.1.4.17) decreases the cellular level of cAMP and cGMP and regulates the function of platelets such as platelet aggregation and adhesion. Human platelets contain 3 forms of PDE which differ with respect to substrate specificity; PDE I hydrolyzes cAMP and cGMP, equally, PDE III has a higher affinity for cAMP than cGMP, and PDE V is cGMPspecific. [1][2][3][4][5][6] Recently, highly specific inhibitors for each of the PDE isoforms have been developed for therapeutic uses. These inhibitors which elevate cAMP and/or cGMP level(s) of the platelets and inhibit platelet aggregation and adehesion have therapeutic possibilities such as a drug for angina treatment and thrombosis.7-12) Thus, a simple and sensitive method has been needed for the measurement of PDE activity to develop and estimate the blocking effect of the inhibitors.We previously reported the HPLC measurement of PDE activity in the rat cerebral cortex using (3,4-dimethoxyphenyl)glyoxal (DMPG) as a fluorescence derivatization reagent.13) This method is highly selective for guaninecontaining compounds, and does not require a special substrate which is radioactive or fluorogenic, such as H]-cGMP, 14,15) 2Ј-O-anthraniloyl-cGMP and 2Ј-O-methylanthraniloyl-cGMP. 16,17) This paper extends the study to the assay method for human platelet PDE activity under optimal conditions for the enzyme reaction and the assessment of PDE-inhibitory potency of PDE.
MATERIALS AND METHODSReagents and Solutions GTP, GDP, GMP, cGMP, guanine and guanosine were purchased from Seikagaku Kogyo (Tokyo, Japan). 8-Azaguanine, dipyridamole, 3-isobutyl-1-methylxanthine (IBMX), eburnamenine-1,4-carboxylic acid ethyl ester (vinpocetine) and 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) were from Sigma (U.S.A.). N-cyclohexyl-N-methyl-4-(1,2-dihydro-2-oxo-6-quinolyloxy)butyramide (cilostamide) was from Wako (Osaka, Japan). DMPG was prepared as previously described.18) All other chemicals were of reagent grade.Water was purified by a Milli-Q II system (Japan Millipore, Tokyo, Japan). DMPG (0.1 M) was dissolved in dimethylsulfoxide-water (2 : 3, v/v). Sample Preparation Human platelet PDE samples were prepared according to the method of Radomski et al.
19)Blood samples from healthy volunteers were collected in siliconized tubes containing 0.1 vol. of 3.13% (w/v) citrate (VENOJECT II, Terumo) and then centrifuged at 200ϫg for 10 min at 4°C. Platelet-rich plasma (PRP) was then removed, EDTA-2Na (7.7 mM) was added, and PRP was centrifuged at 2000ϫg for 20 min at 4°C. The platelet pellets were washed in Hepes-Tyrode buffer (129 mM NaCl, 8.9 mM NaHCO 3 , 2.8 mM KCl, 0.8 mM KH 2 PO 4 , 0.8 mM MgCl 2 , 5.6 mM glucose and 10 mM Hepes, pH 7.4) containing EDTA-2Na and then resuspended in 1 ml of 10 mM Hepes buffer (pH 7.4) containing 0.32 M sucrose followed by homogenization by sonication twice for 5 s. The homogenate was used as the source PDE samples.The protein concentration in the enzyme sample was determined by the method of Smith et al. with bovine serum albumin ...
