Photoaffinity labelling of cyclic GMP-inhibited phosphodiesterase (PDE III) in human and rat platelets and rat tissues: effects of phosphodiesterase inhibitors

• Published in: European journal of pharmacology Vol. 268; no. 1; pp. 105 - 114
• Main Authors: Mary Tang, K., Jang, Elliott K., Haslam, Richard J.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.06.1994; Elsevier
• Subjects: 3',5'-Cyclic-GMP Phosphodiesterases - antagonists & inhibitors; 3',5'-Cyclic-GMP Phosphodiesterases - metabolism; Affinity Labels - metabolism; Animals; Biological and medical sciences; Blood Platelets - drug effects; Blood Platelets - enzymology; Cyclic AMP; Cyclic AMP - metabolism; Cyclic GMP - metabolism; Cyclic GMP-inhibited phosphodiesterase (PDE III); Cytosol - drug effects; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Phosphodiesterase inhibitor; Phosphodiesterase Inhibitors - pharmacology; Phosphorus Radioisotopes; Photoaffinity labelling; Platelet; Rats; Rats, Wistar; Vertebrates: blood, hematopoietic organs, reticuloendothelial system; Phosphodiesterase inhibitor; Cyclic AMP; Platelet; Photoaffinity labelling; Cyclic GMP-inhibited phosphodiesterase (PDE III); Human; Rat; Enzyme; 3',5'-Cyclic-GMP phosphodiesterase; Rodentia; Enzyme inhibitor; Esterases; Phosphoric diester hydrolases; Tissue; Vertebrata; Mammalia; Enzymatic activity; Animal; Hydrolases
• ISSN: 0922-4106; 0014-2999
• DOI: 10.1016/0922-4106(94)90125-2

Ultraviolet irradation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not 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 (IC 50 = 13 ± 2 nM), lixazone (IC 50 = 22 ± 4 nM), milrinone (IC 50 = 56 ± 12 nM), cilostamide (IC 50 = 70 ± 9 nM), siguazodan (IC 50 = 117 ± 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC 50 = 3950 ± 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [ 32P]cGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets 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 of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated 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 increased platelet cAMP to the same maximum extent, cilostamide and IBMX 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 inhibit photolabelling of PDE III and to increase platelet cAMP.