The present study investigated the role of nitric oxide (NO)/cGMP signal transduction in the M(3) muscarinic acetylcholine receptor (mAChR)-stimulated increase in aquaporin-5 (AQP5) levels in the apical plasma membrane (APM) of rat parotid glands. Pretreatment of rat parotid tissue with the NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium inhibited both acetylcholine (ACh)- and pilocarpine-induced increases in AQP5 in the APM. NO donors [3-morpholinosydnonimine (SIN-1) and (S)-nitroso-N-acetylpenicillamine (SNAP)] mimicked the effects of mAChR agonists. A selective protein kinase G inhibitor [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg-3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823)] and an NO synthase inhibitor (N(6)-imminoethyl-L-lysine) blocked SIN-1- and SNAP-induced increases in AQP5 in the APM. A calmodulin kinase II inhibitor [(8)-5-isoquinolinesulfonic acid, 4-[2-(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (KN-62)] decreased the pilocarpine-induced increase of AQP5 in the APM. Using diaminofluorescinein-2 diacetate, enhanced NO synthase activity was detected in isolated parotid acinar cells after ACh-treatment. Treatment with dibutyryl cGMP, but not dibutyryl cAMP, induced an increase in AQP5 levels in the APM. BAPTA-AM inhibited the cGMP-induced increase in AQP5 in the APM. Pretreatment of the tissues with a myosin light chain kinase inhibitor [(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9)] inhibited a mAChR-stimulated increase in AQP5 levels in the APM. Although there was a significant ACh-induced increase in AQP5 in the APM in the absence of extracellular Ca(2+), the maximal effect of ACh on the AQP5 levels in the APM occurred in the presence of extracellular Ca(2+). These results suggest that NO/cGMP signal transduction has a crucial role in Ca(2+) homeostasis in the mAChR-stimulated increase in AQP5 levels in the APM of rat parotid glands.