The resolution of dopamine and β1- and β2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex

The stimulation of adenylate cyclase by dopamine and various β-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the β-adrenergic receptors coupled to adenylate cyclase could be classified as either β₁ and β₂ subtypes in the different regions studied.The stimulation of adenylate cyclase by the β-adrenergic agonist, (−)isoproterenol (5 × 10⁻⁶M), was completely blocked by the specific β-adrenergic antagonist, (−)alprenolol (10⁻⁵ M), but not by the dopaminergic antagonist, fluphenazine (10⁻⁵ M), whereas the stimulation of adenylate cyclase by (−)epinephrine (10⁻⁴ M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (−)epinephrine effect was always blocked in the combined presence of (−)alprenolol and fluphenazine. The adenylate cyclase stimulation by (−)epinephrine which is not blocked by (−)alprenolol was due to interaction of (−)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum.Regional differences in the affinity of β-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10⁻⁵ M). In the cerebellum the potency order was (±)protokylol> (±)hydroxybenzylisoproterenol> (±)isoproterenol> (−)epinephrine> (±)salbutamol> (−)norepinephrine, indicating the presence of a β₂-adrenergic receptor. In the cerebral cortex the potency order was (−)isoproterenol> (±)protokylol> (±)hydroxybenzylisoproterenol> (−)epinephrine= (−)norepinephrine((±)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a β₁-adrenergic receptor in these two regions. (±)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex.The ratio of the antagonist potencies of (±)practolol and (±)butoxamine preferential β₁- and β₂-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a β₂ subtype of receptor coupled to adenylate cyclase in the former and β₁ subtypes in the latter two regions. The comparison between the affinities of a series of β-adrenergic agonists and antagonists for the β-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized β₂-adrenergic receptors in lung and β₁-adrenergic receptor in heart substantiated this conclusion.     

[I]4-Aminobenzyl-5′-N-methylcarboxamidoadenosine ([I]AB-MECA) labels multiple adenosine receptor subtypes in rat brain

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A₁, A_2A, A_2B, and A₃ receptors, have been cloned and characterized. The agonist ligand, [¹²⁵I]AB-MECA ([¹²⁵I]4-aminobenzyl-5′-N-methylcarboxamidoadenosine) has high affinity for recombinant A₁ and A₃ receptors [Olah et al., Mol. Pharmacol., 45 (1994) 978–982]. Rodent A₃ receptors are relatively insensitive to xanthines; inhibition of A₁ receptors with xanthines allows selective detection of A₃ receptors despite the lack of selectivity of the ligand. We studied whether [¹²⁵I]AB-MECA is useful for localization and characterization of A₃ receptors in rat brain. The autoradiographic distribution of total [¹²⁵I]AB-MECA (400 pM) binding closely resembled the pattern of A₁ receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [¹²⁵I]AB-MECA binding could be attributed to labeling of A₁ receptors. Xanthine amine congener (1 μM) reduced specific [¹²⁵I]AB-MECA binding by >95%, indicating that xanthine-resistant A₃ receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A₃ receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A₁ receptors), residual [¹²⁵I]AB-MECA binding to A_2A receptors was observed in the striatum. Thus, [¹²⁵I]AB-MECA labels primarily A₁ and A_2A adenosine receptors in rat brain.     

Adenosine stimulates stellation of cultured rat cortical astrocytes

We investigated the effect of adenosine on astrocyte morphology by using cell cultures prepared from the cerebral cortices of neonatal rats. Cultured rat cortical astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but differentiated into process-bearing stellate cells in response to adenosine (1–1000 μM). Adenosine-induced astrocyte stellation was abolished by treatment with microtubule inhibitors, colchicine and paclitaxel, indicating the involvement of cytoskeletal elements. The effect of adenosine was mimicked by other adenosine receptor agonists, and blocked by adenosine receptor antagonists and guanosine 5′-O-(2-thiodiphosphate), indicating that the effect of adenosine is mediated by G protein-coupled adenosine receptors. Although adenosine receptors are known to be linked to adenylate cyclase or phospholipase C, adenosine did not change intracellular cyclic AMP level nor intracellular Ca²⁺ concentration in astrocytes. Alternatively, adenosine-induced stellation was abolished by tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, suggesting that adenosine causes astrocyte stellation through tyrosine dephosphorylation. Adenosine may function as a factor regulating astrocyte differentiation.     

