THE EFFECT OF CYCLIC-AMP AND CYCLIC-GMP PHOSPHODIESTERASE INHIBITORS ON THE SUPEROXIDE BURST OF GUINEA-PIG PERITONEAL-MACROPHAGES

1 The cyclic nucleotide phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP PDE) and guanosine 3':5'-cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2 The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDEI/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 muM and 145 +/- 17 mum respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 mum, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 mum prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 mum salbutamol. 3 The increase in superoxide generation elicited by FMLP (10(-9) - 10(-5) M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 mum IBMX. 4 Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin. Selective inhibitors of PDE IV inhibited the particulate cyclic AMP PDE activity (IC50s rolipram 1.5 +/- 0.3 mum, Ro 20-1724 4.1 +/- 0.6 mum) as did the non-selective inhibitor IBMX (IC50 22 +/- 8 mum). The macrophage particulate PDE activity was resistant to inhibition by the PDE III inhibitor SK&F 94836 and the PDE I/V inhibitor, zaprinast. The cytosolic calcium/calmodulin stimulated cyclic AMP hydrolytic activity was inhibited by zaprinast (IC50 - calcium/calmodulin 123 +/- 39 mum; + calcium/calmodulin IC50 17.7 +/- 6.3 muM). 5 The results indicate that guinea-pig peritoneal macrophages contain a type IV cyclic AMP PDE which is predominantly membrane associated and a predominantly cytosolic calcium/calmodulin stimulated cyclic AMP PDE. Functional studies suggest that both of these PDE activities contribute to cyclic AMP hydrolysis and regulation of superoxide generation in these cells. Inhibition of spontaneous superoxide generation, but not that stimulated by FMLP, suggests that the activity of PDE inhibitors is subject to functional antagonism but that this can be overcome by enhancing cyclic AMP formation.