Extracellular Vesicles: An Additional Compartment for the Second Messenger, cyclic Adenosine Monophosphate (cAMP)

Abstract

RationaleCyclic AMP is generated by adenylyl cyclase to initiate intracellular signaling leading to diverse cellular and tissue responses. Compartmentalization of the cAMP signal within the cellular environment is critical to maintain signaling specificity; however, cAMP is also released into the surrounding extracellular space. Many cell types release extracellular vesicles that encapsulate a variety of functional proteins and RNAs/miRNAs; however, little is known about whether they encapsulate signaling molecules, such as cyclic adenosine monophosphate (cAMP), which could contribute to the extracellular cAMP pool.MethodsTo test the hypothesis that endothelial cells release extracellular vesicles containing cAMP, media was collected from unstimulated aortic and pulmonary endothelial cells and subject to low‐speed and subsequently high‐speed centrifugation. To determine whether cAMP in these extracellular vesicles increased upon cell stimulation, pulmonary microvascular endothelial cells (PMVEC) were stimulated over time to increase cellular cAMP and extracellular vesicles analyzed for cAMP levels. Finally, isoproterenol and rolipram was perfused through the isolated rat lung to determine whether extracellular vesicles isolated from the intact organ also contain cAMP.ResultsWhile cAMP was detected in extracellular vesicles derived from various endothelial cell types, cAMP was higher in PMVECs compared to systemic endothelial cells (aorta) or endothelial cells from pulmonary conduit vessels, pulmonary artery endothelial cells. Further, stimulation of PMVECs with agents that increase near membrane cAMP (isoproterenol or forskolin) in the presence of the phosphodiesterase inhibitor, rolipram, led to an increase in cAMP in extracellular vesicles over time (upto 60 minutes). In PMVECs, cell lysates showed a maximum increase in cAMP 15 minutes after treatment, while the maximum increase in extracellular vesicle‐cAMP was not observed until 60 minutes after treatment. Extracellular vesicles from the perfusate of isoproterenol and rolipram stimulated isolated lungs contained elevated cAMP compared to unstimulated controls.ConclusionExtracellular vesicles released from both the systemic as well as pulmonary vasculature contain cAMP, and cAMP within extracellular vesicles can be increased upon cell stimulation. While cAMP is rapidly packaged into extracellular vesicles within 5‐minute of cell stimulation, the maximal increase is delayed. Isolated lungs can also be stimulated to release extracellular vesicles with increased cAMP. Thus, extracellular cAMP while already accepted to be free in the cytosol is also encapsulated within extracellular vesicles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.