Extracellular vesicles: another compartment for the second messenger, cyclic adenosine monophosphate

Abstract

Rationale Cyclic 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. Methods To 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. Results While 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. Conclusion Extracellular 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.

Purine Metabolites in Tumor-Derived Exosomes May Facilitate Immune Escape of Head and Neck Squamous Cell Carcinoma

“…The functional relevance of such encapsulation of purines into exosomes has been previously discussed. For example, it has been suggested that exosomes might provide an additional compartment for cAMP signaling [ 14 ]. Additionally, adenosine-rich exosomes were previously shown to promote adenosine A 2B R-mediated angiogenesis [ 11 ].…”

“…While the presence of ectonucleotidases on the exosome surface has been well documented, little is known about the presence of other components of the purinergic signaling pathway in the exosome cargo. Sayner et al [ 14 ] reported that exosomes derived from pulmonary microvascular endothelial cells as well as exosomes collected from the perfusate of isolated rat lungs contained cAMP that mediated cellular signaling. Our recently published data indicated that TEX produced by an HNSCC cell line contained adenosine and inosine and induced adenosine A 2B R-mediated responses in recipient endothelial cells [ 11 ].…”

A novel secreted-cAMP pathway inhibits pulmonary hypertension via a feed-forward mechanism

“…After β-adrenergic stimulation, cAMP is secreted from cardiomyocytes, and via Is cAMP extruded to the extracellular space only through cyclic nucleotide transporters? In a recent study, cAMP has been found to be encapsulated within extracellular vesicles from pulmonary microvascular endothelial cells 30 . Extracellular vesicles from pulmonary microvascular endothelial cells and from isolated lung perfusate display an increase in their cAMP content following stimulation of intracellular cAMP formation 30 .…”

“…In a recent study, cAMP has been found to be encapsulated within extracellular vesicles from pulmonary microvascular endothelial cells 30 . Extracellular vesicles from pulmonary microvascular endothelial cells and from isolated lung perfusate display an increase in their cAMP content following stimulation of intracellular cAMP formation 30 . Therefore, mechanisms other than cAMP transport might contribute to the extracellular cAMP content.…”

Role of exosome-associated adenosine in promoting angiogenesis

“…While it is well recognized that exosomes encapsulate functional proteins and nucleic acids, it is currently unclear whether purine metabolites are encapsulated within exosomes. Sayner et al [20] recently reported that EVs encapsulate cAMP to provide an additional second messenger compartment. Our preliminary data show that exosomes not only encapsulate cAMP but also adenosine and adenosine metabolites (inosine, hypoxanthine, and xanthine).…”