Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.