Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors.

The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A(2B) adenosine receptors expressed in Chinese hamster ovary (hA(2B)CHO), in human embryonic kidney 293 (hA(2B)HEK-293) and at endogenous A(2B) receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A(2B) adenosine radioligand [(3)H]-N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide ([(3)H]-MRE 2029F20) revealed a single class of binding sites in hA(2B)CHO, hA(2B)HEK-293 and HMC-1 cells with K(D) (nM) of 1.65+/-0.18, 2.83+/-0.34, 2.62+/-0.27 and B(max) (fmol/mg protein) of 36+/-4, 475+/-50 and 128+/-15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA(2B)HEK-293 cells using [(3)H]-MRE 2029F20, showed a rank order of potency typical of the A(2B) receptors with K(i) values in the range 3.2-28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA(2B)CHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA(2B)CHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA(2B)CHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC(50) value of 2.9+/-0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A(2B) receptors.

Aberrant A2A receptor function in peripheral blood cells in Huntington's disease.

A2A adenosine receptors specifically found on striatal medium spiny neurons play a major role in sensory motor function and may also be involved in neuropsychiatric and neurodegenerative disorders. One hypothesis concerning Huntington's disease (HD) proposes that an imbalance of the cortico-striatal pathway, due to the mutation in the HD gene, leads to striatal vulnerability. An A2A receptor dysfunction has been previously demonstrated in striatal cells engineered to express mutant huntingtin. Here we tested whether a similar dysfunction (i.e., the binding and functional parameters of A2A adenosine receptors) is present in peripheral blood cells (platelets, lymphocytes, and neutrophils) of subjects carrying the mutant gene. This study involved 48 heterozygous and three homozygous patients compared with 58 healthy subjects. Moreover, we selected seven at-risk mutation carriers. A2A receptor density and function are substantially increased in peripheral blood cells from both patients and subjects at the presymptomatic stage. In the neutrophils of the three homozygous HD subjects receptor dysfunction was higher than in heterozygotes. These data indicate the existence of an aberrant A2A receptor phenotype in the peripheral blood cells of subjects carrying the HD mutation. Future studies will assess whether this parameter can be exploited as a peripheral biomarker of Huntington's disease.

Elevated expression of A3 adenosine receptors in human colorectal cancer is reflected in peripheral blood cells.

PURPOSE: Adenosine is a ubiquitous nucleoside that accumulates at high levels in hypoxic regions of solid tumors, and A(3) adenosine receptors have been recently demonstrated to play a pivotal role in the adenosine-mediated inhibition of tumor cell proliferation. In the present work, we addressed the question of the putative relevance of A(3) subtypes in colorectal adenocarcinomas. EXPERIMENTAL DESIGN: Seventy-three paired samples of tumor and surrounding peritumoral normal mucosa at a distance of 2 and 10 cm from the tumor and blood samples obtained from a cohort of 30 patients with colorectal cancer were investigated to determine the presence of A(3) receptors by means of binding, immunocytochemistry, and real-time reverse transcription-polymerase chain reaction studies. RESULTS: As measured by receptor binding assays, the density of A(3) receptor was higher in colon carcinomas as compared with normal mucosa originating from the same individuals (P < 0.05). Overexpression of A(3) receptors at the protein level was confirmed by immunohistochemical studies, whereas no changes in A(3) mRNA accumulation in tumors as compared with the corresponding normal tissue were revealed. The overexpression of A(3) receptors in tumors was reflected in peripheral blood cells, where the density was approximately 3-fold higher compared with healthy subjects (P < 0.01). In a cohort of 10 patients studied longitudinally, expression of A(3) receptors in circulating blood cells returned to normal after surgical resection for colorectal cancer. CONCLUSIONS: This study provides the first evidence that A(3) receptor plays a role in colon tumorigenesis and, more importantly, can potentially be used as a diagnostic marker or a therapeutic target for colon cancer.

