Adenosine A1-receptor stimulation of inositol phospholipid hydrolysis and calcium mobilisation in DDT1 MF-2 cells

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Abstract
1 . The effect of adenosine receptor-stimulation on inositol phospholipid hydrolysis and calcium mobilization has been investigated in the hamster vas deferens smooth muscle cell line DDT1 MF-2. 2 . Adenosine receptor stimulation increased the accumulation of total [3H]-inositol phosphates in DDT1 MF-2 cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was N6-cyclo-pentyladenosine > 5′-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine. 3 . The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (KD 1.2 nm), PD 115,199 (KD 39 nm) and 8-phenyltheophylline (KD 31 nm). 4 . The inositol phosphate response to 2-chloradenosine (10 μm) was not significantly altered when the extracellular Ca2+ ion concentration was reduced from 2.4 mm to 1.2 mm or 0.6 mm. Under calcium-free conditions, however, a reduced but still significant response to 2-chloroadenosine was evident (39 ± 10% of the response in calcium-containing medium). 5 . The 5-lipoxygenase inhibitor AA861 (10 and 100 μm) inhibited the inositol phosphate response to 2-chloroadenosine by 40 ± 9% and 60 ± 4% respectively. The cyclo-oxygenase inhibitor, indomethacin, however, was without significant effect at 1 μm. 6 . 2-Chloroadenosine stimulated an increase in intracellular free Ca2+ ion concentration in fura-2 loaded DDT1 MF-2 cells in calcium-free medium containing 0.1 mm EGTA, which could be inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 μm). 7 . These data suggest that adenosine A1-receptor stimulation results in inositol phospholipid hydrolysis and calcium mobilization from intracellular stores in DDT1 MF-2 cells.