Activation of a chimeric soluble adenylyl cyclase induces endothelial cell gap formation without disrupting the cortical actin rim

Abstract

Endothelial cells express the type 6 adenylyl cyclase (AC), which generates a membrane cAMP pool that stabilizes cortical actin and strengthens the endothelial cell barrier. However, certain bacterial toxins, such as the Pseudomonas aeruginosa toxin ExoY, are soluble ACs, that when introduced into endothelial cells produce a cytosolic cAMP pool that disrupts the endothelial cell barrier. Since cortical actin is an important cAMP effector, we sought to determine whether activation of a soluble AC disrupts this rim as a prerequisite to gap formation. To test this idea, retrovirus was prepared to express a green fluorescent protein fused to β-actin in pulmonary microvascular endothelial cells (PMVECs). These cells were then infected with an adenovirus expressing a mammalian soluble AC construct (sAC I/II). sACI/II is catalytically inactive, unless it is stimulated by forskolin. Live cell confocal imaging was performed in the presence or absence of forskolin. Forskolin activates both endogenous transmembrane ACs and the sACI/II in live cells. Forskolin did not disrupt the PMVEC barrier or alter membrane actin in control cells. In contrast, forskolin induced gap formation in sACI/II expressing cells. However, forskolin-induced gap formation was not accompanied by reorganization of the cortical actin rim. Thus, sACI/II generates a cytosolic cAMP pool that does not directly influence membrane cortical actin. Supported by HL-60024 & HL-66299.