Characterization of guinea-pig eosinophil phosphodiesterase activity: Assessment of its involvement in regulating superoxide generation

Abstract

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O − 2) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 ± 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics ( K m = 1.3 and 31.4 μM) and was unaffected by cGMP ( ic 50 > 100 μ M) or CaCl 2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 ( ic 50 = 0.92 ± 0.43 μ M) , rolipram ( ic 50 = 0.20 ± 0.04 μ M) and denbufylline ( ic 50 = 0.20 ± 0.01 μ M) , potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin ( ic 50 = 0.11 ± 0.02 μ M) and AH-21-132 ( ic 50 = 2.57 ± 0.02 μ M) . Eosinophil cAMP PDE was resistant to SK&F 94120 ( ic 50 > 1000 μM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active ( ic 50 = 35.33 ± 10.74 μ M) . O − 2 release from resting cells was potently inhibited by rolipram ( ic 50 = 0.05 ± 0.03 μ M) and denbufylline ( ic 50 = 0.06 ± 0.04 μ M) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin ( ic 50 = 8.0 ± 2.7 μ M) . AH-21-132 ( ic 50 > 10 μ M) , SK&F 94120 ( ic 50 > 10 μ M) and zaprinast ( ic 50 > 10 μ M) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 μM isoprenaline, potently increased intracellular accumulation ( ec 50 = 0.45 ± 0.16 and 0.28 ± 0.08 μ M , respectively). Trequinsin and AH21–132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 μM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-201724-inhibited cAMP PDE plays a role in regulating eosinophil. O − 2 generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting. O − 2 suggests the existence of a barrier impeding access to the enzyme.