Pro- and anti-inflammatory macrophages express a sub-type specific purinergic receptor profile.

Extracellular nucleotides act as danger signals that orchestrate inflammation by purinergic receptor activation. The expression pattern of different purinergic receptors may correlate with a pro- or anti-inflammatory phenotype. Macrophages function as pro-inflammatory M1 macrophages (M1) or anti-inflammatory M2 macrophages (M2). The present study found that murine bone marrow-derived macrophages express a unique purinergic receptor profile during in vitro polarization. As assessed by real-time polymerase chain reaction (PCR), Gαs-coupled P1 receptors A2A and A2B are upregulated in M1 and M2 compared to M0, but A2A 15 times higher in M1. The ionotropic P2 receptor P2X5 is selectively upregulated in M1- and M2-polarized macrophages. P2X7 is temporarily expressed in M1 macrophages. Metabotropic P2Y receptors showed a distinct expression profile in M1 and M2-polarized macrophages: Gαq coupled P2Y1 and P2Y6 are exclusively upregulated in M2, whereas Gαi P2Y13 and P2Y14 are overexpressed in M1. This consequently leads to functional differences between M1 and M2 in response to adenosine di-phosphate stimulation (ADP): In contrast to M1, M2 showed increased cytoplasmatic calcium after ADP stimulation. In the present study we show that bone marrow-derived macrophages express a unique repertoire of purinergic receptors. We show for the first time that the repertoire of purinergic receptors is highly flexible and quickly adapts upon pro- and anti-inflammatory macrophage differentiation with functional consequences to nucleotide stimulation.

Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. CD affects people worldwide, primarily in tropical areas. The central nervous system (CNS) is an essential site for T. cruzi persistence during infection. The protozoan may pass through the blood-brain barrier and may cause motor and cognitive neuronal damage. Once in the CNS, T. cruzi triggers immune responses that the purinergic system can regulate. Treatment for CD is based on benznidazole (BNZ); however, this agent has negative side-effects and is toxic to the host. For this reason, we investigated whether resveratrol (RSV), a potent antioxidant and neuroprotective molecule, would modulate purinergic signaling and RSV alone or in combination with BNZ would prevent changes in purinergic signaling and oxidative damage caused by T. cruzi. We infected mice with T. cruzi and treated them with RSV or BNZ for 8 days. Increases in ATP and ADP hydrolysis by NTPDase in the total cortex of infected animals were observed. The treatment with RSV in infected group diminished ATP, ADP, and AMP hydrolysis compared to infected group. The combination of RSV + BNZ decreased AMP hydrolysis in infected animals compared to the INF group, exerting an anti-inflammatory effect. RSV acted as a neuroprotector, decreasing adenosine levels. Infected animals presented an increase of P2X7 and A2A density of purine receptors. RSV reduced P2X7 and A2A and increased A1 density receptors in infected animals. In addition, infected animals showed higher TBARS and reactive oxygen species (ROS) levels than control. RSV diminished ROS levels in infected mice, possibly due to antioxidant properties. In short, we conclude that resveratrol could act as a neuroprotective molecule, probably preventing inflammatory changes caused by infection by T. cruzi, even though the mice experienced high levels of parasitemia.

Purinergic Receptor Expression and Cellular Responses to Purinergic Agonists in Human Prostate Cancer Cells.

BACKGROUND: Anticancer activity of extracellular nucleotides has been investigated in many types of cancer. Herein, the effects of extracellular nucleotides and the receptor profile for these nucleotides on prostate cancer (PCa) were elaborated. MATERIALS AND METHODS: PCa cell lines representing different stages of PCa were used. The effects of ATP and adenosine on PCa growth and migration on different extracellular matrix proteins were examined by MTT and wound-healing assays. Purinergic receptor profiling was carried out by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A growth-inhibitory effect of ATP and adenosine was observed on all PCa cell lines tested. Several ATP-recognized P2 receptors and adenosine receptors were commonly expressed in PCa cell lines. Neither ATP nor adenosine had any significant effect on PCa migration. CONCLUSION: ATP and adenosine had an antiproliferative effect on PCa cells without affecting their motility, indicating their potential as a novel therapy for PCa.

