Characterization of Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells.
A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.
Sandberg
M
M
Medizinische Universitätsklinik, Klinische Forschergruppe, Würzburg, Federal Republic of Germany.
Butt
E
E
Nolte
C
C
Fischer
L
L
Halbrügge
M
M
Beltman
J
J
Jahnsen
T
T
Genieser
H G
HG
Jastorff
B
B
Walter
U
U
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000201:enzymology; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology*
D010975:Platelet Aggregation Inhibitors; D013873:Thionucleotides; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D004004:Dichlororibofuranosylbenzimidazole; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP