In Vivo Evidence that Nonneuronal β-Adrenoceptors as Well as Dopamine Receptors Contribute to Cyclic AMP Efflux in Rat Striatum

Abstract: We applied in vivo microdialysis to assess the effects of dopaminergic and β-adrenergic receptor stimulation on cyclic AMP efflux in rat striatum under chloral hydrate anesthesia. Dopamine (up to 1 mM) infused for 20 min through the probe did not increase cyclic AMP, whereas both the selective dopamine D₁ agonist SKF 38393 and D₂ antagonist sulpiride produced modest increases. It is interesting that the β-adrenoceptor agonist isoproterenol produced a marked increase (204.7% of basal level at 1 mM) which was antagonized by the β-adreno-ceptor antagonist propranolol. Pretreatment with a glial selective metabolic inhibitor, fluorocitrate (1 mM), by a 5-h infusion through the probe attenuated basal cyclic AMP efflux by 30.3% and significantly blocked the response to isoproterenol. By contrast, striatal injection of a neuro-toxin, kainic acid (2.5 μg), 2 days before the dialysis experiment did not affect basal cyclic AMP or the response to isoproterenol, but blocked the response to SKF 38393. These data demonstrate that β-adrenoceptors as well as dopamine receptors contribute to cyclic AMP efflux in rat striatum in vivo. They also suggest that basal and β-adre-noceptor-stimulated cyclic AMP efflux are substantially dependent on intact glial cells.     

Effects of Phenylalanine on the Release of Endogenous Dopamine from Rat Striatal Slices

We examined the effect of phenylalanine (50-400 microM) on the electrically stimulated release of endogenous 3,4-dihydroxyphenylethylamine (dopamine or DA) from superfused rat striatal slices. In the absence of tyrosine, phenylalanine (25 microM) partially sustained DA release, but less well than an equimolar concentration of tyrosine. In the presence of tyrosine (50 microM), phenylalanine (in concentrations of greater than or equal to 200 microM) inhibited DA release into the superfusate. This inhibition was not associated with changes in tissue levels of tyrosine or DA, nor was it mimicked by addition of high concentrations of tyrosine or leucine to the medium. We conclude that phenylalanine is a less effective precursor of DA in rat striatum than tyrosine and that it can also act to inhibit DA synthesis, depending on its concentration.     

Resveratrol Protects Rat Striatal Slices Against Anoxia-induced Dopamine Release

Incubation of rat striatal slices in anoxic medium caused significant alterations in dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) outputs; while DA release increased several times, 50% decline in DOPAC output was observed under this condition. Tissue ATP level, on the other hand, was decreased 40% by anoxia. Presence of resveratrol in the medium decreased anoxia-induced DA release in a concentration-dependent manner. Enhanced DA output, however, was declined slightly by epicatechine and catechine, and not altered significantly by morin hydrate and quercetin dehydrate which are other penolic compounds present in the red wine. In contrary to DA output, anoxia-induced decline in tissue ATP level was not ameliorated by resveratrol. In addition to anoxia, resveratrol, as observed with DA uptake blocker nomifensine, also reduced DA release stimulated by ouabain. Efficiencies of both resveratrol and nomifensine to attenuate ouabain-induced DA output, however, were closely dependent on ouabain concentration in the medium. These results indicate that some phenolic compounds, particularly resveratrol decrease anoxia-induced DA output and appear promising agents to improve the alterations occurred under anoxic-ischemic conditions.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

Activation of β-Adrenergic Receptors Stimulates Release of an Inhibitory Transmitter from Astrocytes

Activation of beta-adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP-mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2'-dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the beta-adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS-receptor-mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.     

Ischemia-Induced Changes in Extracellular Lévels of Striatal Cyclic AMP: Role of Dopamine Neurotransmission

Dopamine has been demonstrated to be involved in the development of ischemic neuronal damage in the striatum. This detrimental effect of dopamine may involve activation of second messenger systems, such as the cyclic AMP (cAMP) cascade, which may enhance the susceptibility of striatal neurons to ischemia. In the present study, we have evaluated the relationship between ischemia-induced changes in cAMP and dopamine neurotransmission. Microdialysis probes were implanted in both striata, and a D1 antagonist (SCH-23390, 100 microM) was administered through one probe and modified Ringer's solution through the other. After a stabilization period, rats (n = 6) were subjected to 20 min of ischemia by two-vessel occlusion plus hypotension. Extracellular samples were collected from both striata, before, during, and after ischemia, and analyzed for cAMP by radioimmunoassay. Ischemia induced a significant increase in extracellular cAMP (means +/- SE, fmol/microliter; baseline: 4.35 +/- 1.1, ischemia: 12.2 +/- 1.98), which was also observed at 4 h of recirculation (mean level of 8.45 +/- 1.14). Treatment with the D1 antagonist significantly inhibited the rise in extracellular cAMP during ischemia and recirculation. These results indicate that an ischemia-induced surge in dopamine and activation of D1 receptors are involved in the generation of cAMP during ischemia and recirculation. Because activation of the adenylate cyclase cascade may modulate the effects of glutamate, generation of cAMP through this pathway may play a role in facilitating the injurious effects of dopamine during ischemia.     