Effects of perinatal exposure to lead (Pb) on purine receptor expression in the brain and gliosis in rats tolerant to morphine analgesia.

The aim of the present study was to investigate the molecular effects of perinatal exposure to lead (Pb) on protein and mRNA expression of purine receptors: P2X4, P2X7, adenosine receptor A1; and astrocytes (GFAP mRNA expression) and on microglia activation (Iba1 mRNA expression) in several structures of the mesolimbic system (striatum, hippocampus, prefrontal cortex) in rats expressing tolerance to the antinociceptive effect of morphine. Rat mothers were orally treated with 0.1% lead acetate from conception, through gestation, and postnatally, as well as to offspring up to day (PND) 28; subsequently molecular studies were conducted on adult (PND 60) male rats. Morphine tolerance developed more strongly in rats perinatally exposed to Pb. The analysis revealed a significant up-regulation of protein and mRNA P2X4 receptor expression in the striatum and prefrontal cortex but not in the hippocampus; P2X7 protein and mRNA receptor expression in the striatum and hippocampus, but not in the prefrontal cortex; A1 protein receptor expression in all investigated structures and A1 mRNA expression in the striatum and hippocampus; Iba1 mRNA expression in the striatum and hippocampus; and GFAP mRNA expression in the striatum and prefrontal cortex. Immunohistochemical analysis has also revealed significant alterations. Strong expressions of P2X4, P2X7, A1 receptors, astrocytes and microglia activation were observed in the hippocampus in Pb and/or morphine treated rats. The higher expression of purine receptors and glial cell activation are important markers of neuroinflammatory processes. Therefore, we conclude that Pb-induced neuroinflammation may be responsible for the intensification of morphine tolerance in the Pb-treated rats. Additionally, the dysregulation of A1 adenosine receptors, mainly in the hippocampus, may also be involved in the intensification of morphine tolerance in Pb-treated rats. Our study demonstrates the significant participation of environmental factors in addictive process; additionally, it shows the necessity of modification of addictive disorder with neuroprotective agents.

Physiological and immune-biological characterization of a long-term murine model of blunt chest trauma.

Blunt chest trauma causes pulmonary and systemic inflammation. It is still a matter of debate whether the long-term course of this inflammatory response is associated with persistent impairment of lung function. We hypothesized that an increase of inflammatory biomarkers may still be present at later time points after blunt chest trauma, eventually, despite normalized lung mechanics and gas exchange. Anesthetized spontaneously breathing male C57BL/6J mice underwent a blast wave-induced blunt chest trauma or sham procedure. Twelve and 24 h later, blood gases and lung mechanics were measured, together with blood, bronchoalveolar lavage (BAL), and tissue cytokine concentrations (multiplex cytokine kit); heme oxygenase 1 (HO-1), activated caspase-3, Bcl-xL, and Bax expression (Western blotting); nuclear factor-κB activation (electrophoretic mobility shift assay); nitrotyrosine formation; and purinergic (P2XR4 and P2XR7) receptor expression (immunohistochemistry). Histological damage was assessed by hematoxylin and eosin and periodic acid-Schiff staining. High-resolution respirometry allowed assessing mitochondrial respiration in diaphragm biopsies. Chest trauma significantly increased tissue and BAL cytokine levels, associated with a significant increase in HO-1, purinergic receptor expression, and tissue nitrotyrosine formation. In contrast, lung mechanics, gas exchange, and histological damage did not show any significant difference between sham and trauma groups. Activation of the immune response remains present at later time points after murine blunt chest trauma. Discordance of the increased local inflammatory response and preserved pulmonary function may be explained by a dissociation of the immune response and lung function, such as previously suggested after experimental sepsis.

