Cloning and expression of the A2a adenosine receptor from guinea pig brain

A full-length complementary DNA (cDNA) clone encoding the guinea pig brain A₂ adenosine receptor has been isolated by polymerase chain reaction (PCR) and low-stringency-hybridization screening of a guinea pig brain cDNA library. This cDNA contains a long open reading frame encoding a 409-amino acid-residue protein which is highly homologous to the A₂ adenosine receptors previously cloned from other species. Hydrophobicity analysis of the deduced protein sequence reveals seven hydrophobic regions, characteristic of a member of the G-protein-coupled receptor superfamily. Radioligand binding assay and functional (GTPase and cAMP) assays of the receptor, transiently expressed in mammalian cells, demonstrate typical characteristics of the A₂ type adenosine receptor. The messenger RNA (mRNA) of this A₂ receptor is found in the brain, heart, kidney and spleen. Receptor autoradiography with [³H]CGS21680, a specific A₂ agonist, and in situ hybridization with A₂ cRNA probe in guinea pig brain indicate that the receptor is expressed exclusively in the caudate nucleus. The pharmacological profile and anatomical distribution of this receptor indicate that it is of the A_2a subtype. This work represents the first cloning of an A_2a receptor in a rodent species, offers a complete pharmacological characterization of the receptor and provides an anatomical comparison between binding profile and gene expression of the receptor.Abbreviations ADAC adenosine amine congener - BA N⁶-benzyladenosine - bp nucleotide base pair - cAMP cyclic adenosine 3,5-monophosphate - CCPA 2-chloro-N⁶-cyclopentyladenosine - CGS 21680 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamido adenosine hydrochloride - CHA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclopentyladenosine - CPX 8-cyclopentyl-1,3-dipropylxanthine - DME Dulbecco's modified Eagle's medium - DMPX 3,7-dimethyl-1-propargylxanthine - DPMX 1,3-dipropyl-7-methylxanthine - DPX 1,3-dipropyl-8-(2-amono-4-chlorophenyl)xanthine - FCS fetal calf serum - IBMX 3-isobutyl-1-methylxanthine - KHB Kreb-HEPES buffer - MECA 5-N-methylcarboxamidoadenosine - NECA 5-N-ethylcarboxamidoadenosine - D-PBS Dulbecco's phosphate buffered saline - PCR polymerase chain reaction - R-PIA R(–)-N⁶-(2-phenylisopropyl)adenosine - SSPE sodium chloride-sodium phosphate-EDTA buffer - TM transmembrane domain - XAC xanthine amine congener Special issue dedicated to Dr. Bernard W. Agranoff     

Mobilization of Storage Pool Dopamine and Late Ipsilateral Augmentation of Striatal Dopamine Synthesis in the Trained Circling Rat

Rats were infused intraventricularly with [3H]tyrosine over a 20-min period during various times while circling. 3,4-Dihydroxyphenylethylamine (dopamine) and dihydroxyphenylacetic acid (DOPAC) levels were measured using HPLC with electrochemical detection and fractions were collected for tritium monitoring. During the first 20 min of circling, the specific activity of dopamine was increased by 290% in striatum contralateral to the circling direction whereas DOPAC specific activity was increased 50% on the same side. This differential change in relative specific activity suggests that unlabeled storage pool dopamine was mobilized to DOPAC during circling. Synthesis of dopamine and DOPAC in contralateral striatum returned to baseline levels as turning slowed (50-70 min). When turning ceased, there was an increase in ipsilateral striatal dopamine synthesis during the 20-min period following circling. We hypothesize that this ipsilateral increase represents either a "stop" signal following circling or a release of inhibition of ipsilateral nigral neurons.     

ADP-Ribosylation by Cholera Toxin of Membranes Derived from Brain Modifies the Interaction of Adenylate Cyclase with Guanine Nucleotides and NaF

We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.     

Unilateral Activation of Caudate Tyrosine Hydroxylase During Voluntary Circling Behavior

We trained rats to circle for a sucrose water reward and found that this behavior is associated with a unilateral increase in the activity of caudate tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis. The increase in tyrosine hydroxylase activity occurs in caudate contralateral to the circling direction and the change is transient, increasing during the first 20 min of circling but then plateauing and falling as turning slows. Enhanced synthetic capacity is followed by increases in the contents of dopamine and dihydroxyphenylacetic acid in the contralateral caudate nucleus. These observations are the first evidence for specific activation of a neurotransmitter synthetic enzyme during voluntary motor behavior.     