Evidence for selective loss of brain dopamine- and histamine-stimulated adenylate cyclase activities in rabbits with aging

Dopamine-stimulated adenylate cyclase activity in striatum and both dopamine-and histamine-stimulated adenylate cyclase activity in hypothalamus, frontal cortex and anterior limbic cortex declined by about 50% as rabbits aged from 5.5 months to 5.5 years of age. These changes were primarily in maximal response to amine although an additional component involving decreased affinity in the case of dopamine may also be present. In contrast, dopamine-stimulated adenylate cyclase of retina and both basal and guanyl-5′-yl-imidodiphosphate (Gpp(NH)p)-stimulated activity in these regions were not altered with age. There was no measurable decrease in the old animals in either dopamine or norepinephrine concentration in striatum, anterior limbic cortex or retina, or in choline acetylase activity or [³H]quinuclidinylbenzilate binding in striatum, anterior limbic cortex or frontal cortex. It is proposed that selective age-dependent decreases in transmitter receptors coupled to adenylate cyclase occur in the absence of or independent from neuronal cell loss, as evidenced by the retention of the other biochemical markers.     

Localization and characterization of epidermal growth-factor receptors in the developing rat medial septal area in culture

The presence and binding properties of epidermal growth-factor receptors (EGF-Rs) in different cell types purified from the rat medial septal area in culture were investigated. We report that astrocytes, oligodendrocytes and neurons from this area possess EGF-Rs while microglia do not. EGF-binding sites are detectable on astrocytes derived from the medial septum of both embryonic and neonatal rats. Scatchard analysis of the data for astrocytes from the fetal rats show that EGF specifically binds to both high- (K_d = 7.21 × 10⁻¹⁰ M, B_max = 3602 receptors/cell) and low-affinity (K_d = 3.99 × 10⁻⁸ 10⁻⁸ M, B_max = 6,265 receptors/cell) receptors on these cells. On the other hand, astrocytes purified from neonatal tissue possess a greater number of high-affinity receptors (B_max = 10,938 receptors/cell) when compared with the embryonic astroglia. With time in culture, the number of both types of receptors on neonatal astrocytes decreases. Oligodendrocytes also possess high- and low-affinity EGF-Rs with dissociation constants of 3.25 × 10⁻¹⁰ M and 3.85 × 10⁻⁸ M, respectively. The number of receptors on oligodendrocytes is significantly lower than those on neonatal astrocytes (B_max = 1185 and 25,081 receptors/cell for high- and low-affinity binding sites, respectively). Finally, neurons from this area also exhibit two different EGF-R types with dissociation constants similar to those described for astrocytes. As the number of receptors/neuron (B_max = 136 and 1159 receptors/cell for high- and low-affinity binding sites, respectively) appears to be extremely low, it is possible that EGF specifically binds only to a subpopulation of neurons from this area. These studies demonstrate which cell types in the developing medial sepal area posses EGF-Rs and provide a detailed characterization of these binding sites. These EGF-R-bearing cells may be potential targets for this growth factor or for transforming growth factor α in this brain area.     

Agonists of A1 and A2A adenosine receptors attenuate methamphetamine-induced overflow of dopamine in rat striatum

The effect of adenosine receptor agonists on the release of striatal dopamine (DA), induced by repeated doses of methamphetamine (MTH), was evaluated. Rats received three injections of MTH (5 mg/kg i.p.) at 2-h intervals. The release of DA in the striatum was measured by a microdialysis in freely moving animals. The agonist of adenosine A₁ receptor, N⁶-cyclopentyladenosine (CPA) and the agonist of adenosine A_2A receptor, 2-[p-(carboxy-ethyl)phenylethylamino]-5′-N-ethylcarboxyamidoadenosine (CGS 21680), either of them being infused locally into the striatum at concentrations of 50 and 100 μM, produced decreases in the extracellular DA level during exposure to MTH, and a weaker effect on the levels of DOPAC and HVA. The above effects were reversed by the specific antagonists of adenosine A₁ and A_2A receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 3,7-dimethyl-1-propargylxanthine (DMPX), respectively. Our results indicate that both the A₁ and A_2A adenosine receptors appear to be involved in reducing the excessive release of DA in the striatum; furthermore, they suggest a neuroprotective role of adenosine in MTH neurotoxicity.     

Modification of opioid agonist binding by pertussis toxin

Membrane fractions prepared from rat striate, cortex and midbrain were treated with pertussis toxin, which has been shown to adenosine diphosphate (ADP)-ribosylate the GTP-binding protein G_i, reducing its coupling with receptors. In striatal membranes, treatment with 40 μg toxin per mg membrane protein labeled 60% of the G_i present and 70% of another G protein, G_o; this treatment reduced binding of the opioid agonist [³H]d-Ala²-d-Leu⁵-enkephalin ([³H]DADLE) 20–50%, with the decrease largerly reflecting a decreased affinity. In cortex, toxin treatment reduced [³H]DADLE binding by 35–70%, corresponding to ADP-ribosylation of 50% of G_i and 40% of G_o. In midbrain, [³H]DADLE binding was unaffected by toxin treatment that ADP-ribosylated 86% of the G_i and 72% of the G_o. These results provide further evidence that opioid receptors are associated with GTP-binding proteins in striatum and cortex, where they have also been shown to inhibit adenylate cyclase. Despite the presence of G_i and G_o in midbrain, however, there appears to be no coupling between them and opioid receptors.     

Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes

Our previous study has demonstrated that astrocytes derived from the rat frontal cortex contain 5-hydroxytryptamine (5-HT)₇ receptors positively coupled to adenylyl cyclase. In this study, we observed a desensitization of 5-HT₇ receptors induced by a treatment with agonists (0.1, 1, and 10 μM, 0.5 to 3.5 h). Maximum responses, but not the EC₅₀ values, in the concentration–response curve of 5-HT-induced cyclic AMP formation were decreased after pretreatment with 5-HT. Pretreatment with 5-carboxamidotryptamine (5-CT) elicited a potent desensitization of 5-HT-induced cyclic AMP formation. Neither 2-methyl-5-HT nor α-methyl-5-HT caused the desensitization. When the astrocytes were treated with isoproterenol, N-ethylcarboxamidoadenosine, and dibutyryl cyclic AMP (all of which increase intracellular cyclic AMP levels), 5-HT-induced cyclic AMP responses were not affected. Conversely, adenylyl cyclase activity mediated by either isoproterenol or N-ethylcarboxamidoadenosine was attenuated by pretreatment with each of these agonists, but not 5-HT. In addition, our study showed that the administration of 5-HT, 5-CT, and 8-hydroxy-2-(di-n-propylamino)tetralin to the astrocytes stimulated cyclic AMP formation both in the presence and absence of forskolin, whereas in neuron-rich cultures of the frontal cortex, these agonists did not change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. Neurons may predominantly contain 5-HT_1A receptors that are negatively coupled to adenylyl cyclase. These results suggest that 5-HT₇ receptors are highly expressed in astrocytes but not in neuronal cells, and that pretreatment with their agonists results in a homologous desensitization of the receptors.     

Mouse cerebellar GABAB participation in the expression of acute ethanol-induced ataxia and in its modulation by the cerebellar adenosinergic A1 system