Blunted coronary vasodilator response to uridine adenosine tetraphosphate in post-infarct remodeled myocardium is due to reduced P1 receptor activation.

We previously demonstrated that uridine adenosine tetraphosphate (Up4A) exerts a potent vasodilator effect in the healthy porcine coronary vasculature. Since the coronary microvascular effects of Up4A after myocardial infarction (MI) are unknown, the present study investigated the response to Up4A in coronary microvessels from post-MI remodeled porcine myocardium, and the involvement of purinergic receptor subtypes. Coronary small arteries (diameter ∼150 µm) were dissected from the apex of Sham-operated swine and swine in which MI had been produced 5 weeks earlier by transient (2h) occlusion of the left circumflex coronary artery, and mounted on Mulvany wire myographs. Up4A (10(-9)-10(-5)M) produced coronary vasodilation that was reduced in MI as compared to Sham-operated swine. Up4A-induced vasodilation was reduced by P1 blockade with 8-phenyltheophylline in Sham-operated swine and to a lesser extent in MI, while the attenuation by the A2A receptor blocker SCH58261 was similar in Sham-operated and MI swine. Up4A-induced vasodilation remained unaffected by non-selective P2 receptor antagonist PPADS, but was attenuated by selective P2X1 and P2Y1 receptor antagonists MRS2159 and MRS2179, albeit to a similar extent in Sham-operated and MI swine. These responses were paralleled by similar mRNA expression levels of A2A, P2X1 and P2Y1 receptors in MI compared to slaughterhouse control swine. Finally, attenuation of Up4A-induced coronary vasodilation by nitric oxide synthase inhibition was not attenuated in MI as compared to Sham-operated swine. In conclusion, blunted coronary vasodilation in response to Up4A in MI swine is most likely due to reduced activation of P1, rather than P2, receptors and does not involve a loss of NO bioavailability.

Purinergic receptors expressed in human skeletal muscle fibres.

Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls showed similar distribution of receptors. The current study demonstrates that purinergic receptors are located intracellularly in human skeletal muscle fibres. The similar cellular localization of receptors in healthy and diabetic subjects suggests that diabetes is not associated with an altered distribution of purinergic receptors in skeletal muscle fibres. We speculate that the intracellular localization of purinergic receptors may reflect a role in regulation of muscle metabolism; further studies are nevertheless needed to determine the function of the purinergic system in skeletal muscle cells.

Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.

Biphenotypic acute leukemia (BAL) is a relatively rare subtype of acute leukemia characterized by the presence of both myeloid and lymphoid cell surface antigens. We have now screened for transforming genes in BAL blasts with the use of the focus formation assay with a retroviral cDNA expression library constructed from malignant blasts isolated from a BAL patient. Some of the retroviral inserts recovered from transformed foci were found to encode wild-type purinergic receptor P2Y, G protein coupled, 8 (P2RY8). The oncogenic potential of P2RY8 was confirmed with the in vitro focus formation assay as well as with an in vivo tumorigenicity assay in nude mice. A variety of luciferase-based reporter assays revealed that P2RY8 increased both the trans-activation activities of CREB and Elk-1 as well as the transcriptional activities of the serum response element and enhancer-promoter fragments of the c-Fos and c-Myc genes. Quantitation of P2RY8 mRNA in CD34(+) cells of bone marrow showed that P2RY8 expression is frequently increased in leukemia patients, especially in those with refractory disease. Our data thus reveal an abundant expression of P2RY8 in leukemic cells and its unexpected role in the pathogenesis of acute leukemia.

P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl-D-aspartate-glutamate and acetylcholine receptors during neuronal differentiation.

