GABA Alters GABAA Receptor mRNAs and Increases Ligand Binding

Abstract: Adenosine A_2a receptors have been localized to GABAergic striatopallidal neurons, but their functional role is unknown. To address this question, the modulation of endogenous GABA release by adenosine A_2a receptors was examined in slices of rat globus pallidus. The selective adenosine A_2a receptor agonist CGS-21680 (3.0–10 n M ) significantly increased electrically stimulated release (overflow) of GABA, with 10 n M CGS-21680 resulting in a 44% increase compared with the control. Both the nonselective adenosine receptor antagonist 8-phenyltheophylline (10 μ M ) and the selective A_2a receptor antagonist KF-17837 (100 n M ) abolished the CGS-21680-induced increase in GABA overflow. Higher concentrations of CGS-21680 (0.10–1.0 μ M ) decreased GABA overflow by ˜25%.8-Phenyltheophylline (10 μ M ) antagonized these effects, whereas KF-17837 (100 n M ) did not, suggesting actions of CGS-21680 on other adenosine receptors at these concentrations. These results demonstrate that activation of adenosine A_2a receptors augments electrically stimulated release of GABA from globus pallidus slices and suggest a mechanism by which adenosine may modulate GABAergic output from the striatopallidal efferent system.     

A2A Adenosine Receptors Inhibit ATP-Induced Ca2+ Influx in PC12 Cells by Involving Protein Kinase A

Abstract: The regulatory role of A_2A adenosine receptors in P₂ purinoceptor-mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2- p -(2-carboxyethyl)-phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680), a specific agonist of the A_2A adenosine receptor, the extracellular ATP-evoked rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5-trisphosphate production evoked by ATP were not affected by CGS-21680 treatment. However, ATP-evoked Ca²⁺ influx was inhibited following CGS-21680 stimulation. The CGS-21680-mediated inhibition occurred independently of nifedipine-induced inhibition of the [Ca²⁺]_i rise. The CGS-21680-induced inhibition was completely blocked by reactive blue 2. The CGS-21680 effect was mimicked by forskolin and dibutyryl-cyclic AMP and blocked by Rp -adenosine 3',5'-cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A_2A adenosine receptors leads to inhibition of P₂ purinoceptor-mediated Ca²⁺ influx through ATP-gated cation channels and involves protein kinase A.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

Photoaffinity Labeling with 2-[2-(4-Azido-3-[125I]-Iodophenyl)ethylamino]adenosine and Autoradiography with 2-[2-(4-Amino-3-[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain

Abstract: The A_2a adenosine receptor agonist 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [¹²⁵I]APE, discriminates between high- and low-affinity conformations of A_2a adenosine receptors. In this study, [¹²⁵I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [¹²⁵I]APE binding is consistent with that expected of an A_2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [¹²⁵I]APE binding to these brain regions is abolished by 1 µ M 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 n M 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [¹²⁵I]APE to the corresponding arylazide results in [¹²⁵I]AzPE. The rank-order potency of compounds to compete for [¹²⁵I]AzPE binding in the dark is CGS-21680 > d -( R )- N ⁶-phenylisopropyladenosine > N ⁶-cyclopentyladenosine, indicating that it also is an A_2a-selective ligand. Specific photoaffinity labeling by [¹²⁵I]AzPE of a single polypeptide (42 kDa) corresponding to A_2a adenosine receptors is reduced 55 ± 4% by 100 µ M guanosine 5'- O -(3-thiotriphosphate) and 91 ± 1.3% by 100 n M CGS-21680. [¹²⁵I]APE and [¹²⁵I]AzPE are valuable new tools for characterizing A_2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.     

Inhibition of Voltage-Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells

Abstract: The role of the A_2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ could be inhibited with CGS21680, an A_2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A_2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp -adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A_2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca²⁺ concentration induced by 70 m M K⁺, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca²⁺ influx without VSCC activity after cobalt or 70 m M K⁺ pretreatment. These data suggest that the A_2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A_2A receptors induce inhibition of VSCCs as well as ATP-induced Ca²⁺ influx, the two inhibitory effects are clearly distinct from each other.     