The possible modulation and of co-modulation by the cerebellar GABA_B and adenosine A₁ receptors of ethanol-induced motor impairment were investigated in the mice using rotorod performance as the test response. Direct cerebellar microinfusion of GABA_B agonist, baclofen, and antagonist, phaclofen, into the permanently cannulated mice, produced a dose-dependent accentuation and attenuation, respectively, of ethanol-induced motor impairment. The baclofen and phaclofen exhibited accentuation and attenuation, respectively, via GABA_B receptors linked to pertussis toxin-sensitive G protein. A co-modulation by the cerebellar adenosine A₁ receptors was also observed because intracerebellar microinfusion of adenosine agonists N⁶-cyclohexyladenosine (CHA), 5′-N-ethylcarboxamidoadenosine (NECA), and 2-p-(2-carboxyethyl)-phenyl-ethylamino-5′-N-ethylcarbox-amidoadenosine (CGS-21680), and antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), also accentuated and attenuated, respectively, ethanol-induced motor impairment. The accentuation of ethanol-induced motor impairment by baclofen was further enhanced after the intracerebellar microinfusion of CHA, suggesting a co-modulation by the co-localized adenosine A₁ receptors. A similar response was observed after the intracerebellar microinfusion of adenosine A₁ = A₂ agonist NECA and the several-fold higher dose of adenosine A₂-selective agonist CGS-21680. Ethanol-induced motor impairment was markedly blocked by intracerebellar A₁-selective antagonist, DPCPX, as well as by the intracerebellar pertussis toxin pretreatment suggesting again a co-modulation by the adenosine A₁ receptors and the involvement of pertussis toxin-sensitive G protein, respectively. The almost 25-fold higher dose of CGS-21680 to accentuate and DPCPX to attenuate, respectively, ethanol-induced motor impairment together with the reported cerebellar localization of adenosine A₁ subtype only, suggested A₁ receptor activation by NECA and CGS-21680. The functional similarity between GABA_B and adenosine A₁, receptors associated with their anatomical co-localization on the cerebellar granule cells, mainly axons and axonal terminals, may suggest a possible common adenylate cyclase catalytic unit as the basis of modulation of ethanol's motor impairment by these two receptor mechanisms.     

Biogenic amine-sensitive adenylate cyclases in primary culture of neuronal or glial cells from mesencephalon

Primary cultures of virtually pure mesencephalic neurons (5 days) or glials (4 weeks) from 14-day-old mouse embryo were obtained using appropriate medium.Membranes prepared from neuronal cells contained mainly serotonin and β₁-adrenergic-sensitive adenylate cyclases. However, a low but significant classical dopamine-sensitive adenylate cyclase activity (D₁ receptor) was detected. Contrasting with the data obtained from a previous study on striatal neurons¹⁴ no adenosine-sensitive adenylate cyclase was found on mesencephalic neurons. Study on the additive effects of the 3 biogenic amines-sensitive adenylate cyclases indicated that: (1) all neuronal cells having dopamine receptors possess β₁-adrenergic receptors (no additivity); (2) β₁-adrenergic and serotonin receptors on the one hand, and dopamine and serotonin receptors on the other hand, were coupled with independent adenylate cyclase systems localized either on two different domains of the same cell or on different cells (complete additivity).Membranes prepared from primary mesencephalic cultures of glial cells contained a mixture of β₁- and β₂-adrenergic receptor subtypes coupled with an adenylate cyclase (70% and 30%, respectively). No dopamine- or serotonin-sensitive adenylate cyclase was detected on mesencephalic glial cells.     

Specific Binding of [H] phencyclidine in rat central nervous tissue: further characterization and technical considerations

The interaction of phencyclidine (PCP) with its specific receptor sites in the central nervous system has been further characterized. Kinetic association and dissociation rate constants of 2.9 × 10⁶ M⁻¹ and 4.8 × 10⁻¹ were determined, yielding a kinetic K_D of 1.6 × 10⁻⁷ M, in agreement with the K_D previously determined at equilibrium. Permissible separation time of 13 s was calculated from the kinetic data, well above the actual separation time of less than 10 s in the rapid filtration assay. Presoaking of filters in 0.01% poly-l-lysine eliminated displacable [³H]PCP adsorption to filter material. Binding data obtained via centrifigation assays was identical to that obtained with the rapid filtration method. Stereospecificity of the PCP receptor was demonstrated by the finding that (+)-ketamine is four-fold more potent than (−)-ketamine in displacing specifically bound [³H]PCP. Several proteolytic enzymes including trypsin, papain and thermolysin potently inactivated PCP receptors. Detailed regional distribution studies showed highest density of PCP receptors in subicular cortex and hippocampus, intermediate levels in hypothalamus, striatum, frontal cortex and cerebellum, lower levels in brainstem and spinal cord, and negligible levels in corpus callosum, a white- matter control area. Benzomorphan opiates with PCP-like behavioral effects interact with the PCP receptor. These data support the pharmacological relevance of the PCP receptor site as demonstrated by the rapid filtration method.     

