Comparison of the responses of freshly isolated and cultured human monocytes and P388D₁ cells to agents affecting cyclic AMP metabolism

We have studied the effects of beta-adrenergic agonists, PGE₁, MIX, and insulin on the metabolism and function of freshly isolated and cultured human monocytes and P388D₁ cells. Human monocytes were isolated from fresh defibrinated blood by centrifugation through Ficoll/Hypaque, followed by adherence to plastic tissue culture dishes. Immediately after the adherence step, basal monocytic cyclic AMP was 0.61 ± 0.06 pmol/10⁶ cells (mean ± S.E. for eight different preparations). The cyclic AMP content of fresh monocytes was transiently increased no more than fivefold by incubation with 0.1 mM ISO together with 0.1 mM MIX. Cellular cyclic AMP was elevated 70-fold by 10 μM PGE₁ with 0.1 mM MIX. The basal cyclic AMP of monocytes cultured for 7 days was 3.77 ± 0.86 pmol/10⁶ cells (seven preparations). Their responses to ISO, MIX, and PGE₁ were similar to those of fresh monocytes. Insulin (0.2 μM) had no effect on cyclic AMP of either fresh or cultured monocytes. The cyclic AMP content of “macrophage-like” P388D₁ cells grown in monolayer culture was elevated ninefold by 10 μM PGE₁ and 50-fold by 10 μM PGE₁ plus 0.1 mM MIX. However, 0.1 mM ISO did not increase cyclic AMP, even in the presence of MIX. Insulin (0.2 μM) did not change cyclic AMP in the presence or absence of PGE₁. In sonicated P388D₁ cells, adenylyl cyclase activity was increased fivefold by 10 μM PGE₁ but was insensitive to 0.5 mM EPI, 0.5 mM ISO, or 0.2 μM insulin. Insulin (0.1 μM) had no effect on incorporation of [¹⁴C]glucose into glycogen by any of the cell types studied. Also, insulin (0.25 nM to 1.25 μM) did not influence performance of ADCC by human monocytes. We conclude that the adenylyl cyclases of human monocytes and P388D₁ cells respond strongly to PGE₁ but weakly to beta-adrenergic agonists. Since there was no effect of insulin on monocytic cyclic AMP, glycogen metabolism, or antibody-dependent effector function, we suggest that human monocytic insulin-binding sites are not coupled to physiologically important effector systems.