Rapid calcium‐dependent reduction of intraendothelial cAMP: a trigger to increase vascular permeability?

Further understanding of vascular endothelial barrier modulation will better enable clinical control of fluid balance and aid drug delivery. Acute inflammatory mediators increase intracellular Ca2+ very rapidly in vascular endothelium, leading to breakdown of the barrier. Investigations both in vivo and in cell culture show that this response is strongly inhibited by increased intracellular cAMP. Ca2+ and cAMP signalling pathways are subject to potential crosstalk through Ca2+-regulated adenylate cyclase 6 (AC6) and Ca2+-dependent phosphodiesterase I as well as interaction at converging downstream effectors which regulate endothelial cell contraction and endothelial junctions. Ca2+-sensitive fluorophores enable rapid optical-based measurement of intracellular Ca2+. However, understanding pathway crosstalk has been hampered by poor temporal resolution of cAMP measurement. Previous studies of cAMP in endothelium used enzyme immunoassays and yielded limited temporal information (Cioffi et al. 2002; Baumer et al. 2008). Fluorescence resonance energy transfer (FRET) has been used previously to determine changes in cAMP concentration but use of the regulatory and catalytic subunits of protein kinase A (PKA) was problematic (Adams et al. 1991). A new technique enables FRET measurement based on a single cAMP binding domain and yields a fast, sensitive indicator that has no catalytic or targeting domains to interfere with cellular processes (Nikolaev et al. 2004). The latter research group has now used their technique to investigate endothelium. The study by Werthmann et al. (2009) in this issue of The Journal of Physiology uses the new FRET assay to measure cAMP in real time using cultured endothelial cells stimulated with thrombin and pre-stimulated with isoproterenol to elevate cAMP. The data clearly demonstrate that the rapid initial Ca2+ rise is closely followed by transient suppression of cAMP concentration. This thrombin-induced fall in cAMP was abolished by prior transfection with siRNA to downregulate AC6, thus directly implicating Ca2+-inhibited AC6 in rapid cAMP regulation. While the study yields important new data on early intracellular signals after thrombin stimulation, questions remain about the link between these mechanisms and endothelial barrier regulation. For example, while both the Ca2+ increase and subsequent cAMP decrease reach respective peaks in seconds, the permeability increase of thrombin-stimulated endothelial monolayers that can be measured (in the absence of pre-stimulation of cAMP) does not peak for about 10 min in vivo and can last an hour or more in cell culture (Baumer et al. 2009) even though the present study suggests cAMP levels return toward pre-thrombin levels in 1.5 min. Several potential pathways lead from Ca2+ and cAMP to modulation of the adhesion and contraction of endothelial cells via both PKA and small GTPases (Adamson et al. 2008). Rho family GTPases RhoA and Rac1 regulate the actin cytoskeleton and endothelial barrier and are potential targets of modulated cAMP pathways; cAMP has been shown to activate Rac1 via activation of Ras family GTPase Rap1 through the GTP exchange factor (GEF) Epac. Further investigations will require measurement of changes in these GTPases under conditions where the time course of cAMP is followed. This will require use of inflammatory agents such as bradykinin and platelet-activating factor that do not stimulate the RhoA pathway in intact microvessels as much as the thrombin response in cell culture (Adamson et al. 2003). Also the role of an increase in cAMP in both cAMP pre-stimulated and non-pre-stimulated cells that followed the initial reduction about 60 s after thrombin application needs evaluation. It is notable that pre-stimulation with isoproterenol or forskolin was required in the experiments of Werthmann et al. to demonstrate a thrombin-induced decrease in cAMP concentration. As elevated cAMP blocks permeability increase, any thrombin-induced change in permeability could not be measured under the same conditions that cAMP concentration was measured in this study. Thus, an important goal will be to determine whether cAMP can be monitored at lower concentrations in the absence of pharmacological stimulation or whether such changes can be measured in vivo, where endothelium may be in a different state perhaps having higher basal cAMP. The recent collaborative study by the two Wurzburg groups using thrombin to stimulate a drop in trans-endothelial resistance may point the way using a variety of cell culture and intact endothelial barriers (Baumer et al. 2009). A focus on signalling pathways, changes in permeability and possible spatial imaging of cAMP will enable further investigation of vascular permeability regulation.