Temporally resolved cAMP monitoring in endothelial cells uncovers a thrombin‐induced [cAMP] elevation mediated via the Ca<sup>2+</sup>‐dependent production of prostacyclin

Werthmann, Ruth C.; Bünemann, Moritz

doi:10.1113/jphysiol.2010.200121

Cited by 11 publications

(14 citation statements)

References 36 publications

(65 reference statements)

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“…26 If this calcium influx is attenuated, the increase in permeability that characterizes frog microvessels after plasma protein removal is attenuated, even in the continued absence of plasma protein. A local increase in calcium ion concentration close to the peripheral actin band weakens adhesion mechanisms, 34,88 just the opposite to the S1P1 action described above. These observations suggest that the function of the glycocalyx as a permeability barrier may depend as much on the organization of anchoring points to the cell cytoskeleton and the modulation of associated signaling pathways as on the organization within the matrix itself.…”

Section: Endothelial Surface Layer Structurementioning

confidence: 87%

Endothelial Glycocalyx: Permeability Barrier and Mechanosensor

2011

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Endothelial cells are covered with a polysaccharide rich layer more than 400 nm thick whose mechanical properties limit access of circulating plasma components to endothelial cell membranes. The barrier properties of this endothelial surface layer are deduced from the rate of tracer penetration into the layer and the mechanics of red and white cell movement through capillary microvessels. This review compares the mechanosensor and permeability properties of an inner layer (100–150 nm, close to the endothelial membrane) characterized as a quasi-periodic structure which accounts for key aspects of transvascular exchange and vascular permeability with those of the whole endothelial surface layers. We conclude that many of the barrier properties of the whole surface layer are not representative of the primary fiber matrix forming the molecular filter determining transvascular exchange. The differences between the properties of the whole layer and the inner glycocalyx structures likely reflect dynamic aspects of the endothelial surface layer including tracer binding to specific components, synthesis and degradation of key components, activation of signaling pathways in the endothelial cells when components of the surface layer are lost or degraded, and the spatial distribution of adhesion proteins in microdomains of the endothelial cell membrane.

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Section: Endothelial Surface Layer Structurementioning

confidence: 87%

Endothelial Glycocalyx: Permeability Barrier and Mechanosensor

2011

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“…Also, some observations could be erroneous due to the employed cAMP sensor molecule. For example, when thrombin induced cAMP was examined in endothelial cells, PKA-based sensor, which releases an active PKA catalytic subunit upon cAMP binding displayed a very different cAMP time course compared to Epac1-camp (Werthmann et al, 2011). PKA overexpressed as a sensor could activate PDEs and alter cAMP time course.…”

Section: Discussionmentioning

confidence: 99%

Real-Time Monitoring of Intracellular cAMP During Acute Ethanol Exposure

Gupta

Qualls‐Creekmore

Yoshimura

2013

Alcohol Clin Exp Res

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Background In previous studies we have shown that ethanol enhances the activity of Gs-stimulated membrane-bound adenylyl cyclase (AC). The effect is AC isoform specific and the type 7 AC (AC7) is most responsive to ethanol. In this study, we employed a fluorescence resonance energy transfer (FRET) based cAMP sensor, Epac1-camps, to examine real-time temporal dynamics of ethanol effects on cAMP concentrations. To our knowledge, this is the first report on real-time detection of the ethanol effect on intracellular cAMP. Methods Hela cells were transfected with Epac1-camps, dopamine D1A receptor, and one isoform of AC (AC7 or AC3). Fluorescent images were captured using a specific filter set for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and FRET, respectively and FRET intensity was calculated on a pixel-by-pixel basis to examine changes in cAMP. Results During 2-minute stimulation with dopamine (DA), the cytoplasmic cAMP level quickly increased, then decreased to a plateau, where the cAMP level was higher than the level prior to stimulation with DA. Ethanol concentration dependently increased cytoplasmic cAMP in cells transfected with AC7, while ethanol did not have effect on cells transfected with AC3. Similar trends were observed for cAMP at the plasma membrane and in the nucleus during 2-minute stimulation with DA. Unexpectedly, when cells expressing AC7 were stimulated with DA or other Gs protein-coupled receptor’s ligand plus ethanol for 5 seconds, ethanol reduced cAMP concentration. Conclusion These results suggest that ethanol has two opposing effects on the cAMP generating system in an AC isoform specific manner, the enhancing effect on AC activity and the short lived inhibitory effect. Thus, ethanol may have a different effect on cAMP depending on not only AC isoform but also the duration of exposure.

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“…However, only the advent of imaging technologies first by fluorescence (Tsien and Tsien, 1990) and later by FRET (Miyawaki, 2003) allowed the discovery of the full spectrum of spatiotemporal patterns in second-messenger concentrations. These studies revealed not only that concentrations in calcium as well as cAMP can show complex patterns of oscillations but that these may be interlinked by various intracellular mechanisms such as Ca 2ϩ -regulated phosphodiesterases and adenylyl cyclases (Zaccolo and Pozzan, 2003;Landa et al, 2005;Harbeck et al, 2006;Willoughby and Cooper, 2006;Kim et al, 2008;von Hayn et al, 2010;Ni et al, 2011;Werthmann et al, 2011). Oscillations have also been de- 326 scribed for the activity of protein kinase C (Violin et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…Although it has been possible to generate images of primary cells or of functional units such as thyroid follicles or pancreatic islets isolated from mice transgenically expressing second-messenger sensors Calebiro et al, 2009;Mironov et al, 2009;von Hayn et al, 2010;Werthmann et al, 2009Werthmann et al, , 2011, the high degree of background fluorescence calls for several improvements for in vivo microscopy, including red-shifted sensors and FRET analysis by multiphoton and second harmonic generation microscopy (Provenzano et al, 2009). In addition, BRET studies have been performed in cells isolated from transgenic mice expressing luciferaselabeled ␤ 2 -adrenergic receptors and GFP2-labeled ␤-arrestin2 (Audet et al, 2010).…”

Section: Discussionmentioning

confidence: 99%