Adenosine A2a Receptor-Mediated Modulation of Striatal Acetylcholine Release In Vivo

Abstract: To determine the functions of striatal adenosine A_2a receptors in vivo, the effects of a selective agonist, 2-[4-(2-carboxyethyl)phenethylamino]-5'- N -ethylcarboxamidoadenosine hydrochloride (CGS 21680), and an antagonist, ( E )-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837), on acetylcholine release were investigated in the striatum of awake freely moving rats using microdialysis. Intracerebroventricular injection of CGS 21680 (10 µg) increased acetylcholine release in striatum and KF17837 (30 mg/kg p.o.) antagonized the CGS 21680-induced acetylcholine elevation. To investigate the contribution of dopaminergic and GABAergic neurons on A_2a receptor-mediated acetylcholine release, the effects of CGS 21680 were studied by using dopamine-depleted rats in the presence or absence of GABA antagonists. In the dopamine-depleted striatum, the intrastriatal application of CGS 21680 (0.3–30 µ M ) increased extracellular acetylcholine, which was significantly greater than that in normal striatum. The CGS 21680-induced elevation of acetylcholine release was still observed in the presence of GABA antagonists bicuculline (30 µ M ) and 2-hydroxysaclofen (100 µ M ) and was similar in both normal and dopamine-depleted striatum. These results suggest that A_2a agonist stimulates acetylcholine release in vivo, and this effect of A_2a agonist is modulated by dopaminergic and GABAergic neurotransmission.     

Dual Signalling by the Adenosine A2a Receptor Involves Activation of Both N- and P-Type Calcium Channels by Different G Proteins and Protein Kinases in the Same Striatal Nerve Terminals

Abstract: Many G_s-linked receptors have been reported to use multiple signalling pathways in transfected cells but few in their normal cell environment. We show that the adenosine A_2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of G_s, adenylyl cyclase, protein kinase A, and P-type calcium channels; the other is mediated by a cholera toxin-insensitive G protein, protein kinase C, and N-type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A_2a receptor antagonist KF17837. This demonstrates that the A_2a receptor activates two signalling systems in striatal cholinergic neurons.     

Adenosine A2b Receptors Mediate an Increase in Interleukin (IL)-6 mRNA and IL-6 Protein Synthesis in Human Astroglioma Cells

Abstract: The cytokine interleukin (IL)-6 has recently been demonstrated to play a role in the pathology of Alzheimer's disease (AD). The mechanisms leading to increased IL-6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the level of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL-6 gene expression in the brain. Here we show that the mixed A₁ and A₂ agonist 5'-( N -ethylcarboxamido)adenosine (NECA) induces an increase in IL-6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A₁-specific agonists R -phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A_2a-specific agonist CGS-21680 shows only marginal effects. Increased levels of mRNA are already found within 30 min after NECA treatment. The A_2a-selective antagonists 8-(3-chlorostyryl)caffeine and KF17837 [( E )-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine], which have also some antagonistic properties at A_2b receptors, and the nonspecific adenosine antagonist 8-phenyltheophylline were equipotent at inhibiting the NECA-induced increase in IL-6 protein synthesis, whereas the specific A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine is much less potent. The results indicate that adenosine A_2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A_2A receptor on the A₁ receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A_2A receptor agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low-affinity binding site for the A₁ receptor agonist 2-chloro- N ⁶-cyclopentyladenosine (CCPA). This effect was blocked by the A_2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C-mediated heterologous desensitisation of the A₁ receptor by the A_2A receptor.     

Activation of Phosphodiesterase IV During Desensitization of the A2A Adenosine Receptor-Mediated Cyclic AMP Response in Rat Pheochromocytoma (PC12) Cells

Abstract: Prolonged activation of an A_2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A_2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A_2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)-selective inhibitor (Ro 20-1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A_2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A_2A-selective agonist (CGS21680) leads to increased PDE4 activity in a dose-dependent manner, which can be blocked by an A_2A-selective antagonist [8-(3-chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4-immunoreactive bands (72 and 79 kDa) were increased significantly during A_2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A_2A desensitization could be blocked by a protein kinase A (PKA)-selective inhibitor (H89) and was not observed in a PKA-deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA-dependent pathway plays a critical role in dampening the signal of the A_2A receptor.     

Role of Histidine Residues in the Adenosine A2a Receptor Ligand Binding Site

Abstract: The pH dependency of the binding of ligands to adenosine A_2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two- to fourfold increase in affinity was observed for CGS-21680, 5'- N -ethylcarboxamidoadenosine, adenosine, 3'-deoxyadenosine, 5'-deoxyadenosine, inosine, and N ⁶-methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2'-deoxyadenosine, although 2'-methoxyadenosine binding was pH dependent. This effect on the affinity of CGS-21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2'-hydroxyl group of adenosine agonists.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

GABAB Receptor-Mediated Effects in Synaptosomes of Lethargic (lh/lh) Mice

Abstract: Previously, we have shown a significant increase in number of GABA_B receptor binding sites in neocortex and thalamus of lethargic ( lh/lh ) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABA_B receptors would inhibit [³H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age-matched male lh/lh and +/+ mice were similar in uptake of [³H]GABA. In the neocortical preparation, baclofen dose-dependently inhibited [³H]GABA release evoked by 12 m M KCl, an effect mediated by GABA_B receptors. The maximal inhibition ( I _max) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC₅₀ (3 µ M ) was unchanged. In the thalamic preparation, the effect of baclofen (50 µ M ) was 58% less robust in lh/lh mice. Other effects mediated by GABA_B receptors (inhibitions in Ca²⁺ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABA_B receptor-mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABA_B receptors on GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.     

