Effects of Arachidonic Acid on Dopamine Synthesis, Spontaneous Release, and Uptake in Striatal Synaptosomes from the Rat

Abstract: Arachidonic acid (AA) markedly stimulated, in a dose-dependent manner, the spontaneous release of [³H]dopamine ([³H]DA) continuously synthesized from [³H]tyrosine in purified synaptosomes from the rat striatum. As estimated by simultaneous measurement of the rate of [³H]H₂O formation (an index of [³H]tyrosine conversion into [³H]DOPA), the AA response was associated with a progressive and dose-dependent reduction of [³H]DA synthesis. In contrast to AA, arachidic acid, oleic acid, and the methyl ester of AA (all at 10⁻⁴ M ) did not modify [³H]DA release. The AA (3 × 10⁻⁵ M )-evoked release of [³H]DA was not affected by inhibiting AA metabolism, with either 5,8,11,14-eicosatetraynoic acid or metyrapone, suggesting that AA acts directly and not through one of its metabolites. AA also inhibited in a dose-dependent manner [³H]DA uptake into synaptosomes, with a complete blockade observed at 10⁻⁴ M . However, AA (10⁻⁴ M ) still stimulated [³H]DA spontaneous release in the presence of either nomifensine or other DA uptake inhibitors, indicating that AA both inhibits DA reuptake and facilitates its release process. Finally, the AA (10⁻⁴ M )-evoked release of [³H]DA was not affected by protein kinase A inhibitors (H-89 or Rp -8-Br-cAMPS) but was markedly reduced in the presence of protein kinase C inhibitors (Ro 31-7549 or chelerythrine).     

5-Hydroxytryptamine Stimulates [3H]Dopamine Release from the Fish Retina

Abstract: Serotonin (5-hydroxytryptamine, 5-HT; 0.5 μM and above) stimulated the release of [³H]dopamine ([³H]DA) from particulate fractions of the carp ( Cyprinus carpio ) retina. The 5-HT effect was dose- and Ca²⁺-dependent, and was structurally specific. A similar response was not elicited by the other indoles (5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 5-hydroxytrypto-phan, or 5-hydroxyindoleacetic acid) examined. An increase in [³H]DA release was elicited by addition of 5-HT agonists (5-methoxytryptamine, 5-methoxy- N,N- dimethyltryptamine, and tryptamine), but not antagonized by three 5-HT antagonists (metergolin, methysergide, and spiperone). Either DA alone or noradrenaline (0.5 m M ) produced a large increase in [³H]DA release from the particulate fractions, but this action was Ca²⁺-independent. Further, no significant release of [³H]γ-aminobutyric acid could be evoked by 5-HT (0.5 mM) under similar experimental conditions. Taken together, the present data suggest that 5-HT stimulates [³H]DA release from the fish retina through a specific receptor-mediated mechanism on dopaminergic terminals, but not through an exchange or nonspecific phenomenon.     

Protein Kinase FA/Glycogen Synthase Kinase-3α After Heparin Potentiation Phosphorylates τ on Sites Abnormally Phosphorylated in Alzheimer''s Disease Brain

In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [³H]dopamine ([³H]DA) release and neurotensin (NT)-induced facilitation of [³H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4-aminopyridine increased basal [³H]DA release and decreased K⁺-evoked [³H]DA release, whereas apamin was without effect. K⁺-evoked [³H]DA release was decreased by ω-conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [³H]DA release to blockade of various ion channels and suggest the involvement of N-type, L-type, and non-L-non-N-type, but not T-type, voltage-sensitive calcium channels in K⁺-evoked release. Phorbol 12-myristate 13-acetate increased both spontaneous and K⁺-evoked [³H]DA release, suggesting a modulatory action of protein kinase C on DA release in this system. Unexpectedly, however, the effects of the phorbol ester were not counteracted by the protein kinase C inhibitors H7, staurosporine, or polymyxin B. NT-induced facilitation of K⁺-evoked [³H]DA release was insensitive to most of the ion channel blockers, except cadmium (64% decrease in NT effect), suggesting that the corresponding potassium' and calcium channels were not involved in the effect of NT on [³H]DA release in this system. The NT effect was totally suppressed by phorbol ester treatments, indicating a possible desensitization of the corresponding transduction mechanisms after protein kinase C activation.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.     