In Vivo Measurement of Extracellular Cyclic AMP in the Brain: Use in Studies of β-Adrenoceptor Function in Nonanesthetized Rats

Microdialysis measurement of extracellular cyclic AMP (cAMP) in the cerebral cortex of conscious rats was evaluated as a method for assessing central beta-adrenoceptor function in vivo. Extracellular levels of the nucleotide were found to average 3 pmol/ml under baseline conditions. Local infusion of the beta-agonists norepinephrine (NE) and isoproterenol produced rapid (3 min) and marked (three- to sevenfold) dose-dependent increases in extracellular cAMP, which were potentiated by the phosphodiesterase inhibitor rolipram, and blocked by the beta-antagonist timolol. Responses to both catecholamines underwent rapid desensitization (6-9 min) and recovered within several hours. Time-course studies revealed that the baseline cAMP level underwent a gradual increase and then a decrease over the course of a single 8-h run, and peaked at 24 h postimplantation. Responses to NE were stable for the first 24 h after implantation, then increased at 48 and 120 h. The causes of the latter changes may include reactions to novelty, local inflammatory responses, and/or reactions of adjacent glial cells to implantation. Overall, the results indicate that the microdialysis-cAMP method can be extended to nonanesthetized animals and may be a useful tool for studying neurotransmission at central adenylate cyclase-coupled membrane receptors during various behavioral states.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Heterogeneity of β-Adrenoceptors in Guinea-Pig Brain: Radioligand Binding and Cyclic Nucleotide Generation

Abstract: In this report, we have examined the radioligand binding and second messenger signalling characteristics of β-adrenoceptors in the guinea-pig brain. [¹²⁵I]lodocyanopindolol ([¹²⁵I]ICYP)-labelled sites in the cerebellum and cerebral cortex were of similar densities ( B _max 34 and 24 fmol·mg⁻¹) and affinities ( K _D 20 and 55 p M ), respectively. Analysis of competition for [¹²⁵I]ICYP binding in the cerebellum was compatible with the presence of a β₂-adrenoceptor. In this tissue, isoprenaline evoked a cyclic AMP stimulation, and also potentiated cyclic GMP accumulations evoked in the presence of a nitric oxide donor, consistent with mediation via a β₂-adrenoceptor. The [¹²⁵I]ICYP binding profile in the cerebral cortex did not comply with those previously described for β-adrenoceptor subtypes, and isoprenaline failed to alter significantly cyclic AMP accumulation in the cerebral cortex, hippocampus, or neostriatum, even in the presence of forskolin or a phosphodiesterase inhibitor. Isoprenaline was also without effect on cyclic GMP accumulation or phosphoinositide turnover in the cerebral cortex. These results suggest that the guinea-pig cerebellum expresses a functional β₂-adrenoceptor coupled to cyclic AMP generation, and potentiation of cyclic GMP accumulation. However, the guinea-pig cerebral cortex displays binding sites that exhibit β-adrenoceptor-like pharmacology but fail to show functional coupling to cyclic AMP, cyclic GMP, or phosphoinositide signalling systems.     

Inositol Phospholipid Hydrolysis and Potentiation of Cyclic AMP Formation by Noradrenaline in Rat Cerebral Cortex Slices Are Not Mediated by the Same α-Adrenoceptor Subtypes

A pharmacological study was undertaken to determine whether the noradrenaline-stimulated breakdown of inositol phospholipids and the potentiation of isoprenaline-stimulated cyclic AMP by noradrenaline in rat cerebral cortex slices are mediated by the same alpha-receptor subtype. The rank order of potency of a range of alpha 1 and alpha 2 antagonists suggests that both responses may involve an alpha 1 receptor, but there were several differences between the pharmacological profiles for the two systems. Although in both cases, all selective alpha 1 antagonists were more potent than alpha 2 antagonists, the rank orders and the absolute potencies differed for the two responses. The inhibition of the inositol phosphate response was characterised by a high alpha 1/alpha 2 antagonist ratio, and in most cases, Hill slopes of inhibition were consistent with the involvement of a single receptor site. Inhibition of the cyclic AMP response had a much lower alpha 1/alpha 2 antagonist ratio and generally exhibited Hill slopes less than one. Evidence has been provided suggesting that adenosine is involved in the potentiation of cyclic AMP and that other, as yet unidentified, factors may also be involved. Even in the absence of an adenosine component, the results presented support the suggestion that the potentiation due to noradrenaline is mediated by a receptor whose identity does not easily fit with the currently accepted classification of alpha adrenoceptors.     

