preincubated with 10 microM cytochalasin B (a drug that induces depolymerization of F-actin). These findings suggest that serotonin, dopamine, or noradrenaline may associate with actin filaments in vivo.     

Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

Desipramine Binding: Relationship to Central and Sympathetic Noradrenergic Activity

We examined the effects of treatments affecting norepinephrine release on the number of norepinephrine reuptake recognition sites as reflected by desipramine binding. To do this, we used manipulations having similar presynaptic but contrasting postsynaptic effects. Presynaptic inhibition by 6-hydroxydopamine lesion or by clonidine, and postsynaptic receptor stimulation by isoproterenol, reduced desipramine binding. Presynaptic stimulation by d-amphetamine and postsynaptic receptor blockade by prazosin increased desipramine binding. Similar effects and binding properties were seen in cerebral cortex, heart, and soleus muscle. After unilateral noradrenergic lesions, reduction in desipramine binding correlated with reduction in norepinephrine uptake. These results show that norepinephrine reuptake appears to be regulated by transmitter release regardless of effects on postsynaptic transmission, and that this regulation is analogous in the central and sympathetic nervous systems.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)