INFLUENCE OF ISOPROTERENOL AND PHOSPHODIESTERASE INHIBITORS ON PLATELET-ACTIVATING-FACTOR BIOSYNTHESIS IN THE HUMAN NEUTROPHIL

Increasing cellular levels of cAMP inhibit eicosanoid production in th e 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, al one or in combination with the phosphodiesterase inhibitor 3-isobutyl- 1-methylxanthine (IBMX), was used to increase cAMP in neutrophils. The incorporation of [H-3]acetate into PAF and the synthesis of leukotrie nes in response to ionophore A23187 were significantly inhibited by 10 muM isoproterenol. The inhibitory effect on PAF was potentiated by th e addition of 10 muM IBMX. The effects of IBMX were mimicked by rolipr am, an inhibitor of the cAMP-specific phosphodiesterase IV. Mass spect rometric analysis of the PAF molecular species in stimulated neutrophi ls indicated that the combination of isoproterenol and IBMX inhibited (> 50%) ionophore- and fMLP-induced production of PAF. To better under stand the mechanism involved in the inhibition of PAF formation, the m ajor biosynthetic steps were examined in the presence and absence of a maximally effective concentration of isoproterenol and IBMX. Isoprote renol alone or in the presence of IBMX had no measurable effect on the ionomycin-induced increase in cytosolic calcium concentration, as ass essed by fura-2 fluorescence. Treating intact neutrophils with a combi nation of isoproterenol and IBMX did not inhibit acetyl-transferase ac tivity when assayed in a subsequent broken cell preparation. Finally, increasing cellular cAMP with these drugs did not influence the abilit y of the neutrophil to catabolize PAF. Phospholipase A2-like activity was assayed in the whole cell by measuring the mobilization of phospho lipase A2 products, PAF, lyso PAF, and arachidonic acid, from cellular phosphoglycerides. Treatment of neutrophils with isoproterenol and IB MX significantly reduced the production of lyso PAF and PAF from 1-alk yl-2-arachidonoyl sn-glycero-3-phosphocholine. Similarly, increasing c ellular levels of cAMP inhibited the cell's ability to mobilize arachi donic acid upon cell activation. These data suggest that increasing ce llular levels of cAMP leads to the inhibition of PAF and leukotriene b iosynthesis, at least in part, by regulation of phospholipase A2 activ ity.