PHOTOAFFINITY-LABELING OF CYCLIC GMP-INHIBITED PHOSPHODIESTERASE (PDE-III) IN HUMAN AND RAT PLATELETS AND RAT-TISSUES - EFFECTS OF PHOSPHODIESTERASE INHIBITORS

Ultraviolet irradation of human platelet cytosol in the presence of P- 32-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 a nd 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibit ed phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dep endent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We ha ve now shown that although photolabelling of platelet PDE III was inhi bited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was n ot affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: trequinsin ( IC50 = 13 +/- 2 nM), lixazinone (IC50 = 22 +/- 4 nM), milrinone (IC50 = 56 +/- 12 nM), cilostamide (IC50 = 70 +/- 9 nM), siguazodan (IC50 = 117 +/- 29 nM) and 3-isobutyl 1-methyluanthine (IBMX) (IC50 = 3950 +/- 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative m eans of investigating their actions at a molecular level that avoids t he need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [P-32]cGMP showed that the 110 kDa PDE III pr esent in human material was replaced by a 115 kDa protein, labelling o f which was also blocked by PDE III inhibitors. Heart and other rat ti ssues contained much less of this putative 115 kDa PDE III than rat pl atelets. In contrast, the 80 kDa protein was labelled much less in pla telets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication o f different patterns of cyclic nucleotide action. We compared the abil ities of phosphodiesterase inhibitors to block the photolabelling of P DE III in human platelet cytosol and to increase the iloprost-stimulat ed accumulation of cAMP in intact platelets. Whereas trequinsin (EC(50 ) = 19 +/- 3 nM), lixazinone (EC(50) = 122 +/- 8 nM), milrinone (EC(50 ) = 5320 +/- 970 nM) and siguazodan (EC(50) = 18880 +/- 3110 nM) all i ncreased platelet cAMP to the same maximum extent, cilostamide and IBM X increased cAMP further, indicating that they inhibited a PDE isozyme in addition to PDE III. Differences in membrane permeability of these compounds may account for discrepancies between their abilities to in hibit photolabelling of PDE III and to increase platelet cAMP.