title: Inhibition and stimulation of photoreceptor phosphodiesterases by dipyridamole and M&B 22,948.
abstract: Few high affinity inhibitors of the photoreceptor phosphodiesterases have been identified. We show here that dipyridamole and M&B 22,948 (Zaprinast), potent inhibitors of the cGMP-binding, cGMP-specific phosphodiesterase (PDE), also inhibit trypsin- or transducin-activated bovine rod and cone photoreceptor phosphodiesterases at submicromolar concentrations. Dixon plots demonstrated that the inhibition of trypsin-activated rod PDE was competitive, with Ki values of 140 nM for M&B 22,948 and 380 nM for dipyridamole. Both of these drugs were much more potent than other PDE inhibitors, including isobutylmethylxanthine (IBMX). These results reinforce the suggestion that the photoreceptor and the cGMP-binding, cGMP-specific PDE are closely related. In addition, the high affinity and selectivity of these agents should make them useful for probing the regulation and function of PDE in the photoreceptor. At low substrate concentrations, the effects of these drugs on basal unactivated PDE activity were similar to those seen with trypsin- or transducin-activated PDE. At millimolar substrate concentrations, however, the effects of the drugs were biphasic; PDE activity was stimulated at drug concentrations from 1 to 10 microM and inhibited at higher concentrations. Stimulation was not observed with IBMX. This stimulation of activity apparently was not an allosteric effect caused by direct binding of the dipyridamole and M&B 22,948 to the high affinity noncatalytic cGMP binding sites on the PDEs; whereas no cooperativity of cGMP binding to this site has been demonstrated, the drugs actually stimulated the binding of low concentrations of cGMP to this site. In addition, whereas preincubation with cGMP and cGMP analogs blocked the stimulation exerted by the drugs, they did so only at much higher concentrations than those necessary for saturation of the high affinity noncatalytic cGMP site. Because the stimulation can only be seen at higher substrate levels than are thought to exist in the photoreceptor, only the inhibitory effects of the drugs are likely to be pharmacologically relevant. However, the stimulation exerted by these drugs may point to a hitherto unknown allosteric interaction between the catalytic and regulatory sites on the PDE or to a previously unrecognized regulatory site.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle 98195.
authors.forename: P G
authors.initials: PG
authors.lastname: Gillespie
authors.forename: J A
authors.initials: JA
authors.lastname: Beavo
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D004176:Dipyridamole / Q000494:pharmacology*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D010726:Phosphodiesterase Inhibitors; D010786:Photoreceptor Cells / Q000201:enzymology*; D011688:Purinones / Q000494:pharmacology*; D051381:Rats; D012374:Rod Cell Outer Segment / Q000201:enzymology; D014357:Trypsin / Q000494:pharmacology
chemical_list: D010726:Phosphodiesterase Inhibitors; D011688:Purinones; D004176:Dipyridamole; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin; C011145:zaprinast; D006152:Cyclic GMP