A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective 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; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Effects of CGS 21680, a selective A2A adenosine receptor agonist,on cardiac output and vascular resistance in acute heart failure in the anaesthetized rat

1. The effects of CGS 21680, a selective A_2A adenosine receptor agonist, on cardiac output, blood pressure, mean circulatory filling pressure (P_mcf), arterial and venous resistances, heart rate and left ventricular end-diastolic pressure were assessed in rats with acute heart failure by means of coronary artery occlusion.
2. Animals (n=6 in each group) were divided into five groups: group I, sham-operated vehicle-treated (0.9% saline; 0.018 mL min⁻¹); groups II-V, subject to coronary artery occlusion and treated with vehicle (0.9% saline; 0.018 ml min⁻¹) and CGS 21680 (0.1, 0.3 and 1.0 μg kg⁻¹ min⁻¹), respectively. Haemodynamic measurements were taken one hour after completion of surgery, ninety minutes after coronary artery occlusion (except in group I), and fifteen minutes after infusion of saline or CGS 21680.
3. Baseline haemodynamic measurements before occlusion were found not to differ significantly between the different groups of animals. However, after occlusion, cardiac output, rate of rise in left ventricular pressure (+dP/dt) and blood pressure were significantly reduced when compared to corresponding values in sham-operated animals. In addition, occlusion of the coronary artery resulted in a significant elevation in venous resistance, P_mcf and left ventricular end-diastolic pressure as compared to corresponding values in sham-operated animals.
4. Infusion with CGS 21680 at the highest dose significantly reduced blood pressure, arterial resistance and left ventricular end-diastolic pressure when compared to occluded vehicle-treated animals (group II). Administration of CGS 21680 at the highest dose also significantly increased cardiac output (28%) and heart rate (10%) in comparison to occluded vehicle-treated animals. In addition, the highest dose of CGS 21680 significantly reduced P_mcf (9%) and venous resistance (62%) in comparison to occluded vehicle-treated animals. Administration of CGS 21680 did not significantly affect +dP/dt when compared to occluded vehicle-treated animals.
5. The results from the present investigation indicate that occlusion of the coronary artery in rats results in a state of heart failure characterized by reduced arterial pressure and cardiac output, and increased venous resistance, P_mcf and left ventricular end-diastolic pressure. Administration of CGS 21680 to animals with acute heart failure resulted in increased cardiac output which was due to reduced venous resistance, as well as increased heart rate.

     

Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats

Background and Purpose

Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoylhydrazide (LASSBio-1359), on monocrotaline (MCT)-induced pulmonary hypertension in rats.

Experimental Approach

PAH was induced in male Wistar rats by a single i.p. injection of MCT (60 mg·kg⁻¹) and 2 weeks later, oral LASSBio-1359 (50 mg·kg⁻¹) or vehicle was given once daily for 14 days. Echocardiography was used to measure cardiac function and pulmonary artery dimensions, with histological assay of vascular collagen. Studies of binding to human recombinant adenosine receptors (A₁, A_2A, A₃) and of docking with A_2A receptors were also performed.

Key Results

MCT administration induced changes in vascular and ventricular structure and function, characteristic of PAH. These changes were reversed by treatment with LASSBio-1359. MCT also induced endothelial dysfunction in pulmonary artery, as measured by diminished relaxation of pre-contracted arterial rings, and this dysfunction was reversed by LASSBio-1359. In pulmonary artery rings from normal Wistar rats, LASSBio-1359 induced relaxation, which was decreased by the adenosine A_2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A_2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A_2A receptor agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, were very similar.

Conclusion and Implications

In rats with MCT-induced PAH, structural and functional changes in heart and pulmonary artery were reversed by treatment with oral LASSBio-1359, most probably through the activation of adenosine A_2A receptors.     

