Adenosine receptors in colon carcinoma tissues and colon tumoral cell lines: focus on the A(3) adenosine subtype

Adenosine may affect several pathophysiological processes, including cellular proliferation, through interaction with A(1), A(2A), A(2B), and A(3) receptors. In this study we characterized adenosine receptors in human colon cancer tissues and in colon cancer cell lines Caco2, DLD1, HT29. mRNA of all adenosine subtypes was detected in cancer tissues and cell lines. At a protein levels low amount of A(1), A(2A), and A(2B) receptors were detected, whilst the A(3) was the most abundant subtype in both cancer tissues and cells, with a pharmacological profile typical of the A(3) subtype. All the receptors were coupled to stimulation/inhibition of adenylyl-cyclase in cancer cells, with the exception of A(1) subtype. Adenosine increased cell proliferation with an EC(50) of 3-12 microM in cancer cells. This effect was not essentially reduced by adenosine receptor antagonists. However dypiridamol, an adenosine transport inhibitor, increased the stimulatory effect induced by adenosine, suggesting an action at the cell surface. Addition of adenosine deaminase makes the A(3) agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (Cl-IB-MECA) able to stimulate cell proliferation with an EC(50) of 0.5-0.9 nM in cancer cells, suggesting a tonic proliferative effect induced by endogenous adenosine. This effect was antagonized by 5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo [1,5-c] pyrimidine (MRE 3008F20) 10 nM. Cl-IB-MECA-stimulated cell proliferation involved extracellular-signal-regulated-kinases (ERK1/2) pathway, as demonstrated by reduction of proliferation with 1,4-diamino-2,3-dicyano-1,4-bis-[2-amino-phenylthio]-butadiene (U0126) and by ERK1/2 phosphorylation. In conclusion this study indicates for the first time that in colon cancer cell lines endogenous adenosine, through the interaction with A(3) receptors, mediates a tonic proliferative effect.     

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression.

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.     

Developmental regulation of neuronal survival by adenosine in the in vitro and in vivo avian retina depends on a shift of signaling pathways leading to CREB phosphorylation or dephosphorylation

Previous studies have shown a cAMP/protein kinase A-dependent neuroprotective effect of adenosine on glutamate or re-feeding-induced apoptosis in chick retina neuronal cultures. In the present work, we have studied the effect of adenosine on the survival of retinal progenitor cells. Cultures obtained from 6-day-old (E6) or from 8-day-old (E8) chick embryos were challenged 2 h (C0) or 1 day (C1) after seeding and analyzed after 3-4 days in vitro. Surprisingly, treatment with the selective A2a adenosine receptor agonists N(6) -[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) or 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) promoted cell death when added at E6C0 but not at E6C1 or E8C0. DPMA-induced cell death involved activation of A2a receptors and the phospholipase C/protein kinase C but not the cAMP/protein kinase A pathway, and was not correlated with early modulation of precursor cells proliferation. Regarding cyclic nucleotide responsive element binding protein (CREB) phosphorylation, cultures from E6 embryos behave in an opposite manner from that from E8 embryos, both in vitro and in vivo. While the phospho-CREB level was high at E6C0 cultures and could be diminished by DPMA, it was lower at E8C0 and could be increased by DPMA. Similar to what was observed in cell survival studies, CREB dephosphorylation induced by DPMA in E6C0 cultures was dependent on the Phospholipase C/protein kinase C pathway. Accordingly, cell death induced by DPMA was inhibited by okadaic acid, a phosphatase blocker. Moreover, DPMA as well as the adenosine uptake blocker nitrobenzyl mercaptopurine riboside (NBMPR) modulate cell survival and CREB phosphorylation in a population of cells in the ganglion cell layer in vivo. These data suggest that A2a adenosine receptors as well as CREB may display a novel and important function by controlling the repertoire of developing retinal neurons.     

Adenosine acts as a chemoprotective agent by stimulating G-CSF production: a role for A1 and A3 adenosine receptors

Adenosine, a ubiquitous nucleoside, is released into the extracellular environment from metabolically active or stressed cells. It binds to cells through specific A1, A(2A), A(2B), and A3 G-protein-associated cell-surface receptors, thus acting as a signal-transduction molecule by regulating the levels of adenylyl cyclase and phospholipase C. In this study, we showed that adenosine stimulates the proliferation of murine bone marrow cells in vitro. Pharmacological studies, using antagonists to the adenosine receptors, revealed that this activity was mediated through the binding of adenosine to its A1 and A3 receptors. This result was further corroborated by showing that the two selective A1 and A3 receptor agonists, N-cyclopentyladenosine (CPA) and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-D-ribofuranuronamide (IB-MECA) respectively, induced bone marrow cell proliferation in a manner similar to adenosine. Adenosine's interaction with its A1 and A3 receptors induced G-CSF production, which led to its stimulatory effect on bone marrow cells. These results were confirmed in vivo when we demonstrated that low-dose adenosine (0.25 mg/kg) acted as a chemoprotective agent. When administered after chemotherapy, it restored the number of leukocytes and neutrophils to normal levels, compared with the decline in these parameters after chemotherapy alone. It is suggested that low-dose adenosine, already in clinical use, may also be applied as a chemoprotective agent.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.     