l-Aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes

The role of l -aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well-known excitatory neurotransmitter l -glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca²⁺. At 35 and 50 mM KCl, the overflows of l -aspartate, but not those of l -glutamate, became sensitive to dl -threo-β-benzyloxyaspartic acid ( dl -TBOA), an excitatory amino acid transporter inhibitor. In the presence of dl -TBOA, the 50 mM KCl-evoked release of l -aspartate was still largely external Ca²⁺-dependent. The dl -TBOA insensitive, external Ca²⁺-independent component of the 50 mM KCl-evoked overflows of l -aspartate and l -glutamate was significantly decreased by the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP 37157. The Ca²⁺-dependent, KCl-evoked overflows of l -aspartate and l -glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced l -aspartate and l -glutamate release was completely external Ca²⁺-dependent and never affected by dl -TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [³H] d -aspartate and [³H] l -glutamate. Therefore, our data showing that l -aspartate is released from nerve terminals by calcium-dependent, exocytotic mechanisms support the neurotransmitter role of this amino acid.     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.     

Expression of Excitatory Amino Acid Receptors by Cerebellar Cells of the Type-2 Astrocyte Cell Lineage

Abstract: We have used postnatal rat cerebellar astrocyte-enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type-2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ-[³H]aminobutyric acid ([³H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μ M . Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μ M ). N -Methyl- d -aspartic acid and dihydrokainate (100 μ M ) did not stimulate [³H]GABA release from cultured astrocytes. l -Glutamic acid (20–100 μ M ) stimulated [³H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [³H]GABA release were completely Na⁺ dependent; that of kainate was also partially Ca²⁺ dependent. Kynurenic acid (50–200 μ M ) selectively antagonized the releasing effects of kainic acid and also that of l -glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μ M ). d -[³H]aspartate was taken up by both type-1 and type-2 astrocytes, but only type-2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type-2 astrocytes were also expressed by the immature, bipotential progenitors of type-2 astrocytes and oligodendrocytes.     

Pharmacology and Regional Distribution of the Binding of 6-[3H]Nitro-7-Sulphamoylbenzo[f]-Quinoxaline-2,3-Dione to Rat Brain

Abstract: 6-Nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [³H]NBQX binding to rat brain. The association rate of [³H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0°C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 m M unlabelled l -glutamate. [³H]NBQX bound to a single class of sites with K _D and B _max values of 47 n M and 2.6 pmol mg⁻¹ of protein, respectively. The rank order of inhibition of [³H]NBQX binding by AMPA receptor ligands was NBQX ≫ 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ≥ ( S )-5-fluorowillardiine ≥ AMPA ≫ l -glutamate. The chaotrope KSCN had no effect on the IC₅₀ value of unlabelled NBQX displacement of [³H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [³H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [³H]NBQX to coronal sections showed a distribution compatible with that of [³H]AMPA binding. These data indicate that [³H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.     

THE EFFECT OF DELTA-AMINOLAEVULINIC ACID ON THE UPTAKE AND EFFLUX OF AMINO ACID NEUROTRANSMITTERS IN RAT BRAIN SYNAPTOSOMES

Abstract— δ-Aminolaevulinic acid (δ-ALA) is an omega amino acid structurally similar to γ-aminobutyric acid (GABA) and l -glutamate. We have examined the effect of δ-ALA on the uptake and efflux of radiolabelled GABA and l -glutamate in rat cortical synaptosomes and report: (1) low concentrations of δ-ALA reduced the potassium-stimulated release of [³H]GABA from the synaptosome preparation. This effect was reversed by the GABA receptor antagonist bicuculline. We postulate that GABA release is modulated by a feedback mechanism on presynaptic GABA receptors, and that δ-ALA has agonist activity at these receptors. (2) δ-ALA at high concentrations (0.75-5.0 m m ) stimulated the efflux of l -[¹⁴C]glutamate from preloaded synaptosomes. (3) δ-ALA had no effect on potassium-stimulated release of l -glutamate. (4) Uptake of labelled l -glutamate was inhibited by δ-ALA in a noncompetitive fashion. (5) Synaptosomes did not accumulate [¹⁴C]δ-ALA in the range 0.5-50 δ m . These results are discussed in relation to the control of GABA release from nerve endings, and the role of δ-ALA in the neuropsychiatric manifestations of the acute porphyric attack.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