Heterogeneity of N-Methyl-D-Aspartate Receptors Regulating the Release of Dopamine and Acetylcholine from Striatal Slices

In an attempt to examine some functional characteristics of the N-methyl-D-aspartate (NMDA) receptor complex, the NMDA-evoked effluxes of endogenous dopamine (DA) and [³H]acetylcholine ([³H]ACh) were simultaneously examined in a rat Striatal slice preparation. NMDA induced release of both DA and ACh in a concentration-dependent, Ca²⁺-, Mg²⁺-, and tetrodotoxin-sensitive manner. These release responses were remarkably reduced by long-term pre-treatment with a low concentration of NMDA. an indication of the desensitization of the NMDA receptor. Glycine was potent in reversing the desensitization-related reduction of DA release but failed to reverse the diminution of ACh release in the same slices. Our results indicate that the NMDA receptors regulating the release of DA and ACh are different with respect to their glycine modulatory site. This finding is consistent with a functional heterogeneity of the NMDA receptor complex in the rat striatum.     

Purification and Properties of Bovine Brain Dopamine β-Hydroxylase

Abstract: Dopamine β-hydroxylase (DBH) was purified from bovine brain by a series of steps including extraction with 0.5% Triton X-100, ammonium sulfate fractionation, and serial chromatographies with Concanavalin A (Con A)-Sepharose, Biogel A-1.5 m, DEAE-Sephadex, and phenyl-Sepharose. The overall purification was approximately 4200-fold and the final specific activity was 147 nmol/min/mg protein. Bovine brain DBH was apparently a glycoprotein and interacted with immobilized Con A. Furthermore, the enLyme bound to phenyl-substituted agarose by hydrophobic interaction. An approximate molecular weight was estimated to be 400,000 by gel filtration; the protein eluted earlier than bovine adrenal DBH with a molecular weight estimated to be 290,000. The K_m values toward tyramine and ascorbate were 1.53 and 1.42 mM, respectively, the optimal pH was 5.0 in the presence of 20 mM tyramine as substrate. Immunological titration studies indicated that bovine brain and adrenal DBH had common antigenic sites. Our data showed a close similarity between the bovine brain and adrenal enzymes.     

Inactivation of Cyclic AMP-Dependent Taurine Release from Astroglia

When astroglial cells are exposed to beta-adrenergic agonists for long periods of time (greater than 20 min), transient increases in taurine release and intracellular cyclic AMP (cAMP) are observed. Three phases of taurine release can be distinguished: activation, inactivation, and an elevated steady state. In this article, we present data describing the relationship between intracellular cAMP levels and inactivation of taurine release. To do this, we compared the apparent first-order rate constants for the inactivation of taurine release (ktau) with the apparent first-order rate constant for the decline of intracellular cAMP (kcAMP). We also measured ktau under experimental conditions that were chosen to provide a wide range of intracellular cAMP concentrations or to stimulate release without the involvement of the beta-adrenergic receptor and adenylate cyclase. When taurine release was stimulated with a saturating concentration of isoproterenol, the inactivation of release was significantly faster than the decline of intracellular cAMP. Furthermore, there were no significant differences in ktau measured under any of the experimental conditions used. Thus, inactivation of taurine release does not involve changes in the activity of the beta-adrenergic receptor and adenylate cyclase, i.e., desensitization, and appears to be independent of the intracellular concentration of cAMP. These results indicate that cAMP-mediated events can be regulated by mechanism(s) in addition to those that control receptor-adenylate cyclase interactions and the synthesis of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vitro Release of Endogenous β- Alanine, GABA, and Glutamate, and Electrophysiological Effect of β-Alanine in Pigeon Optic Tectum

The efflux of 20 amino acids, induced by either high K+ concentration or veratrine, was determined in pigeon tectal slices. Ca2+-dependent, K+-induced release of beta-alanine, gamma-aminobutyric acid (GABA), and glutamate was observed. Veratrine caused release of the same amino acids plus glycine in a tetrodotoxin-sensitive manner. beta-Alanine had a strong inhibitory effect on the activity of tectal neurons which was blocked by strychnine but not by bicuculline. The results indicated a transmitter function for beta-alanine in the optic tectum, and were consistent with the previously proposed transmitter role of GABA and glutamate in this structure.     