Methamphetamine induces long-term changes in GABAA receptor alpha2 subunit and GAD67 expression

The present study investigated whether GABA(A) receptor alpha2 subunit and GAD(67) are involved in chronic high dose methamphetamine (METH)-induced sensitization and neurotoxicity. The METH sensitization was established in rats by 7-day pump infusion plus daily injection (25mg/kg/day) and a subsequent 28-day withdrawal period. Behavioral sensitization was assessed by behavioral ratings after challenge with METH (0.5mg/kg). The neurotoxicity was evaluated by the expression of glial fibrillary acidic protein (GFAP). Western blot assay showed that METH sensitization decreases GABA(A) alpha2 subunit and GAD(67) protein levels in the nucleus accumbens (NAc) core and shell, and conversely, these proteins were increased in the caudate. An upregulation of GFAP expression was observed in the caudate, but not in the NAc core and shell. These data suggest that inhibition of GABA transmission in the NAc is related to METH behavioral sensitization, whereas activation of GABA transmission in the caudate is associated with METH-induced neurotoxicity.     

Intra-striatal estradiol in female rats impairs response learning within two hours of treatment

Estradiol treatment administered systemically or directly to the dorsolateral striatum across two days impairs performance on a response task in which rats learn to make a specific body turn to locate food on a maze. Estradiol can act through both slow and rapid signaling pathways to regulate learning impairments, however it is impossible to dissociate the slow from the rapid contributions of estradiol following long exposures. To assess the rapid effects of estradiol on striatum-sensitive learning, we trained rats on a response learning task after either relatively short or long treatments of estradiol infused directly into the striatum. Three-month-old female rats were ovariectomized 21 days before training and received guide cannulae implanted bilaterally into the dorsolateral striatum. For short duration treatments, rats were given bilateral infusions (0.5 μl) of 17β-estradiol-sulfate (0, 5, 50, or 500 nM in aCSF-vehicle) either 2 h or 15 min prior to training. For long duration treatments, rats received a series of estradiol infusions (500 nM) at 48, 24, and 2 h prior to training. Replicating previous findings (Zurkovsky et al., 2007), intra-striatal estradiol treatments given for two days prior to training impaired response learning. Estradiol-induced impairments in performance were also demonstrated 2 h, but not 15 min, after single infusions. Thus, estradiol acts within hours of exposure in the striatum, a structure lacking classical estrogen receptors, to impair response learning.     

Activation of mu opioid receptors in the striatum differentially augments methamphetamine-induced gene expression and enhances stereotypic behavior

Mu opioid receptors are densely expressed in the patch compartment of striatum and contribute to methamphetamine-induced patch-enhanced gene expression and stereotypy. To further elucidate the role of mu opioid receptor activation in these phenomena, we examined whether activation of mu opioid receptors would enhance methamphetamine-induced stereotypy and prodynorphin, c-fos, arc and zif/268 expression in the patch and/or matrix compartments of striatum, as well as the impact of mu opioid receptor activation on the relationship between patch-enhanced gene expression and stereotypy. Male Sprague-Dawley rats were intrastriatally infused with d-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO; 1?μg/μL), treated with methamphetamine (0.5?mg/kg) and killed at 45?min or 2?h later. DAMGO augmented methamphetamine-induced zif/268 mRNA expression in the patch and matrix compartments, while prodynorphin expression was increased in the dorsolateral patch compartment. DAMGO pre-treatment did not affect methamphetamine-induced arc and c-fos expression. DAMGO enhanced methamphetamine-induced stereotypy and resulted in greater patch versus matrix expression of prodynorphin in the dorsolateral striatum, leading to a negative correlation between the two. These findings indicate that mu opioid receptors contribute to methamphetamine-induced stereotypy, but can differentially influence the genomic responses to methamphetamine. These data also suggest that prodynorphin may offset the overstimulation of striatal neurons by methamphetamine.     

Comparison of the In Vitro Receptor Binding Properties of N-[3H]Methylspiperone and [3H]Raclopride to Rat and Human Brain Membranes

The aim of the present investigation was to study and compare the in vitro binding properties of the two radioligands N-[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug-naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5-hydroxytryptamine 2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10-fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half-life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The Ki values were, however, two- to four-fold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.     

Plasminogen activator inhibitor-2 (PAI-2) mRNA is localized in the accumbens nucleus of the mouse brain and is induced in specific brain sites after kainate excitation

Plasminogen activator inhibitor-2 (PAI-2) specifically inhibits plasminogen activators, extracellular fibrinolytic serine proteases that are also implicated in brain plasticity and toxicity. Primarily localized intracellularly, PAI-2 is thought to also counteract apoptosis mediated by a currently undefined intracellular protease. Here we localized PAI-2 mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate excitation. We found that in the normal brain PAI-2 mRNA was confined to an area within the accumbens nucleus shell. After kainate was injected (i.p.), PAI-2 mRNA was substantially and rapidly (within 2 h) induced in neuron-like cells primarily in layers II–III of the neocortex; the cingulate, piriform, entorhinal and perirhinal cortices; the olfactory bulb, nucleus and tubercle; in the accumbens nucleus, shell and core; throughout the caudate putamen and the amygdaloid complex; in the CA1 and CA3 areas of the hippocampus, and in the parasubiculum. These findings suggest that PAI-2 could play a role in the accumbens nucleus as well as in activity-related events associated with olfactory, striatal, and limbic structures.     

RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.