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Activation of A(1) adenosine receptors leads to the inhibition of cAMP accumulation and the stimulation of inositol phosphate accumulation via pertussis toxin-sensitive G-proteins. In this study we have investigated the signaling of the A(1) adenosine receptor in Chinese hamster ovary (CHO) cells, when expressed at approximately 203 fmol/mg (CHOA1L) and at approximately 3350 fmol/mg (CHOA1H). In CHOA1L cells, the agonists N(6)-cyclopentyladenosine (CPA), (R)-N(6)-(2-phenylisopropyl)adenosine, and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited cAMP production in a concentration-dependent manner. After pertussis toxin treatment, the agonist NECA produced a stimulation of cAMP production, whereas CPA and (R)-N(6)-(2-phenylisopropyl)adenosine were ineffective. In CHOAIH cells, however, all three agonists produced both an inhibition of adenylyl cyclase and a pertussis toxin-insensitive stimulation of adenylyl cyclase. All three agonists were more potent at inhibiting adenylyl cyclase in CHOA1H cells than in CHOA1L cells. In contrast, A(1) agonists (and particularly NECA) were less potent at stimulating inositol phosphate accumulation in CHOA1H cells than in CHOA1L cells. After pertussis toxin treatment, agonist-stimulated inositol phosphate accumulation was reduced in CHOA1H cells and abolished in CHOA1L cells. The relative intrinsic activity of NECA in stimulating inositol phosphate accumulation, compared to CPA (100%), was much greater in the presence of pertussis toxin (289.6%) than in the absence of pertussis toxin (155.2%). These data suggest that A(1) adenosine receptors can couple to both pertussis toxin-sensitive and -insensitive G-proteins in an expression level-dependent manner. These data also suggest that the ability of this receptor to activate different G-proteins is dependent on the agonist present.

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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 /G 11 protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G i /G o protein-coupled adenosine A 1 receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A 1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y 2 purinoceptor. 2 The selective adenosine A 1 receptor agonist N 6 -cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC 50 7.1+0.4 nM). CPAmediated increases in MAP kinase activity were blocked by PD 98059 (50 mM; 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 A 1 receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 mM; 6+10% of control). In contrast, daidzein (100 mM), the inactive analogue of genistein had no signi®cant eect (96+12 of control). MAP kinase responses to CPA (1 mM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55+8% inhibition) and LY 294002 (30 mM; 40+5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 mM). 4 Activation of the endogenous P2Y 2 purinoceptor with UTP also stimulated time and concentrationdependent increases in MAP kinase activity in CHO-A1 cells (EC 50 =1.6+0.3 mM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67+3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 mM; 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 mM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 mM). 7 Adenosine A 1 and P2Y 2 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 A 1 receptor and the endogenous P2Y 2 purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, costimulation of the adenosine A 1 receptor and the P2Y 2 purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.