Pre-synaptic nicotinic and D2 receptors functionally interact on dopaminergic nerve endings of rat and mouse nucleus accumbens

The existence of pre-synaptic auto- and hetero receptors which modulate neurotransmitter release is well documented. Emerging evidence show that in some cases these pre-synaptic receptors may also cross-talk with each other. The aim of the present work was to investigate whether acetylcholine receptors (nAChRs) and dopamine (DA) autoreceptors, which are both able to modulate DA release, functionally interact on the same nerve endings. We used rat and mouse nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed to nicotinic and dopaminergic receptor ligands. Both nicotinic agonists and 4-aminopyridine (4-AP) provoked [³H]DA release which was inhibited by quinpirole and blocked by sulpiride and raclopride. Both the inhibitory effect of quinpirole and the stimulatory effect of (−)nicotine did not change when the nAChRs or the DA receptors were desensitized. (−)Nicotine and 4-AP were able to stimulate [³H]DA overflow also in mouse synaptosomes and this overflow was partially inhibited by quinpirole. In the β₂ knockout mice quinpirole was still able to inhibit the [³H]DA overflow elicited by 4-AP. To conclude: in rat and mouse the (−)nicotine evoked-release can be modulated by D₂/D₃ autoreceptors present on the DA terminals and nAChRs function is independent from D₂/D₃ autoreceptors which themselves may function independently from the activation of nAChRs.     

[3H]Homoquinolinate Binds to a Subpopulation of NMDA Receptors and to a Novel Binding Site

Abstract: NMDA receptors mediate several important functions in the CNS; however, little is known about the pharmacology, biochemistry, and function of distinct NMDA receptor subtypes in brain tissue. To facilitate the study of native NMDA receptor subpopulations, we have determined the radioligand binding properties of [³H]homoquinolinate, a potential subtype-selective ligand. Using quantitative receptor autoradiography, NMDA-specific [³H]homoquinolinate binding selectively labeled brain regions expressing NR2B mRNA (layers I–III of cerebral cortex, striatum, hippocampus, and septum). NMDA-specific [³H]homoquinolinate binding was low in brain regions that express NR2C and NR2D mRNA (cerebellar granular cell layer, NR2C; glomerular layer of olfactory bulb, NR2C/NR2D; and midline thalamic nuclei, NR2D). In forebrain, the pattern of NMDA-specific [³H]homoquinolinate binding paralleled NR2B and not NR2A distribution. In addition to NMDA-displaceable binding, there was a subpopulation of [³H]homoquinolinate binding sites in the forebrain, cerebellum, and choroid plexus that was not displaced by NMDA or l -glutamate. In contrast, we found that the derivative of homoquinolinate, 2-carboxy-3-carboxymethylquinoline, markedly inhibited the NMDA-insensitive binding of [³H]homoquinolinate without inhibiting the NMDA-sensitive population. [³H]Homoquinolinate may be useful for selectively characterizing NR2B-containing NMDA receptors in a preparation containing multiple receptor subtypes and for characterizing a novel binding site of unknown function.     

Increase in Chloride-Dependent l-Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment

Abstract: The effect of energy failure on Cl⁻-dependent l -glutamate ( l -Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl⁻-dependent l -[³H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l -[³H]Glu uptake was inhibited by l -Glu, dl -2-amino-4-phosphonobutyrate, l -homocysteic acid, l -cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl⁻. Uptakes of Na⁺-dependent l -[³H]-Glu, [³H]GABA, and [³H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the V _max value without affecting the K _m value. The increased l -[³H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m -chlorophenylhydrazone, and NaCN in normoxic conditions increased l -[³H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl⁻-dependent l -Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.     