The in vitro differentiation of P19 murine embryonal carcinoma cells to neurons resembles developmental stages which are encountered during neuronal development. Three days following induction to neuronal differentiation by retinoic acid, most cells of the P19 population lost expression of the stage specific embryonic antigen (SSEA-1) and expressed the neural progenitor cell specific antigen nestin. Beginning from day 4 of differentiation nestin expression was down-regulated, and expression of neuron-specific enolase as marker of differentiated neurons increased. The molecular mechanisms underlying neuronal differentiation are poorly understood. We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at mRNA transcription and protein levels as well as ATP-induced Ca2+ transients during neuronal differentiation of P19 cells. Gene and protein expression of P2X2, P2X6, P2Y2, and P2Y6 receptors increased during the course of differentiation, whereas P2X3, P2X4, P2Y1 and P2Y4 receptor expression was high in embryonic P19 cells and then decreased following induction of P19 cells to differentiation. P2X1 receptor protein expression was only detected on days 2 and 4 of differentiation. Although P2X5 and P2X7 mRNA transcription was present, no protein expression for this receptor subunit could be detected throughout the differentiation process. In undifferentiated cells, mainly ionotropic P2X receptors contributed to the ATP-induced Ca2+-response. In neuronal-differentiated P19 cells, the ATP-induced Ca2+-response was increased and the metabotropic component predominated. Purinergic receptor function is implicated to participate in neuronal maturation, as cholinergic and glutamate-N-methyl-D-aspartate (NMDA) induced calcium responses were affected when cells were differentiated in the presence of purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin or reactive blue-2. Our data suggest that inhibition of P2Y1 and possibly P2X2 receptors led to a loss of NMDA receptor activity whereas blockade of possibly P2X2 and P2Y2 purinergic receptors during neuronal differentiation of P19 mouse led to inhibition of cholinergic receptor responses.

Alterations in purinoceptor expression in human long saphenous vein during varicose disease.

OBJECTIVES: Varicose veins are dilated tortuous veins of varying tone. Purinergic signalling is important in the control of tone and in mediating trophic changes in blood vessels. The expression of P2 receptors in control and varicose veins will be examined. METHODS: Purinergic signalling in circular and longitudinal smooth muscle of the human long saphenous vein was studied in control and varicose tissues using immunohistochemistry, organ bath pharmacology and electron microscopy. RESULTS: P2X1, P2Y1, P2Y2, P2Y4 and P2Y6 receptors were present on circular and longitudinal smooth muscle. Purine-mediated circular and longitudinal muscle contractions were weaker in varicose veins. Electron microscopy and immunohistochemistry findings support the view that smooth muscle cells change from the contractile to synthetic phenotype in varicose veins, associated with an upregulation of P2Y1 and P2Y2 receptors and a down regulation of P2X1 receptors. CONCLUSIONS: Down regulation of P2X1 receptors on the smooth muscle of varicose veins is associated with loss of contractile activity. Upregulation of P2Y1 and P2Y2 receptors is associated with a shift from contractile to synthetic and/or proliferative roles. The phenotype change in smooth muscle is associated with weakening of vein walls and may be a causal factor in the development of varicose veins.

Purinergic signaling and kinase activation for survival in pulmonary oxidative stress and disease.

Stimulus-induced release of endogenous ATP into the extracellular milieu has been shown to occur in a variety of cells, tissues, and organs. Extracellular ATP can propagate signals via P2 receptors that are essential for growth and survival of cells. Abundance of P2 receptors, their multiple isoforms, and their ubiquitous distribution indicate that they transmit vital signals. Pulmonary epithelium and endothelium are rich in both P2X and P2Y receptors. ATP release from lung tissue and cells occurs upon stimulation both in vivo and in vitro. Extracellular ATP can activate signaling cascades composed of protein kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K). Here we summarize progress related to release of endogenous ATP and nucleotide signaling in pulmonary tissues upon exposure to oxidant stress. Hypoxic, hyperoxic, and ozone exposures cause a rapid increase of extracellular ATP in primary pulmonary endothelial and epithelial cells. Extracellular ATP is critical for survival of these cells in high oxygen and ozone concentrations. The released ATP, upon binding to its specific receptors, triggers ERK and PI3K signaling and renders cells resistant to these stresses. Impairment of ATP release and transmission of such signals could limit cellular survival under oxidative stress. This may further contribute to disease pathogenesis or exacerbation.

