Department of Physiology and Cell Biology, University South Alabama, Mobile, Alabama, United States.
Center for Lung Biology, University South Alabama, Mobile, Alabama, United States.
Am J Physiol Lung Cell Mol Physiol. 2024 Nov 1;327(5):L756-L768. doi: 10.1152/ajplung.00038.2024. Epub 2024 Sep 24.
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 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 expressing ExoY. Surprisingly, in PMVECs, only a small proportion of cGMP is detected within the cell at baseline or following 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, 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 . 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. 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.
III 型分泌系统（TTSS）功能完备的铜绿假单胞菌表达可溶性杂环酶、外切酶 Y（ExoY），可在肺微血管内皮细胞（PMVECs）中生成环核苷酸。在细胞内，环核苷酸信号高度分隔，但这些第二信使也会释放到细胞外空间。尽管内源性腺苷酸环化酶（AC）的激动剂刺激或 ExoY 的存在会增加环核苷酸，但细胞内与细胞外隔间的信号比例仍未解决。此外，尚不清楚原发性感染或用源自原发性感染的无菌培养基上清液处理是否会改变 PMVECs 中β-肾上腺素能激动剂诱导的 cAMP 升高。在此，我们确定 PMVECs 在β-肾上腺素能刺激内源性 AC 后以及在表达 ExoY 的感染后，会将 cAMP 持续释放到细胞外空间。令人惊讶的是，在 PMVECs 中，在基线或在用 感染后，只有一小部分 cGMP 被检测到细胞内，而大部分总 cGMP 被检测到细胞外。因此，仅通过检查细胞内环核苷酸，可能会低估肺内皮细胞生成环核苷酸的能力，因为大部分环核苷酸被递送到细胞外隔室。此外，感染或用原发性感染的无菌培养基上清液处理会抑制β-肾上腺素能 cAMP 反应，而功能性 ExoY 的表达会进一步减弱该反应。这些发现揭示了表达 TTSS 功能完备的 ExoY 感染后，细胞外环核苷酸过多。ExoY 感染会增加细胞内环核苷酸，但也会在细胞外空间中检测到过多的 cAMP 和 cGMP，并揭示了肺内皮细胞生成 cAMP 和 cGMP 的更大能力。感染或用源自原发性感染的无菌培养基上清液处理会抑制肺内皮细胞中β-肾上腺素能诱导的 cAMP 反应，而功能性 ExoY 的表达会加剧这种反应。