Serotonin Stimulation of 5-HT4 Receptors Indirectly Enhances In Vivo Dopamine Release in the Rat Striatum

Abstract: Serotonin (5-HT) applied at 1, 3, and 10 µ M into the striatum of halothane-anesthetized rats by in vivo microdialysis enhanced dopamine (DA) outflow up to 173, 283, and 584% of baseline values, respectively. The 5-HT effect was partially reduced by 1 or 10 µ M GR 125,487, a 5-HT₄ antagonist, and by 100 µ M DAU 6285, a 5-HT_3/4 antagonist, whereas the 5-HT_1/2/6 antagonist methiothepin (50 µ M ) was ineffective. In the presence of tetrodotoxin the effect of 1 µ M 5-HT was not affected by 5-HT₄ antagonists. In addition, tetrodotoxin abolished the increase in DA release induced by the 5-HT₄ agonist ( S )-zacopride (100 µ M ). In striatal synaptosomes, 1 and 10 µ M 5-HT increased the outflow of newly synthesized [³H]DA up to 163 and 635% of control values, respectively. The 5-HT₄ agonists BIMU 8 and ( S )-zacopride (1 and 10 µ M ) failed to modify [³H]DA outflow, whereas 5-methoxytryptamine (5-MeOT) at 10 µ M increased it (62%). In prelabeled [³H]DA synaptosomes, 1 µ M 5-HT, but not ( S )-zacopride (1 and 10 µ M ), increased [³H]DA outflow. DAU 6285 (10 µ M ) failed to modify the enhancement of newly synthesized [³H]DA outflow induced by 5-MeOT or 5-HT (1 µ M ), whereas the effect of 5-HT was reduced to the same extent by the DA reuptake inhibitor nomifensine (1 µ M ) alone or in the presence of DAU 6285. These results show that striatal 5-HT₄ receptors are involved in the 5-HT-induced enhancement of striatal DA release in vivo and that they are not located on striatal DA terminals.     

Chronic Dopamine D2 Receptor Activation Does Not Affect Survival and Differentiation of Cultured Dopaminergic Neurons: Morphological and Neurochemical Observations

Abstract: Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D₂ autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high-affinity uptake for [³H]DA. In rat striatal tissue, activation of D₂ receptors has been shown to inhibit the release of DA. Previously accumulated [³H]DA was released from the cultures upon depolarization in a Ca²⁺-dependent manner. K⁺-evoked [³H]DA release could be inhibited by the selective D₂ receptor agonists LY 171555 and N0437 in a concentration-dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D₂ receptor antagonist sulpiride. These observations are indicative for the expression of functional D₂ receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase-immunoreactive neurons, or the uptake capacity for [³_H]DA. Our data demonstrate that chronic stimulation of DA D₂ receptors does not impair survival or differentiation of cultured fetal dopaminergic neurons.     

Characterization of the Binding of [3H]NS 257, a Novel Competitive AMPA Receptor Antagonist, to Rat Brain Membranes and Brain Sections

Abstract: The binding of [³H]NS 257 {1,2,3,6,7,8-hexahydro-3-(hydroxyimino)- N,N -[³H]dimethyl-7-methyl-2-oxobenzo[2,1- b :3,4- c '] dipyrrole-5-sulfonamide} to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [³H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 m M . In the presence of thiocyanate [³H]NS 257 bound to a single population of binding sites with an affinity of 225 ± 8 n M and a binding site density of 0.61 ± 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [³H]NS 257 for both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and l -glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > l -glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [³H]NS 257 binding sites in rat brain was similar to that of [³H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Opposing Actions of D1 and D2-Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D₁-and D₂-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D₁ or D₂ receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [³H]AA, D₂-receptor stimulation enhanced release of [³H]AA produced by application of the Ca²⁺ ionophore A23187 or of the purinergic agonist ATP. By contrast, D₁-receptor stimulation inhibited [³H]AA release. This inhibitory effect of D₁ receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D₂ and D₁ receptors in striatum, potentiation and inhibition, respectively, of Ca²⁺-evoked AA release.