Abstract

Abstract 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 (9 S ,10 R ,12 R )-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 pyrrolo3,4- i 1,6benzodiazocine-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)-propylphenyl 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.