Increased corticotropin-releasing factor receptors in rat cerebral cortex following chronic atropine treatment

Rats were treated chronically with atropine (14 days, 20 mg/kg/day, s.c.) and corticotropin-releasing factor (CRF) receptors and CRF-mediated adenylate cyclase activity were measured in discrete brain regions. Chronic atropine treatment produced significant increases in muscarinic cholinergic receptors in the frontoparietal cortex (30% increase) and hippocampus (20% increase). No significant changes in the concentration of [¹²⁵I]Tyr^o-rat CRF binding sites were observed in olfactory bulb, cerebellum, striatum and hippocampus. In contrast, there was a significant and selective increase (35%) in CRF receptors in the frontoparietal cortex of atropine-treated rats. However, no significant corresponding changes in the V_max or EC₅₀ of CRF-stimulated adenylate cyclase activity accompanied the upregulation of CRF receptors in the cerebral cortex. These results demonstrate that (1) CRF receptors in rat brain are subject to receptor regulation, (2) the upregulation of CRF receptors occurs as a consequence of chronic muscarinic cholinergic receptor blockade, and (3) this interaction between acetylcholine and CRF may be limited to the cerebral cortex.     

Effect of embryonic knock-down of GABAA receptors on the levels of monoamines and their metabolites in the CNS of the mouse

In vitro evidence indicates that γ-aminobutyric acid (GABA), acting at GABA_A receptors, exerts a positive trophic effect on monoaminergic neurons during embryogenesis. To determine whether in vivo antagonism of GABA_A receptors during embryogenesis interferes with the development of monoaminergic neurons, we used mice in which the number of GABA_A receptors was decreased by 50% by targeted deletion of the β₃ subunit gene of the GABA_A receptor. Levels of serotonin, dopamine, norepinephrine, and the metabolites 3,4-deoxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were measured in the brainstem, cortex, striatum and spinal cord of female adult homozygous null (β₃^−/−) and wild-type (β₃^+/+) mice, as well as progenitor C57BL/6J and Strain 129/SvJ mice. The level of norepinephrine in the spinal cord of β₃^−/− mice was 44% less than that of β₃^+/+ mice, and did not differ in the brainstem, cortex or striatum. This finding suggests that β₃ subunit-containing GABA_A receptors mediate the trophic effects of GABA on a subpopulation of spinally-projecting noradrenergic neurons. In contrast, the levels of serotonin, dopamine or their metabolites were unaffected, suggesting that the development of serotonergic and dopaminergic neurons may require activation of only a small fraction of GABA_A receptors or may not be dependent on β₃ subunit-containing GABA_A receptors. Finally, Strain 129/SvJ and C57BL/6J mice differed with respect to the levels of dopamine and its metabolites in the brainstem, spinal cord and cortex. These differences may need to be considered when assessing the phenotype of gene-targeted mice for which these mice serve as progenitor strains.     

No alterations in the 5-HT1A-mediated inhibition of forskolin-stimulated adenylate cyclase activity in the hippocampal membranes from rats chronically treated with lithium or antidepressants

Summary In order to determine the relevance of 5-HT_1A-related signal transduction in the mode of action of lithium and antidepressants, the effects of long-term treatment with these drugs on the 5-HT_1A-mediated inhibition of forskolin-stimulated adenylate cyclase activity were investigated in the rat hippocampal membranes. Chronic administration of antidepressants altered neither the [³H]8-hydroxy-2-(di-n-propylamino)tetralin ([³H]8-OH-DPAT) binding sites nor the inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT. Long-term treatment with lithium did not affect the inhibitory effect of 5-HT on the forskolin-stimulated adenylate cyclase activity, either. Neither the stimulation by forskolin nor the inhibition by guanyl-5-ylimidodiphosphate (Gpp(NH)p) of adenylate cyclase activity was not influenced by lithium treatment, suggesting that lithium has no effects on the components of adenylate cyclase system distal to the 5-HT_1A receptors.These results indicate that the 5-HT_1A-mediated neural transmission has not such an important relevance in the mechanisms of action of lithium or antidepressants.     

