Barrier stabilizing effects of cAMP as well as of the small GTPase Rac 1 are well established. Moreover, it is generally believed that permeability-increasing mediators such as thrombin disrupt endothelial barrier functions primarily via activation of Rho A. In this study, we provide evidence that decrease of both cAMP levels and of Rac 1 activity contribute to thrombin-mediated barrier breakdown. Treatment of human dermal microvascular endothelial cells (HDMEC) with Rac 1-inhibitor NSC-23766 decreased transendothelial electrical resistance (TER) and caused intercellular gap formation. These effects were reversed by addition of forskolin/rolipram (F/R) to increase intracellular cAMP but not by the cAMP analogue 8-pCPT-2'-O-Methyl-cAMP (O-Me-cAMP) which primarily stimulates protein kinase A (PKA)-independent signaling via Epac/Rap 1. However, both F/R and O-Me-cAMP did not increase TER above control levels in the presence of NSC-23766 in contrast to experiments without Rac 1 inhibition. Because Rac 1 was required for maintenance of barrier functions as well as for cAMP-mediated barrier stabilization, we tested the role of Rac 1 and cAMP in thrombin-induced barrier breakdown. Thrombin-induced drop of TER and intercellular gap formation were paralleled by a rapid decrease of cAMP as revealed by fluorescence resonance energy transfer (FRET). The efficacy of F/R or O-Me-cAMP to block barrier-destabilizing effects of thrombin was comparable to Y27632-induced inhibition of Rho kinase but was blunted when Rac 1 was inactivated by NSC-23766. Taken together, these data indicate that decrease of cAMP and Rac 1 activity may be an important step in inflammatory barrier disruption.

A crucial event in female reproduction occurs at midcycle, when a LH peak induces the final maturation of ovarian follicles. LH signals via a G protein-coupled receptor selectively expressed in the outermost follicular cell layers. However, how LH signals are relayed inside these cells and finally to the oocyte is incompletely understood. Here, we monitored LH signaling in intact ovarian follicles of transgenic mice expressing a fluorescent cAMP sensor. We found that LH stimulation induces 2 phases of cAMP signaling in all cell layers surrounding the oocyte. Interfering with LH receptor internalization abolished the second, persistent cAMP phase and partially inhibited oocyte meiosis resumption. These data suggest that persistent cAMP signals from internalized LH receptors contribute to transmitting LH effects inside follicle cells and ultimately to the oocyte. Thus, this study indicates that the recently proposed paradigm of cAMP signaling by internalized G protein-coupled receptors is implicated in receptor function and is physiologically relevant.

G-protein-coupled receptors (GPCRs) have long been believed to activate G proteins only on the cell surface. However, we have recently shown that, in thyroid cells, the GPCR for the thyroid-stimulating hormone (TSH) can continue stimulating cAMP production after cointernalization with TSH. cAMP signaling by internalized TSH receptors (TSHRs) was persistent, whereas that by cell-surface TSHRs was apparently transient, but the reasons for the transient signaling by cell-surface TSHRs were not investigated. Here, we developed and used fluorescence resonance energy transfer (FRET)-based methods to precisely compare the kinetics of TSH binding and dissociation from cell-surface TSHRs with those of the subsequent termination of cAMP signaling directly in living cells. Our results indicate that both TSH binding to human TSHRs expressed in a human embryonic kidney cell line (HEK 293) and the ensuing cAMP signals are rapidly and fully reversible (t(1/2,off)=2.96±1.04 and 2.70±0.73 min, respectively). The FRET measurement of TSH binding was specific, as shown by the lack of a detectable interaction between TSH and the β(2)-adrenergic receptor expressed in control cells. Enhancing TSHR internalization by β-arrestin 2 overexpression did not modify the reversibility of TSHR-cAMP signaling. These findings strengthen the view that the cointernalization of TSH-TSHR complexes to a signaling compartment present in thyroid, but not in HEK 293 cells, is responsible for persistent cAMP signaling.

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

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.

No abstract

The barrier function of the endothelium is controlled by the second messengers Ca2+ and cAMP that differentially regulate the permeability of endothelial cells. The Ca2+-elevating agent thrombin has been demonstrated to increase endothelial permeability and to decrease cAMP levels as detected via enzyme immunoassays. To study the effects of thrombin on cAMP with high temporal resolution, we utilised the FRET-based cAMP sensor Epac1-camps in single intact human umbilical vein endothelial cells (HUVECs). In these cells, thrombin induced a delayed increase in [cAMP], initiating after about 40 s, with maximum cAMP levels after 130 s of thrombin application. This increase of cAMP levels was Ca2+-dependent, but did not require calmodulin (CaM). Pharmacological approaches revealed that phospholipase A2 (PLA2) activity and cyclooxygenase (COX)-mediated synthesis of prostaglandins was required for the thrombin-induced elevation of [cAMP]. Furthermore, preincubation of HUVECs with a prostacyclin-receptor antagonist significantly reduced the thrombin-induced increase in [cAMP]. We conclude that thrombin causes the synthesis of prostacyclin in endothelial cells and that the subsequent stimulation of Gs-coupled prostacyclin receptors then results in an increase in [cAMP].

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.