Characterization of adenosine receptors in the rat isolated aorta

• 1.1. Adenosine and its analogues relaxed the isolated rat aorta by an endothelium-dependent mechanism with an order of potency of 5′-N-ethylcarboxamidoadenosine (NECA) > 2-(p-(2-carboxyethyl)phenethylamino)-5′-N-ethylcarboxamidoadenosine (CGS 21680) > adenosine = N⁶-(2-(4-aminophenyl)ethyl)adenosine (APNEA = N⁶-cyclopentyladenosine (CPA) > 5′ - methylthioadenosine (MTA), although the maximal response achieved by CGS 21680 was less than that achieved by NECA.
• 2.2. Both 8-sulphophenyltheophylline (8-SPT) and MTA antagonized responses to the adenosine analogues, but there were some anomolous features of this antagonism and NECA was inhibited more powerfully than the other agonists. This suggests that as well as A_2a receptors mediating relaxation, the rat aorta may relax to adenosine analogues by other mechanisms.

     

Functional interaction between pre-synaptic α6β2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum

Background and Purpose

Pre-synaptic nicotinic ACh receptors (nAChRs) and adenosine A_2A receptors (A_2ARs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson''s disease and addiction. Since A_2ARs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A_2ARs and nAChRs in rat striatal dopaminergic terminals.

Experimental Approach

We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro and carried out locomotor behavioural sensitization in rats in vivo.

Key Results

In striatal nerve terminals, the selective A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 inhibited, while the A_2AR antagonist ZM241385 potentiated the nicotine-stimulated [³H]dopamine ([³H]DA) release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [³H]DA release was no longer modulated by A_2AR ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect that no longer persisted after 1 week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal.

Conclusions and Implications

These results provide the first evidence for a functional interaction between nicotinic and adenosine A_2AR in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson''s disease and other dopaminergic disorders.     

Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation

Background and Purpose

We previously reported that adenosine, acting at adenosine A_2A receptors (A_2AR), inhibits osteoclast (OC) differentiation in vitro (A_2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A_2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A_2AR signalling.

Experimental Approach

OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, the A_2AR antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR.

Key Results

A_2AR stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A_2AR stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A_2AR stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A_2AR stimulation inhibited OC expression of differentiation markers by a PKA-mechanism.

Conclusions and Implications

A_2AR activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.     

Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells.

1. An [3H]-adenine pre-labelling methodology was employed to assay cyclic AMP generation by adenosine analogues in Chinese hamster ovary (CHO.A2B4) cells, transfected with cDNA which has been proposed to code for the human brain A2B adenosine receptor, and in guinea-pig cerebral cortical slices. 2. Adenosine analogues showing the following rank order of potency in the CHO.A2B4 cells (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.91) > adenosine (5.69) > 2-chloroadenosine (5.27) > N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.06). The purportedly A2A-selective agonist, CGS 21680, failed to elicit a significant stimulation of cyclic AMP generation at concentrations up to 10 microM in CHO.A2B4 cells. In the guinea-pig cerebral cortex, NECA was more potent than APNEA with pD2 values of 5.91 and 4.60, respectively. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity in CHO.A2B4 cells (ca. 10 fold stimulation of cyclic AMP), while, in comparison, maximal responses to adenosine (32% NECA response), 2-chloroadenosine (61%), and APNEA (73%) were reduced. 4. Antagonists of NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value in CHO.A2B4 cells: guinea-pig cerebral cortex): XAC (7.89: 7.46) > CGS 15943 (7.75: 7.33) > DPCPX (7.16: 6.91) > PD 115,199 (6.95: 6.39) > 8FB-PTP (6.52: 6.55) > 3-propylxanthine (4.63: 4.59). 5. We conclude that, using the agents tested, the A2B adenosine receptor cloned from human brain expressed in Chinese hamster ovary cells exhibits an identical pharmacological profile to native A2B receptors in guinea-pig brain.     

