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.