Adenosine A1 Receptor Inhibition of Glutamate Exocytosis and Protein Kinase C-Mediated Decoupling

Abstract: The adenosine modulation of glutamate exoeytosis from guinea pig cerebrocortical synaptosomes is investigated. Endogenously leaked adenosine is sufficient to cause a partial tonic inhibition of 4-aminopyridine-evoked glutamate release, which can be relieved by adenosine deaminase. The adenosine A₁ receptor is equally effective in mediating inhibition of glutamate exocytosis evoked by 4-aminopyridine (where K⁺-channel activation would inhibit release) and by elevated KC1 (where K⁺-channel activation would have no effect), arguing for a central role of Ca²⁺-channel modulation. In support of this, the plateau phase of depolarization-evoked free Ca²⁺ elevation is decreased by adenosine with both depolarization protocols. No effect of adenosine agonists is seen on membrane potential in polarized or KC1- or 4-aminopyridine-stimulated synaptosomes. The interaction of protein kinase C with the A₁ receptormediated inhibition is examined. Activation of protein kinase C by 4β-phorbol dibutyrate has been shown previously by this laboratory to modulate glutamate release via K⁺-channel inhibition, and is shown here to have an additional action of decoupling the adenosine inhibition of glutamate exocytosis.     

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D₁ receptor positively coupled with adenylyl cyclase, together with the D₃ receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D₃ receptor signaling pathways. In NG108-15 cells transfected with the human D₃ receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [³H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole-induced [³H]thymidine incorporation. This effect was maximal with 100 n M forskolin, occurred after 6–16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D₃ receptor expression up to 135%, but only after 16 h and at concentrations of >1 µ M . Thus, in this cell line, the D₃ receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D₃ receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D₃ receptor can involve both opposite and synergistic interactions with cyclic AMP.     

Activation of Phospholipase A2 by the Nociceptin Receptor Expressed in Chinese Hamster Ovary Cells

Abstract: To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A₂ (PLA₂) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca²⁺-dependent cytosolic PLA₂ (cPLA₂) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA₂, suggesting that phosphorylation of cPLA₂ is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA₂ phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA₂ through the action of PTX-sensitive G proteins and suggest that cPLA₂ is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

Phosphorylation of DARPP-32 Is Regulated by GABA in Rat Striatum and Substantia Nigra

Abstract: In the medium-sized spiny neurons of the striatonigral pathway, a cascade of events involving the activation of dopamine D₁ receptors, an increase in cyclic AMP, and activation of cyclic AMP-dependent protein kinase causes the phosphorylation of DARPP-32 on Thr³⁴, converting DARPP-32 into a powerful inhibitor of protein phosphatase-1. In the present study, the incubation of striatal or substantia nigra slices with GABA also increased the phosphorylation of DARPP-32 on Thr³⁴. GABA did not significantly increase cyclic AMP levels in slices. The phosphorylation of DARPP-32 by GABA was blocked in both brain regions by pretreatment of slices with the GABA_A receptor antagonist, bicuculline, but not with the GABA_B receptor antagonist, phaclofen. Moreover, the threonine phosphorylation of DARPP-32 produced by maximally effective doses of either forskolin (in striatum) or l -3,4-dihydroxyphenylalanine (in substantia nigra) was increased further by GABA. The data are consistent with a model in which GABA increases the phosphorylation state of DARPP-32 by inhibiting dephosphorylation of the protein by the calcium/calmodulin-dependent protein phosphatase, calcineurin.     

Characterization of CB1 Receptors on Rat Neuronal Cell Cultures: Binding and Functional Studies Using the Selective Receptor Antagonist SR 141716A

Abstract: This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [³H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites ( B _max = 139 ± 9 fmol/mg of protein) displaying a high affinity ( K _D = 0.76 ± 0.09 n M ). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 µ M forskolin or isoproterenol with EC₅₀ values in the nanomolar range (4.6 and 65 n M with forskolin and 1.0 and 5.1 n M with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 µ M forskolin with a potency similar to that observed in cortical neurons (EC₅₀ values of 3.5 and 1.9 n M in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.     

Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.