J. Stephen Fink

Molecular cloning of the rat A2 adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum

A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this cDNA in COS cells revealed high affinity (Kd = 38.6 nM) and saturable binding of the A2 adenosine receptor-selective ligand [3H]CGS 21680. Agonist displacement profile of [3H]CGS 21680 binding was consistent with an adenosine receptor of the A2 subtype (NECA greater than (R)-PIA greater than CPA greater than (S)-PIA). In situ hybridization demonstrated that rat A2 adenosine receptor mRNA was co-expressed in the same striatal neurons as D2 dopamine receptor mRNA, and never co-expressed with striatal D1 dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A2 subtype of adenosine receptor in the forebrain. The co-expression of D2 dopamine and A2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior.

Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow–derived cells

Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow-derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP-->WT, A2A-KO-->WT, or WT-->WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO-->WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT-->WT chimera. ATL146e reduced the induction of IL-6, IL-1beta, IL-1ra, and TGF-alpha mRNA in WT-->WT mice but not in A2A-KO-->WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells.

Enhanced CREB phosphorylation and changes in c-Fos and FRA expression in striatum accompany amphetamine sensitization

Expression in striatum of c-Fos, a 35 kDa Fos-related antigen (FRA) and the phosphorylated form of cyclic AMP response element binding protein (phosphoCREB) was assessed using Western blots in rats that developed behavioral sensitization following repeated amphetamine administration. Treatment with d-amphetamine (5 mg/kg) for 5 consecutive days produced behavioral sensitization. Similar to previous observations using chronic cocaine administration, amphetamine sensitized animals had decreased c-Fos and increased FRA proteins in striatum. Supershift analysis with antisera to c-Fos and FRA proteins demonstrated that 4-Fos and the 35 kDa FRA are components of the striatal AP-1 binding complex from sensitized rats. Thus, amphetamine sensitization is accompanied by alterations in the composition of the AP-1 DNA binding complex. An increased amount of phosphoCREB protein was also present in the striatum of amphetamine sensitized rats. These results suggest that alterations in Fos, FRA and CREB transcription factors are common neuronal responses to chronic psychostimulant administration and may contribute to regulation of genes important to the neuroplastic changes underlying psychostimulant sensitization.

The protein tyrosine phosphatase SHP-2 negatively regulates ciliary neurotrophic factor induction of gene expression

Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1-3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6,7] [8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9-11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.

Minireview: Sleep regulation in adenosine A2A receptor-deficient mice

Adenosine is proposed to be an endogenous sleep-promoting substance based on the results of a variety of pharmacologic and behavioral experiments.1 For example, sleep is induced in rats after administration of metabolically stable adenosine analogues, such as N6-l-(phenylisopropyl)-adenosine, adenosine-5′-N-ethylcarboxamide, and cyclohexyladenosine,2,3⇓ which are agonists for adenosine A1 receptor (A1R) or A2A receptors (A2ARs). Caffeine is considered to inhibit sleep by acting as an antagonist of adenosine receptor.4 Adenosine content is increased in the basal forebrain, one of the sleep centers, after sleep deprivation and is proposed to be a sleep substance accumulating in the brain during prolonged wakefulness.5 Most previous studies on sleep regulation by adenosine have focused on the A1R-mediated pathway1,6⇓ because A1R is widely distributed in the CNS, whereas A2AR is localized mainly in the striatum, nucleus accumbens, and olfactory bulb. However, we found that A2AR is also important in sleep regulation by using several A1R and A2AR agonists, including N6-cyclopentyladenosine (CPA) and 2-(4-(2-carboxyethyl)phenylethylamino)-adenosine-5′- N -ethylcarboxamideadenosine (CGS 21680).7-12⇓⇓⇓⇓⇓ CGS 21680 is highly selective for the A2AR (Ki = 14 nmol/L), having a much lower affinity for the A1R (Ki = 2,600 nmol/L), whereas CPA is selective for the A1R (Ki = 0.6 nmol/L) and has a lower affinity for the A2AR (Ki = 462 nmol/L).13,14⇓ We investigated the molecular mechanism of induction of non-REM (NREM) sleep by prostaglandin (PG) D2, which is also known as a potent endogenous sleep-promoting substance.15-17⇓⇓ In the course of this study, Satoh et al.7 found that PGD2 …

Characterization and expression of the human A2a adenosine receptor gene.