Effect of low frequency electromagnetic fields on A2A adenosine receptors in human neutrophils.

The present study describes the effect of low frequency, low energy, pulsing electromagnetic fields (PEMFs) on A2A adenosine receptors in human neutrophils. Saturation experiments performed using a high affinity adenosine antagonist [3H]-ZM 241385 revealed a single class of binding sites in control and in PEMF-treated human neutrophils with similar affinity (KD=1.05+/-0.10 and 1.08+/-0.12 nM, respectively). Furthermore, after 1 h of exposure to PEMFs the receptor density was statistically increased (P<0.01) (Bmax =126+/-10 and 215+/-15 fmol mg-1 protein, respectively). The effect of PEMFs was specific to the A2A adenosine receptors. This effect was also intensity, time and temperature dependent. In the adenylyl cyclase assays the A2A receptor agonists, HE-NECA and NECA, increased cyclic AMP accumulation in untreated human neutrophils with an EC50 value of 43 (40 - 47) and 255 (228 - 284) nM, respectively. The capability of HE-NECA and NECA to stimulate cyclic AMP levels in human neutrophils was increased (P<0.01) after exposure to PEMFs with an EC50 value of 10(8 - 13) and 61(52 - 71) nM, respectively. In the superoxide anion (O2-) production assays HE-NECA and NECA inhibited the generation of O2- in untreated human neutrophils, with an EC50 value of 3.6(3.1 - 4.2) and of 23(20 - 27) nM, respectively. Moreover, in PEMF-treated human neutrophils, the same compounds show an EC50 value of 1.6(1.2 - 2.1) and of 6.0(4.7 - 7.5) nM respectively. These results indicate the presence of significant alterations in the expression and in the functionality of adenosine A2A receptors in human neutrophils treated with PEMFs.

Alteration of A(3) adenosine receptors in human neutrophils and low frequency electromagnetic fields.

The present study was designed to evaluate the binding and functional characterization of A(3) adenosine receptors in human neutrophils exposed to low frequency, low energy, pulsing electromagnetic fields (PEMFs). Great interest has grown concerning the use of PEMF in the clinical practice for therapeutic purposes strictly correlated with inflammatory conditions. Saturation experiments performed using the high affinity and selective A(3) adenosine antagonist 5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([3H]-MRE 3008F20) revealed a single class of binding sites with similar affinity in control and in PEMF treated human neutrophils (K(D)=2.36+/-0.16 and 2.45+/-0.15 nM, respectively). PEMFs treatment revealed that the receptor density was statistically increased (P<0.01) (B(max)=451+/-18 and 736+/-25fmolmg(-1) protein, respectively). Thermodynamic data indicated that [3H]-MRE 3008F20 binding in control and in PEMF-treated human neutrophils was entropy and enthalpy driven. Competition of radioligand binding by the high affinity A(3) receptor agonists, N(6)-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide (Cl-IB-MECA) and N(6)-(3-iodo-benzyl)adenosine-5'-N-methyl-uronamide (IB-MECA), in the absence of PEMFs revealed high and low affinity values similar to those found in the presence of PEMFs. In both experimental conditions, the addition of GTP 100 microM shifted the competition binding curves of the agonists from a biphasic to a monophasic shape. In functional assays Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and their potencies were statistically increased after exposure to PEMFs. These results indicate in human neutrophils treated with PEMFs the presence of significant alterations in the A(3) adenosine receptor density and functionality.

A convenient synthesis by microwave heating and pharmacological evaluation of novel benzoyltriazole and saccharine derivatives as 5-HT(1A) receptor ligands.

A series of novel 1,2,3-4-benzoyltriazole and saccharine derivatives were designed and synthesized by microwave heating. They were evaluated on a battery of receptors, including serotonin 5-HT(1A,) 5-HT(2A) and 5-HT(2C), and the most interesting compounds were further evaluated on dopaminergic D(1), D(2) and adrenergic alpha(1), alpha(2) receptors. Conventional and microwave heating of the reactions were compared. Synthesis by microwave heating gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. All compounds displayed moderate affinity for 5-HT(1A) receptor. The most interesting compound 33 showed a high affinity (K(i)=93 nM) which was combined with no affinity on the other receptors considered.

