No abstract

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 X 10(-4) M isobutylmethylxanthine (IBMX) or 1 X 10(-3) M dibutyryl cyclic AMP (d-cAMP) inhibited total [3H]-AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]-AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d-cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan-treated cells indicated that 5-HETE was the predominant metabolite generated from [3H]-AA, and its production was depressed during treatment with d-cAMP. Phospholipase activity in human monocyte homogenates was not effected by d-cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.

Radiolabeled human peripheral blood monocytes released [3H]arachidonic acid upon challenge with the calcium ionophore A23187 (10 microM), or f-Met-Leu-Phe (FMLP, 1 microM). Chromatographic analysis of [3H]arachidonic acid labeled phospholipids showed that stimulation by FMLP reduced the amount of labeled phosphatidylcholine exclusively. Treatment of the monocytes with 10(-3) M dibutyryl cyclic AMP (d-cAMP) or 5 X 10(-4) M isobutylmethylxanthine (IBMX) substantially inhibited [3H]arachidonic acid release (30%) and depletion from labeled phosphatidylcholine (PC) in FMLP--but not calcium ionophore--stimulated cells. Using the fluorescent probe Indo-1, the FMLP-induced cytosolic calcium increase was unaffected by 10(-3) M dibutyryl cyclic AMP. The results suggest that FMLP-stimulated phospholipase activity is regulated by cyclic AMP, but not by depressing receptor-medicated increases in cytoplasmic free calcium.