Tanaka Toshio

Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor sepharose chromatography☆

Abstract Cilostamide derivatives are potent inhibitors of human platelet aggregation and selectively inhibit human platelet cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase. N -Cyclohexyl- N -(2-hydroxybutyl)-5-[6-1,2,3,4-tetrahydro-2oxoquinolyloxy)]-butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 μg/ml collagen was 5 μM. On the other hand, the concentrations of OPC-13135 producing 50% inhibition of human platelet cyclic AMP phosphodiesterase and cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase were 0.073 and 21.8 μM, respectively. We purified over 480-fold the soluble low K m form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sehparose column as a final step in the purification procedure. The purified protein has a molecular weight of 175,000, determined by gel filtration and is an acidic protein, as determined by isoelectric focussing (pI = 4.9). Kinetic measurements indicated that the enzyme protein had a K m , value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 μM respectively, and a V max value of 85.3 and 19.8 nmole/min/mg protein, respectively. K i value of the OPC-13135 for the enzyme was 0.015 μM and was of competitive fashion against cyclic AMP.

Selective inhibition of cyclic AMP phosphodiesterase from various human tissues by milrinone, a potent cardiac bipyridine

Abstract We observed the effects of milrinone, an inotropic agent prescribed to treat congestive heart failure, on cyclic nucleotide messenger systems in various human tissues in vitro . Cyclic nucleotide phosphodiesterases (PDEs) from the human heart were separated into three isoforms, FI, FII and FIII, by DEAE-cellulose chromatography. Milrinone proved to be a potent and selective inhibitor of human cardiac FIII PDE, a “low K m ” enzyme for cyclic AMP (cAMP-PDE). The IC 50 value for the inhibition of FIII PDE was 0.42 μM, while those of FI and FII PDEs, “high K m ” enzymes, were 38 and 19 μM, respectively. Kinetic studies showed that milrinone inhibited the activity of FIII PDE, competitively with respect to cAMP, and the K i , was 0.15 μM. Milrinone in doses to 100 μM had no effect on human cardiac cAMP-dependent protein kinase and adenylate cyclase. The activity of cAMP-PDEs from human platelets and the aorta, as well as that from heart, were potently inhibited by milrinone, with much the same IC 50 values. Cyclic AMP-PDEs from human kidney, liver and lung were not readily inhibited by milrinone, and the IC 50 values of cAMP-PDEs from these tissues were about 7-30-fold higher than that from heart. On the other hand, papaverine had a relatively lesser selectivity for any of the cAMP-PDEs. All these results suggest that milrinone exerts inotropic effects by inhibiting cAMP-PDE selectively in the human heart tissues and that this compound can be used to evaluate different forms of cAMP-PDEs present in human tissues.

The binding of the calcium channel blocker, bepridil, to calmodulin.

Bepridil had the highest relative potency for inhibition of myosin light chain kinase (MLCK) activated by Ca2+-calmodulin of all the calcium channel blockers we examined. Kinetic analysis indicated that the primary effect of bepridil was mediated through a competitive inhibition of the enzyme activation by interaction with calmodulin and the apparent Ki value of this agent was 2.2 microM. We then examined the binding of bepridil to calmodulin, using the equilibrium column binding technique. [3H]bepridil bound to the calcium-calmodulin complex, but not to calmodulin in the presence of 2 mM EGTA. Scatchard analysis of the binding of bepridil to calmodulin demonstrated that the dissociation constant was 6.2 microM and the calculated number of specific binding sites was about 5 sites per molecule of calmodulin. The concentrations of unlabeled bepridil, W-7, prenylamine, verapamil and diltiazem producing 50% inhibition (IC50) of the binding of [3H]bepridil to calmodulin were 4 microM, 28 microM, 45 microM, 130 microM and 700 microM, respectively. However, nifedipine and nicardipine did not displace [3H]labeled bepridil from calmodulin. There was a good correlation between the displacement of [3H]bepridil from calmodulin and the inhibitory effect on MLCK by these calcium channel blockers and W-7. These results suggest that bepridil binds to calmodulin in the presence of calcium and potently inhibits the phosphorylation of myosin light chain.