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

Synthesis and biological evaluation of 2-aminothiazoles and their amide derivatives on human adenosine receptors. Lack of effect of 2-aminothiazoles as allosteric enhancers

A number of 2-aminothiazoles (2a-e) and their amide derivatives (4-10) were prepared. The 2-aminothiazoles themselves were tested as allosteric enhancers of agonist binding to human adenosine A(1) receptors. In a variety of experimental set-ups the compounds did not show any such effect, in contrast to earlier findings by another research group. Subsequently the 2-aminothiazoles were used as intermediates in the synthesis of a number of amide derivatives of either aromatic (4-6) or aliphatic nature (7-10). Some of the compounds emerged as moderately active antagonists on human adenosine A(1) and/or A(2A) receptors with lower or negligible potency at adenosine A(3) receptors.     

Homeostatic action of adenosine A3 and A1 receptor agonists on proliferation of hematopoietic precursor cells

Two adenosine receptor agonists, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and N6-cyclopentyladenosine (CPA), which selectively activate adenosine A3 and A1 receptors, respectively, were tested for their ability to influence proliferation of granulocytic and erythroid cells in femoral bone marrow of mice using morphological criteria. Agonists were given intraperitoneally to mice in repeated isomolar doses of 200 nmol/kg. Three variants of experiments were performed to investigate the action of the agonists under normal resting state of mice and in phases of cell depletion and subsequent regeneration after treatment with the cytotoxic drug 5-fluorouracil. In the case of granulopoiesis, IB-MECA 1) increased by a moderate but significant level proliferation of cells under normal resting state; 2) strongly increased proliferation of cells in the cell depletion phase; but 3) did not influence cell proliferation in the regeneration phase. CPA did not influence cell proliferation under normal resting state and in the cell depletion phase, but strongly suppressed the overshooting cell proliferation in the regeneration phase. The stimulatory effect of IB-MECA on cell proliferation of erythroid cells was observed only when this agonist was administered during the cell depletion phase. CPA did not modulate erythroid proliferation in any of the functional states investigated, probably due to the lower demand for cell production as compared with granulopoiesis. The results indicate opposite effects of the two adenosine receptor agonists on proliferation of hematopoietic cells and suggest the plasticity and homeostatic role of the adenosine receptor expression.     

Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells

Adenosine, acting at its receptors, particularly A(2A) receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A(2A) receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective A(2A) receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A(2A) receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-alpha (100 U/ml) but markedly attenuated in cells pretreated with IFN-gamma (100 U/ml). Similarly, IL-1 and TNF-alpha treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-gamma treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-alpha-treated cells but not in control or IFN-gamma-treated cells. Both IL-1 and TNF-alpha increased A(2A) receptor mRNA and protein. In parallel with its effect on A(2A) receptor function, IFN-gamma down-regulated A(2A) receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.     

Polydeoxyribonucleotides enhance the proliferation of human skin fibroblasts: involvement of A2 purinergic receptor subtypes.

It is well-known that nucleotides, nucleosides and purine/pyrimidine bases enhance cell proliferation in vitro. Nevertheless, the molecular mechanisms involved in this mitogenic activity is still controversial, since these compounds are reported both to synergize with growth factor, and to act directly on purinergic receptor inducing per se a proliferative response. It was suggested that cell growth enhancement could be mediated by the A2 purinergic receptor activation. Here we report that a polydeoxyribonucleotide (PDRN) and adenosine are able to increase, the growth rate of human skin fibroblasts in primary cultures. The proliferative activity exerted by PDRN was significantly counteracted by the A2 antagonist 3, 7-Dimethyl-1-propargylxanthine (DMPX), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (PD 116,948, DPCPX). Accordingly, the trophic action of PDRN was mimicked by the A2 agonist N6-[2-(3,5-Dimethoxyphenyl)-2-(methylphenyl)-ethyl]adenosine (DPMA), while the A1 agonist N6-Cyclopenthyladenosine (CPA) did not show any effect. In microfluorimetric studies, we observed that PDRN and adenosine increased the concentration of cytosolic calcium ions. The PDRN-evoked calcium rise was dose-dependent and DMPX sensitive. Taken together, our results suggest that PDRN may operate as a pro-drug providing the cultured cells with an effective amount of mitogenic deoxyribonucleotides, deoxyribonucleosides and bases; moreover, cell proliferation enhancement that has been induced by PDRN seems to be mediated, at least in part, by the activation of purinergic receptors of the A2 subtype.     

A highly conserved tryptophan residue in the fourth transmembrane domain of the A1 adenosine receptor is essential for ligand binding but not receptor homodimerization

Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A₁ adenosine receptor (A₁R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A₁R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A₁R, as evident from coimmunoprecipitation and BRET² analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A₁R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.     

