Soluble adenylate cyclase reveals the significance of compartmentalized cAMP on endothelial cell barrier function

Abstract

Within pulmonary microvascular endothelial cells, activation of endogenous adenylate cyclase generates tightly regulated cAMP transitions in the subplasma membrane space. These cAMP fluxes strengthen contacts between adjacent cells to tighten their barrier function. However, pathogenic bacteria have devised a mechanism to transfer toxic adenylate cyclases into eukaryotic cells and generate a cytosolic pool of cAMP that disrupts the barrier. To determine whether membrane or cytosolic cAMP synthesis dominates in control of endothelial cell barrier function, we expressed a soluble mammalian adenylate cyclase chimaera. This chimaera is not constitutively active, but is activated by forskolin. Thus forskolin application increases cAMP in both the plasma membrane and cytosolic compartments. Forskolin induced inter-endothelial cell gaps in cells expressing the soluble adenylate cyclase, demonstrating that the cytosolic cAMP pool dominates over the plasma membrane cAMP pool in control of endothelial cell barrier strength. Indeed, when the soluble chimaera is relocalized to the plasma membrane, the forskolin-stimulated adenylate cyclase activity does not induce gaps. These results therefore support the growing paradigm that membrane and cytosolic cAMP pools target discrete effectors to control different physiological processes.