Haemodynamic effects of a selective adenosine A2A receptor agonist,CGS 21680, in chronic heart failure in anaesthetized rats

1. Recently we demonstrated that the administration of an A_2A adenosine receptor agonist, CGS 21680, to anaesthetized rats with acute heart failure (1 h post-coronary artery ligation) resulted in an increase in cardiac output. In the present investigation, the effects of CGS 21680 on cardiac output, vascular resistance, heart rate, blood pressure and mean circulatory filling pressure (Pmcf) were investigated in anaesthetized rats with chronic heart failure (8 weeks post-coronary artery ligation).
2. Experiments were conducted in five groups (n=6) of animals: sham-operated vehicle-treated (0.9% NaCl; 0.037 mL kg⁻¹ min⁻¹) animals in which the occluder was placed but not pulled to ligate the coronary artery; coronary artery-ligated vehicle-treated animals; and coronary artery-ligated CGS 21680-treated (0.1, 0.3 or 1.0 μg kg⁻¹ min⁻¹) animals.
3. Baseline blood pressure, cardiac output and rate of rise in left ventricular pressure (+dP/dt) were significantly reduced in animals with coronary artery ligation when compared to sham-operated animals. Coronary artery ligation resulted in a significant increase in left ventricular end-diastolic pressure, Pmcf and venous resistance when compared to sham-operated animals.
4. Administration of CGS 21680 at 0.3 and 1.0 μg kg⁻¹ min⁻¹ significantly (n=6; P<0.05) increased cardiac output by 19±4% and 39±5%, and heart rate by 14±2% and 15±1%, respectively, when compared to vehicle treatment in coronary artery-ligated animals. Administration of CGS 21680 also significantly reduced blood pressure and arterial resistance when compared to coronary artery-ligated vehicle-treated animals. Infusion of CGS 21680 also significantly reduced venous resistance when compared to vehicle-treated coronary artery-ligated animals.
5. The results show that heart failure is characterized by reduced cardiac output, and increased left ventricular end-diastolic pressure, venous resistance and Pmcf. Acute treatment with CGS 21680 in animals with chronic heart failure decreased left ventricular end-diastolic pressure and increased cardiac output. This increase in cardiac output was the result of reduced arterial and venous resistances and increased heart rate.

     

Isoform-specific regulation of the Na+-K+ pump by adenosine in guinea pig ventricular myocytes

Aim:

The present study investigated the effect of adenosine on Na⁺-K⁺ pumps in acutely isolated guinea pig (Cavia sp.) ventricular myocytes.

Methods:

The whole-cell, patch-clamp technique was used to record the Na⁺-K⁺ pump current (I_p) in acutely isolated guinea pig ventricular myocytes.

Results:

Adenosine inhibited the high DHO-affinity pump current (I_h) in a concentration-dependent manner, which was blocked by the selective adenosine A₁ receptor antagonist DPCPX and the general protein kinase C (PKC) antagonists staurosporine, GF 109203X or the specific δ isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A₁ receptor agonist CCPA and a specific activator peptide of PKC-δ, PP114. In contrast, the selective A_2A receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and A₃ receptor agonist Cl-IB-MECA did not affect I_h. Application of the selective A_2A receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 and A₃ receptor antagonist MRS1191 also failed to block the effect of adenosine. Furthermore, H89, a selective protein kinase A (PKA) antagonist, did not exert any effect on adenosine-induced I_h inhibition.

Conclusion:

The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of I_h via adenosine A₁ receptors and the PKC-δ isoform.     

