8-pCPT-conjugated cyclic AMP analogs exert thromboxane receptor antagonistic properties.

Membrane-permeable 8-(4-chlorophenylthio)-2'-O-methyl cyclic AMP (8-pCPT-2'-O-Me-cAMP) has been shown to specifically activate cAMP-regulated Epac proteins, without direct effects on protein kinase A and protein kinase G. During isometric tension measurements in thoracic aortic rings from Wistar rats, we observed that 8-pCPT-2'-O-Me-cAMP selectively induced a rightward shift of the concentration response curve for the thromboxane mimetic U46619, without altering the contractile response to noradrenaline. We hypothesised that 8-pCPT-2'-O-Me-cAMP and similar compounds may function as direct thromboxane receptor antagonists. Indeed, in addition to 8-pCPT-2'-O- Me-cAMP, also 8-pCPT-cAMP, 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS) and 8-CPT-adenosine, but not 8-Bromo-2'-O-Me-cAMP, induced rightward shifts of the contractile response to U46619. Likewise, 8-pCPT-2'-O- Me-cAMP and Rp-8-CPT-cAMPS, but not 8-Bromo-2'-O-Me-cAMP, specifically reduced U46619-induced aggregation of human platelets. In addition, 8-pCPT-2'-O-Me-cAMP and Rp-8-CPT-cAMPS completely reversed U46619-induced reduction of intercellular adhesion molecule-1 expression and migration of human coronary artery endothelial cells. Most important, the cAMP analogs that reduced the contractile response to U46619 also concentration-dependently inhibited binding of the thromboxane receptor radioligand [5,6-3H]SQ29548 to human platelets. We conclude that 8-pCPT-conjugated cAMP analogs exert competitive thromboxane receptor antagonistic properties.

Cyclic AMP-dependent and Epac-mediated activation of R-Ras by G protein-coupled receptors leads to phospholipase D stimulation.

The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical G(s)-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation.