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Following earlier observations that increasing the polyunsaturated fatty-acid (PUFA) content of N1E-115 neuroblastoma cells elevated basal and adenosine (Ado)-stimulated intracellular cyclic AMP (cAMP) formation, we carried out studies to determine the mechanism(s) by which PUFA exerted their modulatory effects. Basal increases in cAMP in the PUFA-enriched (PUFA+) cells were evident with short (60 sec) exposure to a phosphodiesterase inhibitor (Ro 20-1724), and increased to a maximum at 20 min; they were not observed in the absence of Ro 20-1724. Forskolin-stimulated cAMP formation in the presence of the Ro compound was 2- to 3-fold higher in the PUFA+ cells. Basal elevations in cAMP were reduced by approximately 70% by exposing the PUFA+ cells to Ado deaminase (ADA) or to an Ado antagonist, and were further increased by inhibiting ADA, which suggested that they could be producing endogenous Ado that activated stimulatory Ado receptors. However, this did not appear to involve PUFA-mediated stimulation of 5'-nucleotidase activity or inhibition of [3H]Ado uptake. Overall, the results of this study indicated that multiple mechanisms are involved in PUFA modulation of cAMP formation.

The regulation of intracellular cyclic AMP (cAMP) formation by adenosine (Ado) and its analogues has been examined in primary cultures of rat-brain astrocytes and neurons. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, basal levels of cAMP ranged from 40-120 pmol/mg protein in both cell types. Levels were not altered by treating the cells with Ado deaminase, which suggested that they did not produce appreciable amounts of endogenous Ado under standard culture conditions. In the astrocytes, microM quantities of agonists increased cAMP up to 30-fold higher than basal values; the relative potencies were typical of an A2 Ado receptor (NECA greater than Ado greater than R-PIA). Neuron-enriched cultures exhibited a maximum fourfold increase in cAMP in response to NECA; this was decreased a further eightfold when the cultures had prolonged exposure to the antimitotic agent, c-Ara, to eliminate greater than 98% of the nonneuronal cells. Low (nM) amounts of the Ado agonists inhibited cAMP formation in both cell types. In the astrocytes, the order of potency of inhibition of isoproterenol-stimulated cAMP formation was typical of an A1 receptor (R-PIA greater than Ado greater than NECA); maximum inhibition was 55-65%. Isoproterenol did not increase cAMP in the neuronal cultures. However, forskolin-stimulated formation was effectively (approximately 50%) inhibited by A1 Ado agonists; inhibition was not affected by prolonged treatment with c-Ara. From this study we tentatively concluded that rat astrocytes and neurons both contain inhibitory A1 Ado receptors, but that the stimulatory "A2" subtype is localized mainly on astrocytes.

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.