Functional characterization of three adenosine receptor types

1 The purpose of the present study was to classify adenosine receptors into A₁ and A₂ subtypes in a wide range of isolated tissues and cell types (rat adipocytes and atria, guinea-pig ileum and atria (A₁); guinea-pig aorta, dog coronary artery and human platelets and neutrophils (A₂)) using the R- and S-diastereoisomers of N-phenylisopropyladenosine (PIA), N-cyclopentyladenosine (CPA), the novel compound, N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR79236), N-[(2-methylphenyl)methyl]adenosine (metrifudil), 2-(phenylamino)adenosine (CV1808), and 2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]-N-ethylcarboxamidoadenosine (CGS21680); N-ethylcarboxamidoadenosine (NECA) was used as a standard.2 Results obtained in all tissue preparations previously reported to contain A₁-receptors could be described by a single rank order of agonist potency: CPA ≥ GR79236, R-PIA ≥ NECA >> S-PIA ≥ metrifudil ≥ CV1808, CGS21680.3 In contrast, two distinct rank orders of agonist potency were observed in preparations previously reported to contain A₂-receptors. In dog coronary artery, human neutrophils and platelets the rank order of potency was: CV1808, CGS21680 ≥ NECA > R-PIA ≥ metrifudil ≥ CPA > GR79236, S-PIA. However, in guinea-pig aorta the rank order was: NECA > metrifudil > R-PIA, CPA > CV1808, GR79236 ≥ S-PIA, CGS21680.4 The results of this study are consistent with the existence of three types of adenosine receptor: A₁-and two subtypes of A₂-receptor. The receptor present in dog coronary artery, human platelets and neutrophils, probably corresponds to the A_2a subtype, whilst that present in the guinea-pig aorta may be of the A_2b subtype.     

Pharmacological classification of adenosine receptors in the sinoatrial and atrioventricular nodes of the guinea-pig

1. The effects of seven agonist and three antagonist adenosine receptor ligands were compared on the guinea-pig sinoatrial (SA) node (isolated right atrium) and atrioventricular (AV) node (perfused whole heart). Single agonist concentration-effect curves were obtained to 5′-N-ethylcarboxamidoadenosine (NECA), R(−)-N⁶-(2-phenylisopropyl)adenosine (R-PIA), N⁶-cyclohexyladenosine (CHA), 2-chloroadenosine (CADO),),S(+)-N⁶-(2-phenylisopropyl)adenosine (L-PIA), 2-phenylaminoadenosine (CV 1808) and N⁶-aminoadenosine (MeAdo). Adenosine and/or NECA curves were obtained in the absence and presence of the antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 9-chloro-2–(2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazolin-5-imine ({"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334","term_text":"CGS15943"}}CGS15943) and N⁶-(endonorbornan-2-yl)-9-methyladenine (N-0861).
2. A formal comparison of the agonist and antagonist potency data was made by fitting the data to a straight line using a least squares procedure based on principal components analysis to account for the variance on both axes. The antagonist affinity estimates made on the two assays did not deviate significantly from the line of identity.
3. The agonist p[A]₅₀ data obtained on the two assays did not deviate from the line of identity, indicating that there were no significant differences in potencies between the two assays. The p[A]₅₀ ratio of R-PIA and S-PIA was 1.24±0.09 in the SA node and 1.36±0.11 in the AV node, indicating no difference in the stereoselectivity of the PIA isomers between the two tissues.
4. The agonist potency and antagonist affinity data obtained are consistent with previous findings showing that the AV and SA node data are pharmacologically indistinguishable and belong to the adenosine A₁-receptor class. No evidence for the reported A₃-receptor was found.

     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM).
2. In the prostatic half of the vas deferens the A₁ selective adenosine receptor agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A₁/A₂ adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC₅₀±s.e.mean values 6.15±0.24, 5.99±0.26 and 5.51±0.24, respectively). The responses to CPA were blocked by the A₁ adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM).
3. In the epididymal half of the vas deferens NECA potentiated (at ⩽100 nM) and inhibited (at ⩾1 μM) electrically-evoked contractions. In the presence of the non-selective α-adrenoceptor antagonist phentolamine (3 μM), the α₁-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC₅₀ values 6.05±0.25, 5.97±0.29 and 5.71±0.27, respectively). CPA (at 10 μM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC₅₀ 6.14±0.67); this effect was antagonized by DPCPX (30 nM, apparent pK_B 8.26±0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 μM), CPA (up to 1 μM) potentiated electrically-evoked contractions.
4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC₅₀ values 7.49±0.62, 7.65±0.74 and 5.84±0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK_B value of 7.64±0.64.
5. The α₁-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 μM) potentiated responses to phenylephrine (⩽1 μM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 μM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 μM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC₅₀ values 7.57±0.54 and 8.08±0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis.
6. These studies are consistent with the action of stable adenosine analogues at prejunctional A₁ and postjunctional A₁-like adenosine receptors. The prejunctional A₁ adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of α₁-adrenoceptor-, but not of ATP-induced contractility.