Inhibition of Striatal GABA Release by the Adenosine A2a Receptor Is Not Mediated by Increases in Cyclic AMP

Abstract: The adenosine A_2a receptor inhibition of potassium (15 m M )-evoked GABA release from striatal nerve terminals has been examined. High extracellular calcium concentrations (4 m M ) reduced the effect of the A_2a receptor agonist CGS-21680 (1 n M ). CGS-21680 inhibited GABA release in the presence of the L-type calcium channel blocker nifedipine, which itself inhibited evoked GABA release (by 16 ± 4%). ω-Conotoxin inhibited the evoked release by 45 ± 4% and prevented the action of CGS-21680. Forskolin and 8-bromo cyclic AMP both stimulated evoked GABA release at low concentrations, but at higher concentrations they abolished the inhibition by CGS-21680 without affecting the evoked release. The nonselective protein kinase inhibitor H-7 inhibited both the evoked release and the inhibition by CGS-21680, whereas the selective protein kinase A and G inhibitor HA-1004 had no effect on either evoked release or the action of CGS-21680. Pretreatment with pertussis toxin did not affect the A_2a receptor-mediated inhibition. Therefore, the effect of A_2a receptor stimulation was not mediated by protein kinases A or G but was inhibited by elevated cyclic AMP levels and mimicked by inhibitors of the N-type calcium channel and protein kinase C.     

Mutagenesis of Rat Dopamine β-Hydroxylase: Examination in Cell-Free System

Abstract: Dopamine β-hydroxylase (DBH; EC 1.14.17.1) exists as membrane-bound and soluble forms in neurosecretory vesicles. The features of DBH required for glycosylation and incorporation into membranes were studied in a cell-free system. Translation of full-length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl-terminal, in contrast to amino-terminal, truncations gave translation products that were glycosylated by microsomal membranes. Site-directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavage site modified. Translation without membranes indicated that the second AUG is not used to initiate translation. The mutant with Glu⁴¹→ Leu⁴¹ and Ser⁴³→ Thr⁴³ yielded only the GH form with membranes, whereas mutation of Ser⁴³→ Ala⁴³ generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membranes on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moiety. The results suggest a role for cleavage of a signal sequence in the formation of different forms of DBH. The possibility that these mutations change the secondary structure near the signal cleavage site, affecting processing, is discussed.     

Direct Effects of Chronic Ethanol Exposure on β-Adrenergic and Adenosine-Sensitive Adenylate Cyclase Activities and Cyclic AMP Content in Primary Cerebellar Cultures

The direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content were investigated in primary cerebellar cultures. By morphological criteria these cultures mainly contain granule cells with some astrocytes, and each cell type appears to contain both beta-adrenergic and adenosine-sensitive adenylate cyclase systems. Chronic treatment of the primary cerebellar cultures with 120 mM ethanol for 6 days caused a reduction in the stimulation of cyclic AMP content by isoproterenol and by the adenosine analogue 2-chloroadenosine. Kinetic analysis indicated that the chronic ethanol treatment decreased maximal activation of adenylate cyclase, as well as increased the EC50 values for norepinephrine and 2-chloroadenosine. Activation of norepinephrine-stimulated adenylate cyclase activity by in vitro ethanol was significantly enhanced after the chronic ethanol exposure. However, the chronic treatment did not alter activation of the 2-chloroadenosine-stimulated enzyme by in vitro ethanol. A similar difference in the response to in vitro ethanol after the chronic treatment was observed when cyclic AMP content of the intact cells was measured. The present data indicate that chronic ethanol exposure causes a selective increase in the sensitivity of adenylate cyclase to ethanol in some brain cells and a more generalized desensitization of receptor-stimulated cyclic AMP production.     

Effects of Lithium on Inositol Phospholipid Hydrolysis and Inhibition of Dopamine D1 Receptor-Mediated Cyclic AMP Formation by Carbachol in Rat Brain Slices

The in vitro and ex vivo effects of lithium on muscarinic cholinergic inositol phospholipid hydrolysis and muscarinic cholinergic inhibition of dopamine D1-receptor-stimulated cyclic AMP formation were examined in rat brain slices. Following chronic lithium feeding, carbachol-stimulated inositol phosphate accumulation was reduced ex vivo in slices of cerebral cortex but not in striatal slices. Lithium (1 mM) in vitro had no direct effect on dopamine D1-receptor-stimulated cyclic AMP formation, but enhanced the inhibitory effect of carbachol on the D1 response, in striatal slices, and this was not significantly altered by prior lithium feeding. Lithium therefore has effects on two discrete muscarinic responses in rat brain which are apparently maintained after chronic exposure to the ion and might be relevant to its antimanic actions.     

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.