Cholangiocytes express an isoform of soluble adenylyl cyclase that is N-linked glycosylated and secreted in extracellular vesicles.

Soluble adenylyl cyclase (sAC)-derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC 85 ) or 75 kD (sAC 75 ) sAC protein band under glucose-sufficient or glucose-deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase-F demonstrated that both sAC 75 and sAC 85 are N-linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo-H further revealed that sAC 75 and sAC 85 carry distinct sugar chains. We observed release of N-linked glycosylated sAC (sAC EV ) in extracellular vesicles under conditions that support intracellular sAC 85 (glucose-sufficient) as opposed to sAC 75 (glucose-deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sAC EV into extracellular vesicles. The intracellular turnover of sAC 85 is extremely short (t 1/2 ~30 min) and release of sAC EV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N-linked glycosylated sAC isoform that is rapidly released into extracellular vesicles.