Adenosine A2A receptor gene expression in the normal striatum and after 6-OH-dopamine lesion

Adenosine A2A receptors are present on enkephalinergic medium sized striatal neurons in the rat and have an important function in the modulation of striatal output. In order to establish more accurately whether adenosine transmission is a generalized phenomenon in mammalian striatum we compared the A2A R expression in the mouse, rat, cat and human striatum. Secondly we compared the modulation of enkephalin gene expression and A2A receptor gene expression in rat striatal neurons after 6-OH-dopamine lesion of the substantia nigra. Hybridization histochemistry was performed with a 35S-labelled radioactive oligonucleotide probe. The results showed high expression of A2A adenosine receptor genes only in the medium-sized cells of the striatum in all examined species. In the rat striatum, expression of A2A receptors was not significantly altered after lesion of the dopaminergic pathways with 6-OH-dopamine even though enkephalin gene expression was up-regulated. The absence of a change in A2A receptor gene expression after 6-OH-dopamine treatment speaks against a dependency on dopaminergic innervation. The maintained inhibitory function of A2A R on motor activity in spite of dopamine depletion could be partly responsible for the depression of locomotor activity observed in basal ganglia disorders such as Parkinson's disease.

Expression of adenosine A2a receptor gene in rat dorsal root and autonomic ganglia

The adenosine A2a receptors (A2aR) play an important role in the purinergic mediated neuromodulation. The presence of A2aR in the brain is well established. In contrast, little is known about their expression in the periphery. The purpose of this study was to investigate the expression of A2aR gene in the autonomic (otic, sphenopalatine, ciliary, cervical superior ganglia and carotid body) and in the dorsal root ganglia of normal rat. Hybridization histochemistry with S35-labelled radioactive oligonucleotide probes was used. An expression of A2aR gene was found in the large neuronal cells of the rat dorsal root ganglia. The satellite cells showed no expression of A2aR gene. In the superior cervical ganglion, isolated ganglion cells expressed A2aR. In the carotid body clusters of cells with a strong A2aR gene expression were found. In contrast, the ciliary and otic ganglia did not expressed A2aR gene, and only few small sized A2aR expressing cells were demonstrated in the sphenopalatine ganglion. The discrete distribution of A2aR gene expression in the peripheral nervous system speaks for a role of this receptor in the purinergic modulation of sensory information as well as in the sympathetic nervous system.

Development of small molecular tools for the cellular study of adenosine receptors

The adenosine receptors are members of the G-protein coupled receptor (GPCR) family which represents the largest class of cell-surface proteins mediating cellular communication. As a result, GPCRs are formidable drug targets and it is estimated that approximately 30% of the marketed drugs act through members of this receptor class. There are four known subtypes of adenosine receptors: A1, A2A, A2B and A3. The adenosine A1 receptor, which is the subject of this presentation, mediates the physiological effects of adenosine in various tissues including the brain, heart, kidney and adipocytes. In the brain for instance, its role in epilepsy and ischemia has been the focus of many studies. Previous attempts to study the biosynthesis, trafficking and agonist-induced internalisation of the adenosine A1 receptor in neurons using fluorescent protein-receptor fusion constructs have been hampered by the sheer size of the fluorescent protein (GFP) that ultimately affected the function of the receptor. We have therefore initiated a research programme to develop small molecule fluorescent agonists that selectively activate the adenosine A1 receptor. Our probe design is based on the endogenous ligand adenosine and the known unselective adenosine receptor agonist NECA. We have synthesised a small library of non-fluorescent adenosine derivatives that have different cyclic and bicyclic moieties at the 6 position of the purine ring and have evaluated the pharmacology of these compounds using a yeast-based assay. This analysis revealed compounds with interesting behaviour, i.e. exhibiting subtype-selectivity and biased signalling, that can be potentially used as tool compounds in their own right for cellular studies of the adenosine A1 receptor. Furthermore, we have also linked fluorescent dyes to the purine ring and discovered fluorescent compounds that can activate the adenosine A1 receptor.

Adenosine diphosphate (ADP) and ADP receptor play a major role in platelet activation/aggregation induced by sera from heparin-induced thrombocytopenia patients

The molecular basis for heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is not yet fully understood. We found that pretreatment of platelets with AR-C66096 (formerly FPL 66096), a specific platelet adenosine diphosphate (ADP) receptor antagonist, at a concentration of 100 to 200 nmol/L that blocked ADP-dependent platelet aggregation, resulted in complete loss of platelet aggregation responses to HIT sera. AR-C66096 also totally inhibited HIT serum-induced dense granule release, as judged by measurement of adenosine triphosphate (ATP) release. Apyrase, added to platelets at a concentration that had only minor effects on thrombin- or arachidonic acid-induced aggregation, also blocked completely HIT serum-induced platelet aggregation. Furthermore, AR-C66096 inhibited platelet aggregation and ATP release induced by cross-linking Fc gamma RIIA with specific antibodies. These data show that released ADP and the platelet ADP receptor play a pivotal role in HIT serum-induced platelet activation/aggregation. The thromboxane receptor inhibitor, Daltroban, had no effect on HIT serum-induced platelet activation whereas GPIIb-IIIa antagonists blocked platelet aggregation but had only a moderate effect on HIT serum-induced dense granule release. Pretreatment of platelets with chondroitinases but not with heparinases resulted in concentration dependent inhibition of HIT serum-induced platelet aggregation. These novel data relating to the mechanism of platelet activation induced by HIT sera suggest that the possibility should be examined that ADP receptor antagonists or compounds that inhibit ADP release may be effective as therapeutic agents for the prevention or treatment of complications associated with heparin therapy.

Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity

A series of N6-bicyclic and N6-(2-hydroxy)cyclopentyl derivatives of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We observed that the most selective, high potency ligands were achieved through N6-adamantyl substitution in combination with 5′-N-ethylcarboxamido or 5′-hydroxymethyl groups. In addition, we determined that 5′-(2-fluoro)thiophenyl derivatives all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compounds were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homology modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes, these compounds might have therapeutic potential.

Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies

Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARalpha, PPARgamma) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n=7 per group). Upon first presentation of dogs, mRNA levels of PPARalpha, PPARgamma, CAR, PXR and RXRalpha in duodenum as well as PPARgamma, CAR, PXR and RXRalpha in colon were not different among groups (P>0.10). Although mRNA abundance of PPARalpha in colon of dogs with FRD was similar in both IBD and CON (P>0.10), PPARalpha mRNA abundance was higher in IBD than CON (P<0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P<0.05) or CON (P<0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P<0.05). Differences in mRNA levels of PPARalpha and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.

Nuclear receptor and nuclear receptor target gene messenger ribonucleic acid levels at different sites of the gastrointestinal tract and in liver of healthy dogs

Nuclear receptors (NR) are ligand-activated transcription factors that regulate different metabolic pathways by influencing the expression of target genes. The current study examined mRNA abundance of NR and NR target genes at different sites of the gastrointestinal tract (GIT) and the liver of healthy dogs (Beagles; n = 11). Samples of GIT and liver were collected postmortem and homogenized, total RNA was extracted and reverse transcribed, and gene expression was quantified by real-time reverse-transcription PCR relative to the mean of 3 housekeeping genes (beta-actin, glyceraldehyde-3-phosphate dehydrogenase, and ubi-quitin). Differences were observed (P < or = 0.05) in the mRNA abundance among stomach (St), duodenum (Du), jejunum (Je), ileum (Il), and colon (Col) for NR [pregnane X receptor (Du, Je > Il, Col > St), peroxisome proliferator-associated receptor gamma (St, Du, Col > Je, Il), constitutive androstane receptor (Je, Du > Il, Col), and retinoid x receptor alpha (Du > Il)] and NR target genes [glutathione-S-transferase A3-3 (Du > Je > St, Il; St > Col), phenol-sulfating phenol sulfotransferase 1A1 (Du, Je > Il, St; Col > St), cytochrome P450 3A12 (Du, Je > St, Il, Col), multiple drug resistance gene 1 (Du, Je, Il, Col > St), multiple drug resistance-associated protein 2 (Je, Du > Il > St, Col), multiple drug resistance-associated protein 3 (Col > St > Il; Du > Je, Il; St > Il), NR corepressor 2 (St > Il, Col), and cytochrome P450 reductase (St, Du, Je > Il, Col)], but not for peroxisome proliferator-associated receptor alpha. Differences (P > 0.05) in mRNA abundance in the liver relative to the GIT were also observed. In conclusion, the presence of numerous differences in expression of NR and NR target genes in different parts of the GIT and in liver of healthy dogs may be associated with location-specific functions and regulation of GIT regions.

Sodium retention and ascites formation in a cholestatic mice model: role of aldosterone and mineralocorticoid receptor?

Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. CONCLUSION: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids.

Expression of adenosine A2a receptors gene in the olfactory bulb and spinal cord of rat and mouse

The expression of adenosine A2a receptors (A2aR) in the mammalian striatum is well known. In contrast the exact distribution of A2aR in other regions of the central nervous system remains unclear. The aim of this study was to investigate the A2aR gene expression in the rat olfactory bulb and spinal cord, two regions which are seldom included in mapping studies. Secondly, we compared the A2aR expression in the rat and in the mouse brain. Hybridization histochemistry was performed with an S35-labelled radioactive oligonucleotide probe. The results show strong expression of A2aR in the mouse and rat striatum in accordance with previous reports. In the olfactory bulb a weak but specific expression of A2aR was found in the granular cell layer in both species. In contrast, no significant expression of the A2aR gene was observed in other parts of the brain or the rat spinal cord. The presence of the A2aR in the mammalian olfactory bulb suggests a functional role for this receptor in olfaction.