Type three secretion system (TTSS)-competent Pseudomonas aeruginosa expressing soluble promiscuous cyclase, exoenzyme Y (ExoY), generates cyclic nucleotides in pulmonary microvascular endothelial cells (PMVECs). Within cells, cyclic nucleotide signals are highly compartmentalized, but these second messengers are also released into the extracellular space. Although agonist stimulation of endogenous adenylyl cyclase (AC) or the presence of ExoY increases cyclic nucleotides, the proportion of the signal that is in the intracellular versus extracellular compartments is unresolved. Furthermore, it is unclear whether P. aeruginosa primary infection or treatment with sterile media supernatants derived from a primary infection alters beta-adrenergic agonist-induced elevations in cAMP in PMVECs. Herein, we determine that PMVECs release cAMP into the extracellular space constitutively, following beta-adrenergic stimulation of endogenous AC, and following infection with P. aeruginosa expressing ExoY. Surprisingly, in PMVECs, only a small proportion of cGMP is detected within the cell at baseline or following P. aeruginosa ExoY infection with a larger proportion of total cGMP being detected extracellularly. Thus, the ability of lung endothelium to generate cyclic nucleotides may be underestimated by examining intracellular cyclic nucleotides alone, since a large portion is delivered into the extracellular compartment. In addition, P. aeruginosa infection or treatment with sterile media supernatants from a primary infection suppresses the beta-adrenergic cAMP response, which is further attenuated by the expression of functional ExoY. These findings reveal an overabundance of extracellular cyclic nucleotides following infection with ExoY expressing TTSS-competent P. aeruginosa.NEW & NOTEWORTHY P. aeruginosa exoenzyme Y (ExoY) infection increases cyclic nucleotides intracellularly, but an overabundance of cAMP and cGMP is also detected in the extracellular space and reveals a greater capacity of pulmonary endothelial cells to generate cAMP and cGMP. P. aeruginosa infection or treatment with sterile media supernatants derived from a primary infection suppresses the β-adrenergic-induced cAMP response in pulmonary endothelial cells, which is exacerbated by the expression of functional ExoY.
Keywords: Pseudomonas aeruginosa ExoY; beta-adrenergic response; compartmentalization; cyclic nucleotides; pulmonary endothelium.