Influence of isoproterenol and phosphodiesterase inhibitors on platelet-activating factor biosynthesis in the human neutrophil.

Abstract

Increasing cellular levels of cAMP inhibit eicosanoid production in the human neutrophil; however, little is known about the effects of cAMP on platelet-activating factor (PAF) biosynthesis in this cell. In the current study, the beta-adrenergic receptor agonist isoproterenol, alone or in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), was used to increase cAMP in neutrophils. The incorporation of [3H]acetate into PAF and the synthesis of leukotrienes in response to ionophore A23187 were significantly inhibited by 10 microM isoproterenol. The inhibitory effect on PAF was potentiated by the addition of 10 microM IBMX. The effects of IBMX were mimicked by rolipram, an inhibitor of the cAMP-specific phosphodiesterase IV. Mass spectrometric analysis of the PAF molecular species in stimulated neutrophils indicated that the combination of isoproterenol and IBMX inhibited (> 50%) ionophore- and fMLP-induced production of PAF. To better understand the mechanism involved in the inhibition of PAF formation, the major biosynthetic steps were examined in the presence and absence of a maximally effective concentration of isoproterenol and IBMX. Isoproterenol alone or in the presence of IBMX had no measurable effect on the ionomycin-induced increase in cytosolic calcium concentration, as assessed by fura-2 fluorescence. Treating intact neutrophils with a combination of isoproterenol and IBMX did not inhibit acetyltransferase activity when assayed in a subsequent broken cell preparation. Finally, increasing cellular cAMP with these drugs did not influence the ability of the neutrophil to catabolize PAF. Phospholipase A2-like activity was assayed in the whole cell by measuring the mobilization of phospholipase A2 products, PAF, lyso PAF, and arachidonic acid, from cellular phosphoglycerides. Treatment of neutrophils with isoproterenol and IBMX significantly reduced the production of lyso PAF and PAF from 1-alkyl-2-arachidonoyl sn-glycero-3-phosphocholine. Similarly, increasing cellular levels of cAMP inhibited the cell's ability to mobilize arachidonic acid upon cell activation. These data suggest that increasing cellular levels of cAMP leads to the inhibition of PAF and leukotriene biosynthesis, at least in part, by regulation of phospholipase A2 activity.