Airway submucosal gland cell (SMGC) secretions are under the control of various neurotransmitters and hormones. Interactions between different pathways, such as those mediated by cAMP and Ca(2+), in controlling mucus or electrolyte secretions are not well understood. Prostaglandin E(2) (PGE(2)) or forskolin has been shown to enhance acetylcholine (ACh)-induced short circuit current (I(SC)) in SMGC mucous cell monolayers. We show that PGE(2), by activating cAMP-dependent protein kinase A (PKA), enhanced ACh-induced, Ca(2+)-mediated current and changes in [Ca(2+)](i) in mucous cells. PGE(2) pretreatment sensitized ACh-induced I(SC) (DeltaI(SC)) by activating endoprostanoid (EP(2)) receptors. PKA inhibitors 14-22 amide PKI (PKI) and Rp-diastereomer (Rp) of cAMPs prevented the effect of PGE(2). Removing external Ca(2+) or pretreatment with the Ca(2+) entry blocker, SKF96365 [1-[beta-(3-(4-methoxyphenyl) propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl] imidazole], shifted the concentration-response relationships for ACh to the right but did not abolish PGE(2)-induced sensitization of the ACh response. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist and Ca(2+) entry blocker, 2-aminoethoxydiphenyl borate, abolished the ACh-induced response. Charybdotoxin, but not iberiotoxin (IbTX), inhibited the ACh-induced DeltaI(SC). Clotrimazole, but not IbTX, inhibited the ACh-induced serosal K(+) current. Under whole-cell patch clamp, ACh-induced K(+) and Cl(-) currents were coincident with increases in [Ca(2+)](i) in single mucous cells. PGE(2) or forskolin pretreatment did not induce current or [Ca(2+)](i) changes but enhanced ACh-induced currents, membrane hyperpolarization, and [Ca(2+)](i) changes. Intra-cellular dialysis with the PKA-catalytic subunit enhanced ACh-induced whole-cell current as well. These findings demonstrate that PGE(2), via EP(2) receptors and the cAMP/PKA pathway, activates Ca(2+) entry-independent mechanisms, possibly by increasing IP(3)-mediated Ca(2+) release, resulting in the sensitization of ACh-induced currents.