ADENOSINE-A(1)-RECEPTOR STIMULATED INCREASES IN INTRACELLULAR CALCIUM IN THE SMOOTH-MUSCLE CELL-LINE, DDT(1)MF-2

1 The effect of a range of adenosine receptor agonists on intracellular free calcium concentration ([Ca2+]i) has been studied in the hamster vas deferens smooth muscle cell line DDT1MF-2. 2 Adenosine receptor agonists elicited a rapid and maintained increase in [Ca2+]i in fura-2 loaded DDT1MF-2 cells. The initial rise could be maintained in the absence of extracellular calcium, whereas the maintained or plateau phase was dependent upon the presence of extracellular calcium and appeared to be associated with calcium influx. The rank order of agonist potencies was N6-cyclopentyladenosine > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine. 3 The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, K(D) 0.14 nM) and 8-phenyltheophylline (K(D) 112 nM). 4 Pretreatment with the 5-lipoxygenase inhibitor AA861 (20 muM) produced only a small (14 +/- 2%) inhibition of the [Ca2+]i response elicted by N6-cyclopentyladenosine (300 nM), in nominally Ca2+-free buffer containing 0.1 mM EGTA. The cyclo-oxygenase inhibitor, indomethacin (2 muM) was without effect. 5 The Ca2+-influx associated with the plateau phase required the continued presence of agonist on the receptor. The antagonist DPCPX (100 nM) attenuated the rise in [Ca2+]i observed when extracellular Ca2+ was re-applied after the cells had been stimulated with N6-cyclopentyladenosine (CPA;300 nM) in experiments initiated in nominally Ca2+-free buffer. 6 Pretreatment with pertussis toxin (200 ng ml-1 for 4 h) inhibited the CPA (100 nM) stimulated intracellular Ca2+ release and Ca2+ influx but was without effect on the response to histamine (100 muM). 7 These data suggest that adenosine A1-receptor activation in DDT1MF-2 cells stimulates release of Ca2+ from intracellular stores and influx of extracellular Ca2+ through Ca2+ entry pathways in the plasma membrane which required the continued presence of agonist on the receptor.