Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells

doi:10.1016/S0014-2999(96)00917-X

European Journal of Pharmacology

Volume 321, Issue 1, 19 February 1997, Pages 77-86

https://doi.org/10.1016/S0014-2999(96)00917-X Get rights and content

Abstract

Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A₁ receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P_2U purinoceptors and CCK_A receptors. In this study we have investigated whether adenosine A₁ receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [³H]arachidonic acid release and total [³H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [³H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [³H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-{1-[3-(2-isothioureido)propyl]indol-3-yl}-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [³H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [³H]arachidonic acid release elicited by PMA. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) augmented the release of [³H]arachidonic acid produced by thrombin. Co-activation of the adenosine A₁ receptor also potentiated thrombin-stimulated [³H]inositol phosphate accumulation. The synergistic interactions between the adenosine A₁ receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [³H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [³H]inositol phosphates and the release of [³H]arachidonic acid through pertussis-toxin-insensitive G-proteins. Experiments using PMA suggest that protein kinase C differentially regulates thrombin receptor activation of phospholipase C and phospholipase A₂. Co-activation of the transfected human adenosine A₁ receptor augments thrombin-stimulated phospholipase C and phospholipase A₂ activity. Finally, the augmentation of phospholipase A₂ activity by the adenosine A₁ receptor is inhibited by selective protein kinase C inhibitors, suggesting the involvement of protein kinase C.

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In transfected Chinese hamster ovary (CHO-A₁) cells the human adenosine A₁ receptor directly stimulates pertussis toxin-sensitive increases in inositol phosphate production and potentiates (synergistically) the inositol phosphate responses mediated by G_q-coupled P2Y₂ purinoceptor and CCK_A receptors. In the present study we have investigated the role of Gβγ subunits in mediating adenosine A₁ receptor effects on phospholipase C activation (both direct and synergistic) by transiently transfecting CHO-A₁ cells with a scavenger of Gβγ subunits: the C-terminus of β-adrenoceptor kinase 1 (βark1 residues 495–689). [ $^{3} H$ ]inositol phosphate responses to the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA; 1 μM) were inhibited (41±1%) in CHO-A₁ cells transiently transfected with the Gβγ scavenger, βark1 (495–689). Expression of βark1 (495–689) protein was confirmed by Western blotting. In contrast, adenosine A₁ receptor-mediated inhibition of forskolin stimulated [ $^{3} H$ ]cyclic AMP accumulation was unaffected by transient expression of βark1 (495–689). βark1 (495–689) expression had no significant effect on the [ $^{3} H$ ]inositol phosphate responses produced by activation of the endogenous P2Y₂ purinoceptor (100 μM UTP; 92±0.8% of control). [ $^{3} H$ ]inositol phosphate accumulation in response to adenosine A₁ receptor activation was also attenuated in CHO-K1 cells co-transfected with the βark1 (495–689) minigene (59±4% inhibition of control response to 1 μM CPA). Finally, transient expression of βark1 (495–689) in CHO-A₁ cells inhibited the augmentation of [ $^{3} H$ ]inositol phosphate responses resulting from co-activation of adenosine A₁ receptors and P2Y₂ purinoceptors. These experiments indicate that Gβγ subunits are involved in the direct coupling the adenosine A₁ receptor to phospholipase C and that they also participate in the augmentation of P2Y₂ purinoceptor-mediated [ $^{3} H$ ]inositol phosphate responses by the adenosine A₁ receptor.

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Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells

Abstract

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol. (Mol. Pharmacol. Sect.)

Trends Cardiovasc. Med.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Int. J. Biochem.

Eur. J. Pharmacol.

Biochem. Pharmacol.

J. Biol. Chem.

Eur. J. Pharmacol. (Mol. Pharmacol. Sect.)

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Transfected adenosine A₁ receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A₂ activity in CHO cells