The crosstalk between Ca2+ and cAMP signals plays a significant role for the regulation of the endothelial barrier function. The Ca 2+ -elevating agent thrombin was demonstrated to increase endothelial permeability and to decrease cAMP levels. Since Ca

The nucleotide receptor P2Y 1 regulates a variety of physiological processes and is involved in platelet aggregation. Using human P2Y 1 -receptors C-terminally fused with a fluorescent protein, we studied the role of potential receptor phosphorylation sites in receptor internalization and ␤-arrestin-2 translocation by means of confocal microscopy. Three receptor constructs were generated that lacked potential phosphorylation sites in the third intracellular loop, the proximal C terminus, or the distal C terminus. The corresponding receptor constructs were expressed in human embryonic kidney (HEK)-293 cells and stimulated with 100 M ADP. Rapid receptor internalization was observed for the wild-type receptor and from those constructs mutated in the third intracellular loop and the proximal C terminus. However, the construct lacking phosphorylation sites at the distal C terminus did not show receptor internalization upon stimulation. The microscopic data were validated by HAtagged receptor constructs using a cell surface enzyme-linked immunosorbent assay. P2Y 1 -receptor stimulated ␤-arrestin-2-yellow fluorescent protein (YFP) translocation followed the same pattern as receptor internalization. Hence, no ␤-arrestin-2-YFP translocation was observed when the distal C-terminal phosphorylation sites were mutated. Individual mutations indicate that residues Ser352 and Thr358 are essential for receptor internalization and ␤-arrestin-2-YFP translocation. In contrast, protein kinase C (PKC)-mediated receptor desensitization was not affected by mutation of potential phosphorylation sites in the distal C terminus but was prevented by mutation of potential phosphorylation sites in the proximal C terminus. P2Y 1 -receptor internalization in HEK-293 cells was not blocked by inhibitors of PKC and calmodulin-dependent protein kinase. Thus, we conclude that P2Y 1 -receptor desensitization and internalization are mediated by different phosphorylation sites and kinases.The P2Y-receptor family comprises eight different subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ), which can further be subdivided into two subgroups based on their G-protein-coupling specificity (von Kü gelgen, 2006;Abbracchio et al., 2006). One group consist of the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptor and couples mainly to G q -proteins, whereas the second group consisting of the P2Y 12 , P2Y 13 , and P2Y 14 receptor mainly couples to G i -proteins. These receptors mediate the action of extracellular nucleotides on cellular signaling. The P2Y 1 receptor is activated by ADP and has been shown to play a major role in the initiation of platelet activation and aggregation (Abbracchio et al., 2006;Gachet, 2006). Besides expression in platelets, the P2Y 1 receptor is expressed in epithelial and endothelial cells and in immune cells and osteoclasts; thus, the receptor offers a diverse therapeutic potential (Jacobson et al., 2002;Abbracchio et al., 2006;Burnstock, 2006). Significant mutagenesis efforts have led to a ...

cAMP and Ca(2+) are antagonistic intracellular messengers for the regulation of vascular smooth muscle tone; rising levels of Ca(2+) lead to vasoconstriction, whereas an increase of cAMP induces vasodilatation. Here we investigated whether Ca(2+) interferes with cAMP signaling by regulation of phophodiesterases (PDEs) or adenylyl cyclases (ACs). We studied regulation of cAMP concentrations by Ca(2+) signals evoked by endogenous purinergic receptors in vascular smooth muscle cells (VSMCs). The fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps allowed the measurement of cAMP levels in single-living VSMCs with subsecond temporal resolution. Moreover, in vitro calibration of Epac1-camps enabled us to estimate the absolute cytosolic cAMP concentrations. Stimulation of purinergic receptors decreased cAMP levels in the presence of the beta-adrenergic agonist isoproterenol. Simultaneous imaging of cAMP with Epac1-camps and of Ca(2+) with Fura 2 revealed a rise of intracellular Ca(2+) in response to purinergic stimulation followed by a decline of cAMP. Chelation of intracellular Ca(2+) and overexpression of Ca(2+)-independent AC4 antagonized this decline of cAMP, whereas pharmacological inhibition of Ca(2+)-activated PDE1 had no effect. AC assays with VSMC membranes revealed a significant attenuation of isoproterenol-stimulated cAMP production by the presence of 2 muM Ca(2+). Furthermore, small interfering RNA (siRNA) knockdown of AC5 and AC6 (the two ACs known to be inhibited by Ca(2+)), significantly reduced the decrease of cAMP upon purinergic stimulation of isoproterenol-prestimulated VSMCs. Taken together, these results implicate a Ca(2+)-mediated inhibition of AC5 and 6 as an important mechanism of purinergic receptor-induced decline of cAMP and show a direct cross talk of these signaling pathways in VSMCs.

A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.