     

Oroxylin A Induces BDNF Expression on Cortical Neurons through Adenosine A2A Receptor Stimulation: A Possible Role in Neuroprotection

Oroxylin A is a flavone isolated from a medicinal herb reported to be effective in reducing the inflammatory and oxidative stresses. It also modulates the production of brain derived neurotrophic factor (BDNF) in cortical neurons by the transactivation of cAMP response element-binding protein (CREB). As a neurotrophin, BDNF plays roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. Adenosine A2_A receptor colocalized with BDNF in brain and the functional interaction between A2_A receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that oroxylin A modulates BDNF production in cortical neuron through the regulation of A2_A receptor system. As ex-pected, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (A2_A receptor agonist) induced BDNF expression and release, however, an antagonist, ZM241385, prevented oroxylin A-induced increase in BDNF production. Oroxylin A activated the PI3K-Akt-GSK-3β signaling pathway, which is inhibited by ZM241385 and the blockade of the signaling pathway abolished the increase in BDNF production. The physiological roles of oroxylin A-induced BDNF production were demonstrated by the increased neurite extension as well as synapse formation from neurons. Overall, oroxylin A might regulate BDNF production in cortical neuron through A2_A receptor stimulation, which promotes cellular survival, synapse formation and neurite extension.     

Adenosine receptors mediate both contractile and relaxant effects of adenosine in main pulmonary artery of guinea pigs

In guinea pig main pulmonary artery precontracted with noradrenaline, adenosine exerted an initial phasic contraction followed by a tonic contraction and a slow relaxation. After selective blockade by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX: 10 nM) of A₁ receptors, adenosine only elicited a rapid relaxation. This initial response was characterized by use of adenosine (AR) and its analogues N⁶-cyclopentyl-adenosine (CPA), R-N⁶-phenyllsopropyladeno-sine (R-PIA), 2-chloroadenosine (CADO), 5-N-ethyl-carboxamidoadenosine(NECA), N⁶-2-(4-aminophenyl) ethyl adenosine (APNEA) and 2-p-((carboxyethyl)phenethylamino)-5-carboxamidoadenosine (CGS 21 680). The order of potency of the adenosine analogues for purine-induced phasic contraction was CPA > R-PIA > NECA = APNEA > AR > CGS 21 680 suggesting the involvement of activation of A₁ type adenosine receptors in the contraction phase. DPCPX antagonized the CPA-induced contraction with a pA₂ = 9.27 ± 0.26, but the Schild plot slope parameter was significantly lower than unity (0.58 ± 0.09). In contrast, in electrically driven guinea pig atrial myocardium (a tissue reported to possess A1 receptors), the DPCPX-CPA antagonism was purely competitive (pA₂ = 8.95 ± 0.06; slope = 0.93 ± 0.06). In the presence of 300 nM DPCPX, the rank order of potency for the purine-induced fast relaxation was NECA > CADO = AR > CGS 21 680 = R-PIA > CPA. The NECA- and adenosine-induced relaxation was influenced neither by 300 nM CP 66 713 (an antagonist at A_2a receptors), nor by endothelial removal and inhibition of nitric oxide synthase (100 M N^G-nitro-L-arginine: L-NOARG). The adenosine-induced relaxation was antagonized by 8-phenyltheophylline (8-PT), a potent A₁/A₂ antagonist. However, the rapid relaxation elicited by adenosine in the presence of 8-PT, was reversed and contraction developed. It is concluded that adenosine causes contraction via dual action on A₁ adenosine receptors and on xanthine-resistant sites. Our experiments with APNEA (a prototypic A₃ receptor agonist) did not support the suggestion that A₃ receptors are implicated in the xanthine-resistant component of adenosine-induced contraction, as DPCPX (300 nM) completely abolished and even reversed the APNEA-induced contraction. In addition, cromolyn (a mast cell stabilizing agent) did not influence the xanthine-resistant contraction induced by adenosine in the presence of DPCPX, 8-PT and dipyridamole (an adenosine uptake inhibitor). On the basis of the rank order of agonist potency, the receptors involved in the adenosine-induced rapid relaxation most likely is of the A_2b subtype. The opposing action of the xanthine-resistant contraction, however, did not allow a definitive pharmacological characterization of the receptor mediating relaxation.     