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.     

Differential effect of adenosine on tumor and normal cell growth: focus on the A3 adenosine receptor

Adenosine is an ubiquitous nucleoside present in all body cells. It is released from metabolically active or stressed cells and subsequently acts as a regulatory molecule through binding to specific A1, A2A, A2B and A3 cell surface receptors. The synthesis of agonists and antagonists to the adenosine receptors and their cloning enabled the exploration of their physiological functions. As nearly all cells express specific adenosine receptors, adenosine serves as a physiological regulator and acts as a cardioprotector, neuroprotector, chemoprotector, and as an immunomodulator. At the cellular level, activation of the receptors by adenosine initiates signal transduction mechanisms through G-protein associated receptors. Adenosine's unique characteristic is to differentially modulate normal and transformed cell growth, depending upon its extracellular concentration, the expression of adenosine cell surface receptors, and the physiological state of the target cell. Stimulation of cell proliferation following incubation with adenosine has been demonstrated in a variety of normal cells in the range of low micromolar concentrations, including mesangial and thymocyte cells, Swiss mouse 3T3 fibroblasts, and bone marrow cells. Induction of apoptosis in tumor or normal cells was shown at higher adenosine concentrations (>100 microM) such as in leukemia HL-60, lymphoma U-937, A431 epidermoid cells, and GH3 tumor pituitary cell lines. It was further noted that the A3 adenosine receptor (A3AR) plays a key role in the inhibitory and stimulatory growth activities of adenosine. Modulation of the A3AR was found to affect cell growth either positively or negatively depending on the concentration of the agonist, similar to the effect described for adenosine. At nanomolar concentrations, the A3AR agonists possess dual activity, i.e., antiproliferative activity toward tumor cells and stimulatory effect on bone marrow cells. In vivo, these agonists exerted anti-cancer effects, and when given in combination with chemotherapy, they enhanced the chemotherapeutic index and acted as chemoprotective agents. Taken together, activation of the A3AR, by minute concentrations of its natural ligand or synthetic agonists, may serve as a new approach for cancer therapy.     

Th1 cytokines regulate adenosine receptors and their downstream signaling elements in human microvascular endothelial cells

We and others have shown that adenosine, acting at its receptors, is a potent modulator of inflammation and angiogenesis. To better understand the regulation of adenosine receptors during these processes we studied the effects of IL-1, TNF-alpha, and IFN-gamma on expression and function of adenosine receptors and select members of their coupling G proteins in human dermal microvascular endothelial cells (HMVEC). HMVEC expressed message and protein for A(2A) and A(2B), but not A(1) or A(3) receptors. IL-1 and TNF-alpha treatment increased message and protein expression of A(2A) and A(2B) receptor. IFN-gamma treatment also increased the expression of A(2B) receptors, but decreased expression of A(2A) receptors. Resting HMVEC and IFN-gamma-treated cells showed minimal cAMP response to the selective A(2A) receptor agonist 2-[2-(4-chlorophenyl)ethoxy]adenosine (MRE0094). In contrast, MRE0094 stimulated a dose-dependent increase in cAMP levels in TNF-alpha-treated cells that was almost completely blocked by the A(2A) receptor antagonist ZM-241385 (4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl]phenol). The nonselective adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine increased cAMP levels in both TNF-alpha- and IFN-gamma-treated cells, but not control cells, and its effect was only partially reversed by ZM-241385 in TNF-alpha-treated cells and not affected in IFN-gamma-treated cells. HMVEC expressed a higher level of G protein beta1 isoform than beta4 isoform. Although none of the cytokines tested affected G(beta1) expression, both IL-1 and TNF-alpha significantly up-regulated G(beta4) expression. These findings indicate that inflammatory cytokines modulate adenosine receptor expression and function on HMVECs and suggest that the interaction between proinflammatory cytokines and adenosine receptors may affect therapeutic responses to anti-inflammatory drugs that act via adenosine-dependent mechanisms.     

Modulation of sodium transport in fetal alveolar epithelial cells by oxygen and corticosterone

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP > UDP >or= UTP > adenosine >or= 2-methylthioadenosine-5'-diphosphate, with alpha,beta-methylene adenosine 5'-triphosphate, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, AMP, UMP, and ATPalphaS inactive] and PCR analysis indicate involvement of P2Y2 and P2Y6 receptors. The signal transduction pathway, which, via the P2Y2 receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on Ca2+/calmodulin-dependent protein kinase II and nuclear factor-kappaB, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1beta and interleukin-6, but not tumor necrosis factor-alpha. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells.     

Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 3',5'-monophosphate and phosphatases

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A(1), A(2a), A(2b), A(3)) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A(2) receptor agonists and was blocked by selective A(2a) and A(2b) receptor antagonists, indicating that adenosine was mediating its effect through A(2) receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A(2) receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.