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 μM; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 μM) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE, receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE₁ receptor number (B_max) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPγS (100 μM) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of G_sα and S49 cyc⁻ reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE₁ receptors and G_s in situ by application of a C-terminal anti-G_sα antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE₁ receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available G_sα within the PGE₁ receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 μM)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE₁ receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.     

Distribution of adenosine receptors in the postmortem human brain: An extended autoradiographic study

Whole-hemisphere sections from six subjects were used in a quantitative autoradiographic study to characterize and to investigate the distribution of adenosine receptors, using [³H]DPCPX, [³H]CGS 21680, and [³H]SCH 58261 as radioligands. [³H]DPCPX-binding showed the pharmacology expected for adenosine A₁ receptors and is therefore taken to mirror adenosine A₁ receptors. Adenosine A₁ receptors were widely distributed, with the highest densities in the stratum radiatum/pyramidale of the hippocampal region CA 1. Adenosine A₁ receptors were nonhomogeneously distributed in nucleus caudatus, globus pallidus, and cortical areas: In the cingulate and frontal cortex the deep layers showed the highest labeling, while in the occipital, parietal, temporal, and insular cortex it was highest in the superficial layers. In addition, we found very high levels of adenosine A₁ receptors in structures known to be important for cholinergic transmission, especially the septal nuclei. The B_max values and K_D values for [³H]DPCPX-binding in stratum radiatum/pyramidale of CA1 and the superficial layer of insular cortex were 598 and 430 fmol/mg gray matter and 9.9 and 14.2 nM, respectively. [³H]CGS 21680-binding was multiphasic, but showed the pharmacology expected for adenosine A_2A receptors and was taken to represent them. Adenosine A_2A receptors were abundant in putamen, nucleus caudatus, nucleus accumbens, and globus pallidus pars lateralis. Specific [³H]CGS 21680-binding was also found in certain thalamic nuclei and throughout the cerebral cortex. The adenosine A_2A receptor antagonist radioligand [³H]SCH 58261 was also found to label these extrastriatal structures. Thus, adenosine A_2A receptors seem to be more widely distributed in the human brain than previously recognized. Synapse 27:322–335, 1997. © 1997 Wiley-Liss, Inc.     

Heterogeneity of binding sites for N-ethylcar☐amido[H]adenosine in rat brain: Effects of N-ethylmaleimide

Binding sites for N-ethylcar☐amido[³H]adenosine (NECA) in rat brain membranes and cryostat sections were examined in relation to their sensitivities to displacement by unlabeled NECA and R(−)-phenylisopropyladenosine (R-PIA). In membrane fractions from cerebral cortex, cerebellum, hippocampus and striatum, nanomolar concentrations of these adenosine receptor agonists displaced [³H]NECA such that R-PIA was more effective than NECA, consistent with the presence of an A₁-adenosine receptor. At concentrations of displacing agent higher than 1 μM, R-PIA was unable to displace [³H]NECA further in cerebral cortex, cerebellum and hippocampus. In striatum, a second R-PIA-sensitive component of [³H]NECA binding was evident which was more sensitive to NECA than to R-PIA, i.e. it showed the characteristics of an A₂-adenosine receptor. In striatum, however, R-PIA was also unable to displace [³H]NECA binding completely. Similar results were obtained in quantitative autoradiographic studies. Preincubation of cryostat sections with N-ethylmaleimide (NEM) abolished both the A₁- and R-PIA-insensitive binding components such that both NECA and R-PIA were able to displace [³H]NECA binding completely. The remaining sites showed IC₅₀ values of 0.13 and 3.68 μM for NECA and R-PIA, respectively. These A₂-like [³H]NECA binding sites had a highly specific distribution in the brain, being concentrated in the striatum, nucleus accumbens and olfactory tubercle. The results indicate the presence in brain tissue of at least 3 classes of [³H]NECA binding sites, an A₁-site, an A₂-site and a third, unidentified R-PIA-insensitive site.     

