Effects of adenosine analogues on basal, prostaglandin E1- and forskolin-stimulated cyclic AMP formation in intact neuroblastoma cells.
We have examined the effects of R-phenylisopropyladenosine (R-PIA) and other adenosine analogues on basal, prostaglandin E1 (PGE1)- and forskolin-stimulated cyclic AMP (cAMP) formation in intact N1E-115 neuroblastoma cells, to determine whether the cells contain A1 adenosine receptors that are negatively coupled with adenylate cyclase. Basal levels of cAMP (68 +/- 7 pmol/mg protein; mean +/- SE, N = 15) were not altered by low concentrations of R-PIA. The apparent lack of inhibition was not due to increases in cAMP due to activation of a stimulatory A2 receptor by endogenously-synthesized adenosine. By comparison, low levels of R-PIA did reduce significantly (P less than 0.05) PGE1-dependent increases in cAMP formation (maximum response to PGE1, 972 +/- 77 pmol cAMP/mg protein; EC50 for PGE1, 0.2 microM). Inhibition was dose dependent, and resulted in a 30-50% maximum reduction in production stimulated by PGE1. Nanomolar concentrations of R-PIA elicited half-maximal inhibition; the inhibitory response was blocked by 8-phenyltheophylline (8-PT). The order of potencies of several adenosine analogues in eliciting this response suggested that inhibition was mediated by an A1 adenosine receptor. Examination of the effects of R-PIA on forskolin-stimulated cAMP formation yielded several interesting findings. First, stimulation by the diterpene by itself was blocked by both adenosine deaminase (ADA) and 8-PT (40 and 25% inhibition respectively). Low concentrations of R-PIA (less than 10(-6) M) had no effect on forskolin-stimulated cAMP production. At higher levels (greater than or equal to 10(-6) M) the analogues acted synergistically with the diterpene, to yield cAMP levels that were up to 3-fold higher than the additive effect of the two agents. Potentiation was stereospecific, Ca2+ dependent, and was blocked by 8-PT. The results of this study suggest that, in N1E-115 neuroblastoma cells, inhibitory A1 receptors are not stimulated in response to non-specific elevations in cAMP, but are associated with specific stimulatory receptors such as those activated by PGE1.
Murphy
M G
MG
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
Byczko
Z
Z
D000241:Adenosine / Q000031:analogs & derivatives; D000243:Adenosine Deaminase / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology*; D000818:Animals; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D010660:Phenylisopropyladenosine / Q000494:pharmacology*; D011983:Receptors, Purinergic / Q000032:analysis* / Q000187:drug effects; D014407:Tumor Cells, Cultured
D011983:Receptors, Purinergic; D005576:Colforsin; D010660:Phenylisopropyladenosine; D000242:Cyclic AMP; D000243:Adenosine Deaminase; D000527:Alprostadil; D000241:Adenosine