Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase

Acute transitions in cytosolic calcium ([Ca²⁺]i) through store-operated calcium entry channels catalyze interendothelial cell gap formation that increases permeability. However, the rise in [Ca²⁺]i only disrupts barrier function in the absence of a rise in cAMP. Discovery that type 6 adenylyl cyclase (AC₆; EC 4.6.6.1) is inhibited by calcium entry through store-operated calcium entry pathways provided a plausible explanation for how inflammatory [Ca²⁺]i mediators may decrease cAMP necessary for endothelial cell gap formation. [Ca²⁺]i mediators only modestly decrease global cAMP concentrations and thus, to date, the physiological role of AC₆ is unresolved. Present studies used an adenoviral construct that expresses the calcium-stimulated AC₈ to convert normal calcium inhibition into stimulation of cAMP, within physiologically relevant concentration ranges. Thrombin stimulated a dose-dependent [Ca²⁺]i rise in both pulmonary artery (PAECs) and microvascular (PMVEC) endothelial cells, and promoted intercellular gap formation in both cell types. In PAECs, gap formation was progressive over 2 h, whereas in PMVECs, gap formation was rapid (within 10 min) and gaps resealed within 2 h. Expression of AC₈ resulted in a modest calcium stimulation of cAMP, which virtually abolished thrombin-induced gap formation in PMVECs. Findings provide the first direct evidence that calcium inhibition of AC₆ is essential for endothelial gap formation.