Abstract: The actions of the neurotransmitter adenosine are mediated by a family of high‐affinity, G protein‐coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans ∼9–10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5′ untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein‐coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.

Coordinate Regulation of Choline Acetyltransferase, Tyrosine Hydroxylase, and Neuropeptide mRNAs by Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor in Cultured Sympathetic Neurons

Abstract: The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine‐mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40–50% of superior cervical ganglion neurons in dissociated culture.

Synergistic interaction between an adenosine antagonist and a D1 dopamine agonist on rotational behavior and striatal c-Fos induction in 6-hydroxydopamine-lesioned rats

The interaction between adenosine and D1 dopamine systems in regulating motor behavior and striatal c-Fos expression was examined in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. These results were compared to the synergistic interaction between D1 and D2 dopamine systems in 6-OHDA rats. Coadministration of the adenosine antagonist 3,7-dimethyl-1-propargylxanthine (DMPX: 10 mg/kg) and the D1 dopamine agonist SKF38393 (0.5 mg/kg) to 6-OHDA-lesioned rats produced significant contralateral rotation and c-Fos expression in the ipsilateral striatum compared to 6-OHDA rats treated with either drug alone. However, the regional pattern of striatal c-Fos activation following treatment of 6-OHDA rats with SKF38393 and DMPX was different from the dorsolateral pattern of striatal c-Fos induction observed after coadministration of D1 and D2 dopamine agonists (SKF38393: 0.5 mg/kg + quinpirole: 0.05 mg/kg). These data are consistent with a functional interaction between D1 dopamine and adenosine systems in the striatum, but suggest that activation of different subsets of striatal neurons underlie the behavioral synergy observed following combined adenosine antagonist-D1 dopamine agonist and combined D1 dopamine agonist-D2 dopamine agonist treatment.

Genetic and pharmacological inactivation of the adenosine A2A receptor attenuates 3‐nitropropionic acid‐induced striatal damage

Adenosine A2A receptor (A2AR) antagonism attenuates 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced dopaminergic neurodegeneration and quinolinic acid‐induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3‐nitropriopionic acid (3‐NP). 3‐NP was administered to A2AR knockout (KO) and wild‐type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3‐NP‐treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3‐NP‐treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3‐NP‐induced striatal damage was observed in A2AR KO mice in a 129‐Steel background. In addition, the effect of pharmacological antagonism on 3‐NP‐induced striatal neurotoxicity was tested by pre‐treatment of C57Bl/6 mice with the A2AR antagonist 8‐(3‐chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3‐NP alone or 3‐NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3‐NP and in five of six mice treated with CSC (20 mg/kg) plus 3‐NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3‐NP. Since the clinical and neuropathological features of 3‐NP‐induced striatal damage resemble those observed in Huntingtons disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntingtons disease patients.

Differential localization of A2a adenosine receptor mRNA with D1 and D2 dopamine receptor mRNA in striatal output pathways following a selective lesion of striatonigral neurons

We have used the suicide transport agent volkensin to produce selective lesions of striatonigral neurons. By in situ hybridization histochemistry unilateral volkensin injections in the substantia nigra decreased the number of D1 receptor mRNA-expressing neurons in the ipsilateral striatum but did not change the number of D2 receptor and A2a adenosine receptor mRNA-expressing neurons. These findings confirm that striatonigral neurons express D1 receptors and suggest that D2-A2a receptor expressing neurons are predominantly localized to other neuronal populations within the striatum.