Expression of functional purinergic receptors in pulmonary neuroepithelial bodies and their role in hypoxia chemotransmission.

Adenine nucleotides act through specific cell surface receptors to invoke a variety of biological responses. Here we show that cells of neuroepithelial bodies (NEB), presumed O2 airway sensors in neonatal hamster lung, express functional P2X receptors (P2X-R). Positive immunostaining was detected in NEB cells using double-label immunohistochemistry with antibodies against P2X2 and P2X3 receptor subunits, which co-localized with serotonin (5-HT), a marker of NEB cells. For electrophysiological characterization of P2X2-R in NEB cells, fresh neonatal hamster lung slice preparation was used. Under whole-cell patch clamp, perfusion with ATP induced a concentration-dependent, non-desensitizing inward current (EC50=12 microM). Perfusion with alpha,beta-methylene ATP also induced a slow-desensitizing inward current (EC50=8.2 microM). Suramin (IC50 ca. 43 microM) and TNP-ATP (IC50 ca. 8 microM) blocked the currents evoked by both ATP and alpha,beta-methylene ATP. Using carbon fiber amperometry we observed that hypoxia and ATP induced 5-HT release from NEB cells and that this release was blocked by suramin. These data suggest that functional P2X2/3 heteromeric receptors are expressed in NEB cells. The possible function of these purinoreceptors in NEB cells could include modulation of hypoxia chemotransmission.

Purinergic receptor expression in the regeneration epidermis in a rat model of normal and delayed wound healing.

This study investigated changes in the protein expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalized. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps, and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day four after wounding. The re-epithelializing wound edges were analyzed immunohistochemically for P2X(5), P2X(7), P2Y(1) and P2Y(2) receptors, and immunostaining of keratinocytes was quantified using optical densitometry. In normal rat epidermis, P2Y(1) and P2Y(2) receptors were found in the basal layer where keratinocytes proliferate; P2X(5) receptors were associated with proliferating and differentiating epidermal keratinocytes in basal and suprabasal layers; P2X(7) receptors were associated with terminally differentiated keratinocytes in the stratum corneum. In the regenerating epidermis of denervated wounds, P2Y(1) receptor protein expression was significantly increased in keratinocytes (P<0.001) but P2Y(1) receptors (P<0.001) compared with untreated denervated wounds. In innervated wounds, NGF treatment enhanced expression in keratinocytes. P2X(5) (P>0.001) and P2Y(1) receptor protein (P<0.001) expression in keratinocytes. P2X(7) receptors were absent in all experimental wound healing preparations. P2X(5), P2X(7), P2Y(1) and P2Y(2) receptor protein expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.

Ligand-gated purinergic receptors are differentially expressed in the adult rat vestibular periphery.

To further characterize the pattern of expression of the ligand-gated purinergic P2X receptors in the peripheral vestibular system, we conducted reverse transcription-polymerase chain reaction amplification of P2X1 and P2X2 messenger RNA extracted from adult rat vestibular ganglia (Scarpa's ganglia) and vestibular end organs. Transcripts encoding P2X1 were found in both Scarpa's ganglia and the end organs, but transcripts encoding P2X2 were found only in the vestibular end organs. These results support previous electrophysiological data, and they provide a more complete understanding of the specific role of purinergic (adenosine-5'-triphosphate) transmission in the vestibular periphery.

Extracellular ATP and ADP activate transcription factor NF-kappa B and induce endothelial cell apoptosis.