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.

Expression, pharmacological profile, and functional coupling of A2B receptors in a recombinant system and in peripheral blood cells using a novel selective antagonist radioligand, [3H]MRE 2029-F20.

In this study, we compared the pharmacological and biochemical characteristics of A(2B) adenosine receptors in recombinant (hA(2B)HEK293 cells) and native cells (neutrophils, lymphocytes) by using a new potent 8-pyrazole xanthine derivative, [(3)H]N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl-oxy]-acetamide] ([(3)H]MRE 2029-F20), that has high affinity and selectivity for hA(2B) versus hA(1),hA(2A), and hA(3) subtypes. [(3)H]MRE 2029-F20 bound specifically to the hA(2B) receptor stably transfected in human embryonic kidney (HEK) 293 cells with K(D) of 2.8 +/- 0.2 nM and B(max) of 450 +/- 42 fmol/mg of protein. Saturation experiments of [(3)H]MRE 2029-F20 binding in human neutrophils and lymphocytes detected a single high-affinity binding site with K(D) values of 2.4 +/- 0.5 and 2.7 +/- 0.7 nM, respectively, and B(max) values of 79 +/- 10 and 54 +/- 8 fmol/mg of protein, respectively, in agreement with real-time reverse transcription polymerase chain reaction studies showing the presence of A(2B) mRNA. The rank order of potency of typical adenosine ligands with recombinant hA(2B) receptors was consistent with that typically found for interactions with the A(2B) subtype and was also similar in peripheral blood cells. 5'-N-Ethyl-carboxamidoadenosine stimulated cAMP accumulation in both hA(2B)HEK293 and native cells, whereas phospholipase C activation was observed in recombinant receptors and endogenous subtypes expressed in neutrophils but not in lymphocytes. MRE 2029-F20 was revealed to be a potent antagonist in counteracting the agonist effect in both signal transduction pathways. In conclusion, [(3)H]MRE 2029-F20 is a selective and high-affinity radioligand for the hA(2B) adenosine subtype and may be used to quantify A(2B) endogenous receptors. In this work, we demonstrated their presence and functional coupling in neutrophils and lymphocytes that play a role in inflammatory processes in which A(2B) receptors may be involved.

Effects of doxazosin and propranolol on A2A adenosine receptors in essential hypertension.

A2A adenosine receptors inhibit neutrophil adhesion and superoxide anion generation. The aim of the present study was to evaluate the effect of antihypertensive treatment with doxazosin or propranolol on the binding and functional parameters of A2A adenosine receptors of lymphocytes and neutrophils in essential hypertensive patients. Two groups of previously untreated, essential hypertensive patients were studied. The mean affinity (K(d)) and density (B(max)) of adenosine receptors, by the A2A selective radioligand [3H]-ZM-241385 binding assays, and EC50, by cAMP assays, were obtained first on no medication and a second time after treatment for up to 13 weeks with doxazosin (13 patients) or propranolol (8 patients). A third group of 15 healthy normotensive volunteers matched by age, sex, and body mass index was used as a control. Binding and functional parameters of the A2A adenosine receptors were significantly higher in the 2 hypertensive groups than in controls (P always <0.0001), both in lymphocyte and neutrophil membranes. After treatment with propranolol, the binding parameters did not change significantly, whereas after treatment with doxazosin, K(d), B(max), and EC50 values returned to control levels. In never-treated essential hypertensive patients, lower affinity, higher density, and impaired function of A2A adenosine receptors are present. The binding and functional parameters of A2A adenosine receptors appear to be normalized after treatment with doxazosin but not with propranolol.

A(3) adenosine receptors in human neutrophils and promyelocytic HL60 cells: a pharmacological and biochemical study.

