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During anesthesia, muscarinic parasympathetic antagonists can be used as a preoperative medication on order to reduce salivary flow, tracheobronchial secretions, and pharyngeal secretions, as well as to decrease the acidity of gastric secretions. We did an in vivo study to examine whether muscarinic receptor subtypes play a role in the exocrine function of the rat submandibular gland. The effects of muscarinic antagonists (pirenzepine, methoctramine, 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) and glycopyrrolate) were examined on secretion from the submandibular gland evoked by acetylcholine in pentobarbital-anesthetized rats. Glycopyrrolate caused less elevation in heart rate than the other anticholinergics. 4-DAMP and glycopyrrolate markedly inhibited the acetylcholine-evoked fluid responses. Pirenzepine showed significantly lower inhibitory potency than 4-DAMP or glycopyrrolate, while methoctramine had even less of an inhibitory effect. Pirenzepine, 4-DAMP and glycopyrrolate significantly inhibited both protein concentration and amylase activity in the acetylcholine-evoked submandibular saliva, while methoctramine did not affect the responses. The reduction of the protein concentration and amylase activity in submandibular saliva caused by pirenzepine, 4-DAMP and glycopyrrolate were inhibited by N^G-nitro-L-arginine methyl ester (L-NAME). Thus, it might be effective to administer anti-cholinergic drugs together with L-NAME as premedication. These results suggest that glycopyrrolate significantly decreases salivary secretion, and has significantly fewer side effects than other muscarinic parasympathetic antagonists. Further controlled studies are required to determine the safety, efficacy and patient tolerance of glycopyrrolate.