Methods: Male Wistar rats were anaesthetized and LPS (Escherichia coli serotype O111:B4) was injected (50 µl, 0.5 mg/ml) in the right SMG duct from an intra-oral approach. The control left SMG duct received the same volume of sterile saline or was left untreated. Twenty four hours later, under anaesthesia, both SMG ducts were cannulated and salivary secretion was stimulated by an infusion of methacholine into the femoral vein before and after infusion of nitric oxide synthase (NOS) inhibitors. Salivas and salivary glands were collected, weighed and analysed. SDS-PAGE, Western-Blotting and immunohistochemistry were performed. In vitro experiments were also conducted using acini clusters from collagenase-digested SMG and previously infused with LPS. Acini were loaded with a calcium-sensitive dye, stimulated with methacholine and fluorescence levels were assessed by confocal microscopy. All experiments were conducted under Home Office licence.
Results: Methacholine induced a greater secretion from LPS-treated glands compared to controls, but the secretion was much reduced following infusion (i.v.) of the inducible NOS inhibitors L-NIL or aminoguanidine (4±1.32 µg/min/kg), compared to contralateral control glands (23±4.25 μg/min/kg; P<0.05) or LPS-treated glands before the inhibitors (41±3.8 μg/min/kg; P<0.001). Immunohistochemistry and Western-Blotting showed an increased expression of iNOS in LPS-treated glands compared with contralateral control glands. In vitro analysis revealed that intracellular calcium responses to methacholine stimulation were higher in cells from LPS-infused glands (P<0.05).
Conclusions: Methacholine-evoked hypersecretion, after 24h of LPS-induced sialadenitis seems to be due to an iNOS-mediated action on acinar cells.
This project was generously funded by the Wellcome Trust.