Prolonged exposure to hypobaric hypoxia transiently reduces GABAA receptor number in mice cerebral cortex

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day×21 days) applied to adult male mice on GABA_A receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [³H]GABA specifically bound to GABA_A receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (B_max control, 4.70±0.19, hypoxic, 3.33±0.10 pmol/mg protein), with no effect on GABA binding sites affinity (K_d control: 159.3±13.3 nM, hypoxic: 164.2±15.1 nM). Decreased B_max values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA_A receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5α-pregnan-3α-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA_A receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.     

MIF-I and postsynaptic receptor sites for dopamine

In an attempt to determine the mechanism by which the tripeptide l-prolyl-l-leucyl-glycine amide (PLG, MIF-I) exerts its antiparkinsonian effect, the action of this substance on various postsynaptic components of striatal dopaminergic nerves was studied. It was shown that injection of rats with MIF-I (1 mg/kg, IP×5, 24 hr intervals) did not alter tyrosine hydroxylase, dopa decarboxylase, choline acetyltransferase and glutamic acid decarboxylase activities in the striatum under the conditions tested. The activities of adenylate cyclase, dopamine-stimulated adenylate cyclase, and guanylate cyclase were not altered in vitro by various concentrations of MIF-I (0.1 to 1000 μM), although VIP and neurotensin had some effect. Also the rate of uptake of ³H-dopamine by rat striatal synaptosomes was unchanged, as was the binding of ³H-dopamine and ³H-spiperone to beef caudate membranes. This series of studies indicates that MIF-I does not act directly on the striatal dopamine postsynaptic receptor under the conditions tested, although it is possible that MIF-I could act indirectly at this or another site in vivo by releasing or activating some other factor.     

Modulation of ethanol-induced motor incoordination by mouse striatal A1 adenosinergic receptor

We have demonstrated that ethanol-induced motor incoordination is modulated by cerebellar adenosine A₁ receptor. This study represents an extension into another important brain motor area, the striatum that, unlike cerebellum, has high density of both A₁ and A_2A receptors. Direct intra-striatal micro-infusion of Ro15-4513 (0.05, 0.5, 1 ng), a partial inverse-agonist of benzodiazepine, significantly and nearly dose-dependently attenuated ethanol-induced motor incoordination indicating mediation of ethanol’s motor incoordination by striatum. Intra-striatal A₁-selective agonist N⁶-cyclohexyladenosine (CHA; 1, 2, 4 ng), A₁ = A_2A non-selective agonist, 5′-N-ethylcarboxamidoadenosine (NECA; 1.5, 3, 6 ng), and A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 25, 50, 100 ng) dose-dependently accentuated and attenuated, respectively, ethanol-induced motor incoordination, strongly suggesting modulation by striatal adenosine A₁ receptor. Intra-striatal DPCPX significantly antagonized not only ethanol-induced motor incoordination but also its potentiation by intra-striatal CHA, R-(+)-N⁶-(2-phenylisopropyladenosine) (R-PIA), or NECA. No change in motor coordination occurred after the highest dose of CHA, R-PIA, or NECA followed by saline. Similarly, the highest intra-striatal dose of Ro15-4513 or DPCPX neither altered motor coordination or locomotor activity indicating relative selectivity of interaction with ethanol. Nearly 25-fold higher dose of A_2A-selective agonist, CGS-21680, compared to CHA was necessary to produce a comparable potentiation of ethanol’s motor incoordination perhaps suggesting a lack of or less significant striatal A_2A involvement. Intra-striatal pertussis toxin (0.5 μg) pre-treatment markedly attenuated ethanol-induced motor incoordination as well as its potentiation by intra-striatal CHA. These results support that striatum is one of the brain motor areas mediating the motor impairing effects of acute ethanol and that the latter’s modulation occurs via A₁-selective receptors coupled to pertussis toxin-sensitive G proteins.