This work compares the pharmacological and biochemical properties of A(3) adenosine receptors in human polymorphonuclear neutrophil granulocytes (PMNs) and promyelocytic HL60 cells. The gene expression of A(3) receptors was examined by reverse transcription-polymerase chain reaction experiments, whereas the amount of A(3) subtype on the plasma membrane was quantified by using the high-affinity and selective A(3) antagonist [(3)H]5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). Saturation experiments reveal a single high-affinity binding site with K(D) values of 2.3 +/- 0.3, 2.6 +/- 0.4 nM, and B(max) values of 430 +/- 35, 345 +/- 31 fmol/mg of protein for PMNs and HL60 cells, respectively. Competition of radioligand binding by adenosine ligands displays a rank order of potency typical of the A(3) subtype. EC(50) values of N(6)-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide (Cl-IB-MECA) for inhibition of cAMP levels via A(3) receptors are in good agreement with the binding data; furthermore, the response is potently inhibited by MRE 3008F20. In contrast, the high micromolar concentrations of Cl-IB-MECA and MRE 3008F20 in stimulating and blocking Ca(2+) mobilization, respectively, are not completely consistent with the involvement of an A(3) receptor. Furthermore, an important finding of this work is that the inhibition of PMNs oxidative burst is predominantly A(2A)-mediated, even though an effect of A(3) subtype could not be excluded. This conclusion is based on potent blockade of Cl-IB-MECA-mediated inhibition of oxidative burst by SCH 58261 and a minor but significant blockade by MRE 3008F20. In conclusion, HL60 cells express A(3) receptors similar to those in PMNs, thus providing a useful model for investigation of biochemical pathways leading to A(3) receptor activation.

Expression of A3 adenosine receptors in human lymphocytes: up-regulation in T cell activation.

The present study investigates mRNA and protein levels of A3 adenosine receptors in resting (R) and activated (A) human lymphocytes. The receptors were evaluated by the antagonist radioligand [3H]5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo-[1,5-c]-pyrimidine ([3H]MRE 3008F20), which yielded Bmax values of 125 +/- 15 and 225 +/- 23 fmol/mg of protein and KD values of 1.79 +/- 0.30 and 1.85 +/- 0.25 nM in R and A cells, respectively. The protein seems to be induced with remarkable rapidity starting at 15 min and reaches a plateau at 30 min. Western blot assays revealed that the up-regulation of the A3 subtype after lymphocyte activation was caused by an increase in an enriched CD4+ cell fraction. Real-time reverse transcription-polymerase chain reaction experiments confirmed the rapid increase of A3 mRNA after T cell activation. Competition of radioligand binding by adenosine ligands displayed a rank order of potency typical of the A3 subtype. Thermodynamic data indicated that the binding is enthalpy- and entropy-driven in both R and A cells, suggesting that the activation process does not involve, at a molecular level, receptor alterations leading to modifications in the A3-related binding mechanisms. Functionally, the up-regulation of A3 adenosine receptors in A versus R cells corresponded to a potency increase of the A3 agonist N6-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide in inhibiting cAMP accumulation (IC50=1.5 +/- 0.4 and 2.7 +/- 0.3 nM, respectively); this effect was antagonized by MRE 3008F20 (IC50=5.0 +/- 0.3 nM). In conclusion, our results provide, for the first time, an in-depth investigation of A3 receptors in human lymphocytes and demonstrate that, under activating conditions, they are up-regulated and may contribute to the effects triggered by adenosine.

Synthesis, radiolabeling, and preliminary biological evaluation of [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7 receptor.

The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X(7) receptor, named [(3)H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine (compound 13), are reported. This compound bound to human P2X(7) receptors expressed in HEK transfected cells with K(D) and B(max) value of 3.46+/-0.1 nM and 727+/-73 fmol/mg of protein, respectively. The high affinity and facile labeling makes it a promising radioligand for a further characterization of P2X(7) receptor subtype.