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.

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.

RationaleCyclic AMP is generated by adenylyl cyclase (AC) to initiate intracellular signaling leading to diverse cellular and tissue responses. Compartmentalization of cAMP within the cellular environment is critical to maintain signaling specificity such that cAMP generated by transmembrane AC is pulmonary endothelial barrier protective, yet cAMP generated by soluble AC is endothelial barrier disruptive; however, cAMP is also released into the surrounding extracellular space. Currently it is not clear whether cAMP generated from the subplasma membrane versus cytosolic compartments of pulmonary endothelial cells is effluxed, what proportion of total cAMP is released into the extracellular space, and the expression of MPRs potentially responsible for this efflux.MethodsRat pulmonary microvascular endothelial cells (PMVECs) were stimulated with agonists (i.e. isoproterenol, 1μM, or forskolin, 100 μM) to increase cAMP in the subplasma membrane compartment in the presence and absence of the phosphodiesterase 4 inhibitor, rolipram (10μM). The cell lysate and media were collected for cAMP analysis by EIA. Alternatively, PMVECs were treated with the bacteria P. aeruginosa expressing the soluble AC exotoxin, ExoY, as part of the type three secretion system (T3SS) at a multiplicity of infection 20:1 to increase cAMP in the cytosolic compartment. P. aeruginosa expressing a mutant ExoY protein, ExoYK81M, was used as bacteria control. Following 6‐hours inoculation, cell lysate and media were collected and cAMP analysis performed by EIA. To determine whether inhibition of MRP4 attenuated cAMP efflux from the subplasma membrane versus cytosolic compartments, cells were pretreated for 5‐minutes with the MRP4 specific inhibitor, MK571 (20μM), prior to stimulation and cell lysate and media collected for cAMP‐EIA analysis. For each experimental condition, cell lysate versus media was normalized for each dish and presented as the proportion of total cAMP detected in the cell versus media. Western blot analysis and RT‐PCR was performed on lung tissue and pulmonary endothelial cells to determine the expression of MRPs.ResultsFollowing 1‐hour stimulation of transmembrane AC using either isoproterenol or forskolin in the presence of rolipram, cAMP increased in PMVECs in both the cell lysate and media. Similarly, 6‐hour inoculation of PMVECs with the T3SS competent P. aeruginosa expressing ExoY, led to an increase in cAMP in both lysate and media samples. This increase in cAMP was absent in P. aeruginosa expressing the mutant form of ExoY, ExoYK81M. Further, MRP4 inhibition with MK571 attenuated cAMP detected in media samples following activation of both transmembrane AC as well as cytosolic ExoY. RT‐PCR confirmed expression of MRP4 in PMVECs and western blot analysis confirmed expression in tissue from rat lung.ConclusionRegardless of whether cAMP was generated within the subplasma membrane or the cytosolic compartment, cAMP efflux into the extracellular space is attenuated by inhibition of MRP4.Support or Funding InformationNIH R01 HL121513This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RationaleThe second messenger cAMP is widely known to regulate proliferation in numerous cell types. Many of these studies implicate cAMP generated by membrane‐bound cyclases (AC1‐9). While these membrane‐bound ACs generate cAMP in the near membrane compartment, cAMP can also be generated in unique subcellular compartments by the soluble adenylyl cyclase (sAC or AC10). Soluble AC localizes to the cytosol as well as organelles, such as the mitochondria, and is uniquely stimulated by bicarbonate to generate cAMP. Recently, AC10 was observed to be overexpressed in prostate carcinoma, suggesting a role for AC10 in cellular proliferation; however, the independent role of bicarbonate in proliferation has not been investigated. Thus, we tested the hypothesis that bicarbonate is required for proliferation of pulmonary endothelial cells.MethodsPulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs) were seeded at 100, 000 cells per well in media with and without bicarbonate. The cells were counted for seven consecutive days to generate growth curves and blood gas analysis performed to determine pH, pO2, pCO2, and HCO3− concentrations of the media. Additionally, a separate set of PMVECs were seeded at 100, 000 cells per well in bicarbonate free media and bicarbonate added back to the media after 24, 48, 72, 96, 120, 144, and 168 hours and counted for five days following bicarbonate add back.ResultsGrowth curves revealed that PMVECs and PAECs seeded in the bicarbonate‐containing media entered into log phase growth followed by a plateau phase at cell confluence; however, PMVECs and PAECs seeded in the bicarbonate free media did not enter log phase growth nor reach confluence. When bicarbonate was added back to PMVECs initially grown in a bicarbonate free environment, the cells proliferated to numbers comparable to PMVECs seeded in bicarbonate containing media throughout the growth curve; however, if PMVECs were incubated in bicarbonate free media for more than 96 hours, these cells did not enter into log phase growth and did not form a confluent monolayer.ConclusionThese data reveal a requirement for bicarbonate for proliferation of pulmonary endothelial cells, since neither PAECs nor PMVECs formed confluent monolayers in the absence of bicarbonate.Support or Funding InformationNIH 121513This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.