Inflammation within the vasculature is associated with endothelial cell (EC) perturbation, loss of vascular ATP-diphosphohydrolase activity, and platelet microthrombus formation with release of ATP and ADP into the micro-environment. The nature and effects of purinergic stimulation of EC under these circumstances remain largely undetermined. ATP and ADP activated EC transcribed mRNA from certain transcription factor NF-kappa B target genes and expressed E-selectin protein on cell membranes. Band shift analysis and reporter assays confirmed the activation of NF-kappa B in response to both ATP and ADP. Apoptosis was shown to occur in response to purinergic signaling, potentially through the activation of P2z/P2x7 receptors. Induction of EC activation responses and apoptosis in response to stimulation with ATP and ADP is associated with activation of NF-kappa B.

Domains of P2X receptors involved in desensitization.

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X1 (or P2X3) receptor had strong desensitization, whereas currents in cells expressing P2X2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X1 receptor of either one of two 34-residue segments from the P2X2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X2 receptor only by providing both these segments of the P2X1 (or P2X3) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

m3 Muscarinic receptor-induced and Gi-mediated heterologous potentiation of phospholipase C stimulation: role of phosphoinositide synthesis.

Agonist activation of thrombin and purinergic receptors endogenously expressed in human embryonic kidney (HEK) cells and of the stably expressed m3 muscarinic acetylcholine receptor (mAChR) induces phospholipase C (PLC) stimulation, with the most pronounced PLC stimulation observed on mAChR activation. These receptor responses were pertussis toxin (PTX) insensitive and nonadditive, suggesting that the receptors share common signaling pathways. Short term (2 min) pretreatment of HEK cells with carbachol (1 mM), but not ATP, followed by agonist washout, caused a long-lasting (> or = 90 min) sensitization of PLC responses. At 30 min after carbachol treatment and washout, mAChR-stimulated PLC activity, measured as formation of either total inositol phosphates or of inositol-1,4,5-trisphosphate, was enhanced by 1.5-2-fold. PLC stimulation by thrombin and purinergic receptors was increased by approximately 3-fold. Furthermore, carbachol pretreatment also enhanced, by approximately 2.5-fold, stimulation of PLC activity on direct activation of G proteins by AIF4- and guanosine-5'-O-(3-thio)-triphosphate in intact and permeabilized cells, respectively. In contrast, PLC activities, measured with exogenous phosphatidylinositol-4,5-bisphosphate [Ptdns(4,5)P2] in HEK cell lysates, were not altered, suggesting that carbachol pretreatment may enhance the cellular level of Ptdlns(4,5)P2. Indeed, the level of Ptdlns(4,5)P2 was found to be increased by approximately 50% in HEK cells 30 min after short term carbachol treatment, whereas the level of phosphatidylinositol was not altered and that of phosphatidylinositol-4-phosphate decreased (by 40-50%). Pretreatment of HEK cells with PTX prevented the m3 mAChR-induced PLC potentiation and reduced the elevation in Ptdlns(4,5)P2 level by approximately 50%. In conclusion, short term agonist activation of m3 mAChRs stably expressed in HEK cells can lead to a longlasting heterologous potentiation of PLC signaling, which processes apparently involve PTX-sensitive G proteins and an enhanced PLC substrate supply.

Transient expression of the recombinant chick brain P2y1 purinoceptor and localization of the corresponding mRNA.

Extracellular ATP acts at specific cell surface purinoceptors to elicit a wide range of physiological responses. We have recently isolated a cDNA for a G-protein-coupled P2 purinoceptor (P2y1) from chick brain. It has been defined as a P2Y-like purinoceptor by the rank order of potency of P2 purinoceptor ligands, determined electrophysiologically in the Xenopus oocyte expression system. Here, we examine the ligand selectivity of this recombinant receptor, expressed transiently in COS-7 cells. The regional distribution of the P2y1 purinoceptor transcript within the one-day-post-hatch chick brain was also determined. It is widely expressed in the cerebellum and telencephalon and in specific nuclei of the mesencephalon and diencephalon, suggesting a neuronal localization of the P21 purinoceptor.

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.