G-protein activation at 5-HT1A receptors by the 5-ht1F ligand LY334370 in guinea-pig brain sections and recombinant cell lines

1. G-protein activation by the 5-ht_1F receptor agonist 5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate ({"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370) was investigated by use of autoradiography of receptor-activated G-proteins in guinea-pig brain sections and [³⁵S]-GTPγS binding responses in cell lines stably expressing human 5-HT_1A (h 5-HT_1A) receptors.
2. {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 (10 μM) caused little or no stimulation of [³⁵S]-GTPγS binding in guinea-pig brain regions enriched in 5-ht_1F binding sites (e.g., claustrum, caudate/putamen and thalamic nuclei), as identified by labelling with 10 nM [³H]-sumatriptan plus 10 nM 5-carboxamidotryptamine (5-CT).
3. Application of {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 (10 μM) to guinea-pig brain sections resulted in an increase of [³⁵S]-GTPγS binding in hippocampus (123±17%), lateral septum (58±14%), dorsal raphe (57±10%), entorhinal (37±11%) and cingulate cortex (28±10%). This distribution fits with the G-protein activation mediated by 5-HT_1A receptors as found with lisuride (10 μM), and labelling of 5-HT_1A receptors by 140 pM [¹²⁵I]-4-(2′-methoxy-phenyl)-1-[2′-(n-2′′-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI).
4. The {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-mediated [³⁵S]-GTPγS response was antagonized by the selective, silent 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635, 1 μM) in each of the brain structures investigated. The distribution pattern of the [³⁵S]-GTPγS binding response and the antagonist profile suggest that the {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-induced response in guinea-pig brain is mediated by 5-HT_1A receptors.
5. The maximal {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-induced [³⁵S]-GTPγS binding response (83 to 94%) in membranes of recombinant C6-glial/h 5-HT_1A and HeLa/h 5-HT_1A cells was close to that of 5-HT, suggesting {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 to exert high intrinsic activity at h 5-HT_1A receptors.
6. In conclusion, in guinea-pig brain sections and recombinant cell lines the 5-ht_1F receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 causes G-protein activation that is mediated by 5-HT_1A receptors. Caution should be taken when employing this ligand as a putative selective 5-ht_1F agonist.

     

Adenosine receptor-induced cyclic AMP generation and inhibition of 5-hydroxytryptamine release in human platelets.

1. We have assessed the effects of adenosine receptor agonists and antagonists on collagen-induced 5-hydroxytryptamine (5-HT) release and cyclic AMP generation in human platelets. 2. 5'-N-ethylcarboxamidoadenosine (NECA) and CGS 21680 elicited accumulations of cyclic AMP with mean EC50 values of 2678 and 980 nM, respectively. The maximal response to CGS 21680 was approximately half that of the response to 10 microM NECA. 3. NECA and CGS 21680 inhibited collagen-induced 5-hydroxytryptamine release with mean EC50 values of 960 and 210 nM, respectively. The maximal response to CGS 21680 was approximately 25% of the response to 10 microM NECA. 4. The A1/A2a-selective adenosine receptor antagonist PD 115,199 was more potent as an inhibitor of NECA-elicited responses than the A1-selective antagonist DPCPX with calculated Ki values of 22-32 nM and > 10 microM, respectively. 5. In the presence of a cyclic AMP phosphodiesterase inhibitor, the effects of CGS 21680 on cyclic AMP accumulation and 5-HT release were enhanced to levels similar to those elicited by 10 microM NECA. In the absence of phosphodiesterase inhibition, CGS 21680 did not antagonise the effects of NECA. Furthermore, endogenous adenosine did not contribute to the effects of CGS 21680 when phosphodiesterase was inhibited. 6. We conclude that an A2a adenosine receptor appears to be involved in the NECA-elicited increases in cyclic AMP levels and inhibition of 5-HT release in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependence of P2-nucleotide receptor agonist-mediated endothelium-independent relaxation on ectonucleotidase activity and A2A-receptors in rat portal vein

