Characterization of guinea-pig eosinophil phosphodiesterase activity. Assessment of its involvement in regulating superoxide generation.
Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O2-) 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 (Km = 1.3 and 31.4 microM) and was unaffected by cGMP (IC50 greater than 100 microM) or CaCl2 (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 (IC50 = 0.92 +/- 0.43 microM), rolipram (IC50 = 0.20 +/- 0.04 microM) and denbufylline (IC50 = 0.20 +/- 0.01 microM), potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin (IC50 = 0.11 +/- 0.02 microM) and AH-21-132 (IC50 = 2.57 +/- 0.02 microM). Eosinophil cAMP PDE was resistant to SK&F 94120 (IC50 greater than 1000 microM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active (IC50 = 35.33 +/- 10.74 microM). .O2- release from resting cells was potently inhibited by rolipram (IC50 = 0.05 +/- 0.03 microM) and denbufylline (IC50 = 0.06 +/- 0.04 microM) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin (IC50 = 8.0 +/- 2.7 microM). AH-21-132 (IC50 greater than 10 microM), SK&F 94120 (IC50 greater than 10 microM) and zaprinast (IC50 greater than 10 microM) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 microM isoprenaline, potently increased intracellular accumulation (EC50 = 0.45 +/- 0.16 and 0.28 +/- 0.08 microM, respectively). Trequinsin and AH-21-132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 microM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-20-1724-inhibited cAMP PDE plays a role in regulating eosinophil .O2- generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting .O2- suggests the existence of a barrier impeding access to the enzyme.
Souness
J E
JE
Dagenham Research Centre, Rhône-Poulenc Rorer Inc., Health Care Division, Essex, U.K.
Carter
C M
CM
Diocee
B K
BK
Hassall
G A
GA
Wood
L J
LJ
Turner
N C
NC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000097:blood*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D000242:Cyclic AMP / Q000097:blood; D004804:Eosinophils / Q000201:enzymology*; D006168:Guinea Pigs; D007424:Intracellular Fluid / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000187:drug effects; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D013481:Superoxides / Q000378:metabolism*
D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D013481:Superoxides; D000242:Cyclic AMP; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram; D007545:Isoproterenol; D002118:Calcium