Selective inhibition by NPT 15392 of lymphocyte cyclic GMP phosphodiesterase

Abstract

Cyclic nucleotide phosphodiesterases were measured in mouse spleen and thymus lymphocyte membranes and soluble fractions and in extracts of canine tracheal smooth muscle. The immunostimulant erythro-9(2-hydroxy,3-nonyl) hypoxanthine (NPT 15392) was found to be a potent and relatively selective inhibitor of mouse lymphocyte cyclic GMP phosphodiesterase, with IC 50 values 15–180 times greater for cyclic AMP than cyclic GMP phosphodiesterases. The greatest inhibition by NPT 15392 was found using 10 μM substrate, and inhibition was greater in membrane than soluble forms of phosphodiesterase. Spleen soluble enzymes were separated by DEAE Bio-Gel A column into six peaks. A major form of cyclic GMP phosphodiesterase was inhibited effectively by NPT 15392 in a competitive manner ( K i = 50 μM). Cyclic AMP phosphodiesterase activity in the same fraction, but representing only a fifth of the total activity, was also inhibited ( K i = 70 μM). Other soluble enzymes were not affected significantly. Membrane bound enzymes were solubilized and separated into three peaks. One with high affinity for cyclic GMP was strongly inhibited ( K i = 10 μM) by NPT 15392. Inosine and isoprinosine were one-tenth to one-hundredth as effective as NPT 15392 as cyclic nucleotide phosphodiesterase inhibitors. Incubation of mouse splenic lymphocytes with NPT 15392 for 48 hr resulted in enzymes with altered responsiveness to the drug in broken cell assays: inhibition of cyclic GMP hydrolysis was enhanced while that of cyclic AMP hydrolysis was decreased. Among three separated and characterized forms of tracheal smooth muscle phosphodiesterase, NPT 15392 inhibited the low K m cyclic GMP phosphodiesterase 6–10 times more effectively than the other enzymes. These data suggest that the immunopharmacologic activities of NPT 15392 may include specific cyclic GMP phospho-diesterase inhibition as one of several possible mechanisms.