1. The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle.
2. At 25°C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-ClATP (2.7±0.5 μM, n=7)>ATP (12.9±1.1 μM, n=9)⩾2-MeSATP (21.9±4.8 μM, n=4). In the presence of αβ-methylene ATP (αβ-MeATP, 200 μM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited.
3. The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 100 μM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP.
4. The A_2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1–100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited.
5. ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to αβ-MeATP (200 μM) but were inhibited by ZM 241385 (100 nM).
6. Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [γ-³²P]-ATP showed that muscular strips rapidly (10–60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57±11% (n=3) by 200 μM αβ-MeATP.
7. These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A_2A-receptors.

     

Evidence for adenosine A2b receptors in the rat pineal gland

We describe the effects of 5′-N-ethylcarboxamidoadenosine (NECA), a mixed A_2a/A_2b adenosine receptor agonist and 2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), a selective A_2a agonist, on cyclic AMP and N-acetylserotonin synthesis in rat pineal gland. NECA, 1 and 10 μM, increased cyclic AMP by 5- and 25-fold and N-acetylserotonin by 40- and 60-fold respectively, whereas CGS 21680 at the same concentrations was ineffective. These results argue for the presence of adenosine A_2b receptors in rat pinealocytes.     

A comparison of EDHF-mediated and anandamide-induced relaxations in the rat isolated mesenteric artery

1. Relaxation of the methoxamine-precontracted rat small mesenteric artery by endothelium-derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration- and endothelium-dependent fashion in the presence of N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) by either carbachol (pEC₅₀ [negative logarithm of the EC₅₀]=6.19±0.01, R_max [maximum response]=93.2±0.4%; n=14) or calcium ionophore A23187 (pEC₅₀=6.46±0.02, R_max=83.6±3.6%; n=8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC₅₀=6.31±0.06, R_max=94.7±4.6%; n=10; with L-NAME: pEC₅₀=6.33±0.04, R_max=93.4±6.0%; n=4).
2. The selective cannabinoid receptor antagonist, SR 141716A (1 μM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 μM) greatly reduced the R_max values for EDHF-mediated relaxations to carbachol (control, 93.2±0.4%; SR 141716A, 10.7±2.5%; n=5; P<0.001) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control, 84.8±2.1%; SR 141716A, 3.5±2.3%; n=6; P<0.001) but caused a 10 fold parallel shift in the concentration-relaxation curve for anandamide without affecting R_max.
3. Precontraction with 60 mM KCl significantly reduced (P<0.01; n=4 for all) relaxations to 1 μM carbachol (control 68.8±5.6% versus 17.8±7.1%), {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control 71.4±6.1% versus 3.9±0.45%) and anandamide (control 71.1±7.0% versus 5.2±3.6%). Similar effects were seen in the presence of 25 mM K⁺. Incubation of vessels with pertussis toxin (PTX; 400 ng ml⁻¹, 2 h) also reduced (P<0.01; n=4 for all) relaxations to 1 μM carbachol (control 63.5±7.5% versus 9.0±3.2%), {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control 77.0±5.8% versus 16.2±7.1%) and anandamide (control 89.8±2.2% versus 17.6±8.7%).
4. Incubation of vessels with the protease inhibitor phenylmethylsulphonyl fluoride (PMSF; 200 μM) significantly potentiated (P<0.01), to a similar extent (∼2 fold), relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (pEC₅₀: control, 6.45±0.04; PMSF, 6.74±0.10; n=4) and anandamide (pEC₅₀: control, 6.31±0.02; PMSF, 6.61±0.08; n=8). PMSF also potentiated carbachol responses both in the presence (pEC₅₀: control, 6.25±0.01; PMSF, 7.00±0.01; n=4; P<0.01) and absence (pEC₅₀: control, 6.41±0.04; PMSF, 6.88±0.04; n=4; P<0.001) of L-NAME. Responses to the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) were also potentiated by PMSF (pEC₅₀: control, 7.51±0.06; PMSF, 8.00±0.05, n=4, P<0.001).
5. EDHF-mediated relaxation to carbachol was significantly attenuated by the K⁺ channel blocker tetraethylammonium (TEA; 1 mM) (pEC₅₀: control, 6.19±0.01; TEA, 5.61±0.01; n=6; P<0.01). In contrast, TEA (1 mM) had no effect on EDHF-mediated relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (pEC₅₀: control, 6.47±0.04; TEA, 6.41±0.02, n=4) or on anandamide (pEC₅₀: control, 6.28±0.06; TEA, 6.09±0.02; n=5). TEA (10 mM) significantly (P<0.01) reduced the R_max for anandamide (control, 94.3±4.0%; 10 mM TEA, 60.7±4.4%; n=5) but had no effect on the R_max to carbachol or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187.
6. BaCl₂ (100 μM), considered to be selective for blockade of inward rectifier K⁺ channels, had no significant effect on relaxations to carbachol or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, but caused a small shift in the anandamide concentration-response curve (pEC₅₀: control, 6.39±0.01; Ba²⁺, 6.20±0.01; n=4; P<0.01). BaCl₂ (1 mM; which causes non-selective block of K⁺ channels) significantly (P<0.01) attenuated relaxations to all three agents (pEC₅₀ values: carbachol, 5.65±0.02; {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, 5.84±0.04; anandamide, 5.95±0.02; n=4 for each).
7. Apamin (1 μM), a selective blocker of small conductance, Ca²⁺-activated, K⁺ channels (SK_Ca), 4-aminopyridine (1 mM), a blocker of delayed rectifier, voltage-dependent, K⁺ channels (K_v), and ciclazindol (10 μM), an inhibitor of K_v and adenosine 5′-triphosphate (ATP)-sensitive K⁺ channels (K_ATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or anandamide responses.
8. Glibenclamide (10 μM), a K_ATP inhibitor and charybdotoxin (100 or 300 nM), a blocker of several K⁺ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50 nM), an inhibitor of large conductance, Ca²⁺-activated, K⁺ channels (BK_Ca), had no significant effect on the relaxation responses, either alone or in combination with apamin (1 μM). Also, a combination of apamin (1 μM) with either glibenclamide (10 μM) or 4-aminopyridine (1 mM) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or anandamide.
9. A combination of apamin (1 μM) with charybdotoxin (100 nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Apamin (1 μM) and charybdotoxin (300 nM) together consistently inhibited the response to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, while apamin (1 μM) and ciclazindol (10 μM) together inhibited relaxations to both carbachol and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. None of these toxin combinations had any significant effect on relaxation to anandamide.
10. It was concluded that the differential sensitivity to K⁺ channel blockers of EDHF-mediated responses to carbachol and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 might be due to actions on endothelial generation of EDHF, as well as its actions on the vascular smooth muscle, and suggests care must be taken in choosing the means of generating EDHF when making comparative studies. Also, the relaxations to EDHF and anandamide may involve activation of cannabinoid receptors, coupled via PTX-sensitive G-proteins to activation of K⁺ conductances. The results support the hypothesis that EDHF is an endocannabinoid but relaxations to EDHF and anandamide show differential sensitivity to K⁺ channel blockers, therefore it is likely that anandamide is not identical to EDHF in the small rat mesenteric artery.

     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.