Inducible nitric oxide synthase increases secretion from inflamed salivary glands.

OBJECTIVE: Salivary gland secretion is dependent on cholinergic stimulation via autonomic nerves and calcium signalling in acinar cells. Secretory dysfunction associated with SS may be partly caused by the damaging effects of increased glandular concentrations of nitric oxide (NO) derived from up-regulation of inducible NO synthase (iNOS) that accompanies glandular inflammation. The present study examines the effects of increased iNOS expression on salivary gland secretory function. METHODS: The inflammogen lipopolysaccharide (LPS) was introduced intraductally into rat submandibular glands, and glandular responsiveness to cholinergic stimulation was determined. RESULTS: LPS provoked a rapid, long-lasting inflammation, increasing gland weight (by almost 20%) and inflammatory cell infiltration at 3 and 24 h. Immunoblotting of glandular homogenates indicated that iNOS expression was increased approximately 4-fold, and immunohistochemistry of frozen tissue sections showed increased iNOS expression in acinar cells. Salivary secretion from inflamed glands was significantly increased in response to low doses of methacholine and accompanied by increased acinar cell calcium signalling in vitro. Prior administration of the iNOS inhibitors, aminoguanidine or L-NIL [L-N6-(1-iminoethyl)-lysine dihydrochloride] abolished increased secretion and acinar cell calcium signalling. CONCLUSIONS: Up-regulation of glandular iNOS expression can increase cholinergically evoked salivary secretion and appears to offset any secretory hypofunction linked with glandular inflammation. It seems unlikely that increased glandular levels of NO are responsible for the secretory hypofunction that accompanies SS.

Selection of housekeeping genes for gene expression studies in the adult rat submandibular gland under normal, inflamed, atrophic and regenerative states.

BACKGROUND: Real-time PCR is a reliable tool with which to measure mRNA transcripts, and provides valuable information on gene expression profiles. Endogenous controls such as housekeeping genes are used to normalise mRNA levels between samples for sensitive comparisons of mRNA transcription. Selection of the most stable control gene(s) is therefore critical for the reliable interpretation of gene expression data. For the purpose of this study, 7 commonly used housekeeping genes were investigated in salivary submandibular glands under normal, inflamed, atrophic and regenerative states. RESULTS: The program NormFinder identified the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative states, and GAPDH in the atrophic state. For normalisation to multiple housekeeping genes, for each individual state, the optimal number of housekeeping genes as given by geNorm was: ACTB/UBC in the normal, ACTB/YWHAZ in the inflamed, ACTB/HPRT in the atrophic and ACTB/GAPDH in the regenerative state. The most stable housekeeping gene identified between states (compared to normal) was UBC. However, ACTB, identified as one of the most stably expressed genes within states, was found to be one of the most variable between states. Furthermore we demonstrated that normalising between states to ACTB, rather than UBC, introduced an approximately 3 fold magnitude of error. CONCLUSION: Using NormFinder, our studies demonstrated the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative groups and GAPDH in the atrophic group. However, if normalising to multiple housekeeping genes, we recommend normalising to those identified by geNorm. For normalisation across the physiological states, we recommend the use of UBC.

Early markers of regeneration following ductal ligation in rat submandibular gland.

Rat submandibular glands can recover their function and secretory protein content following ductal ligation-induced atrophy. Morphological studies have established that following ligation, deligation of the gland allows the regeneration of new salivary gland tissue. However, little is known about changes happening during early regeneration following intra-oral duct ligation, which does not damage the parasympathetic nerves. Glands that had been 2 weeks ligated or 2 weeks ligated + 3 days deligated were compared. Tissue was prepared for histological, immunohistochemical (SMG-B and Ki-67) and immunocytochemical analyses (smooth muscle actin, aquaporin 5). Haematoxylin and eosin staining of deligated glands showed that some acini regained their cytoplasmic volume; moreover, the loss of Alcian blue/periodic acid-Schiff's staining from the lumen of ducts suggested successful deligation. The deligated gland was characterized by atypical acinar-ductal branched structures, which were less frequent in the ligated gland and rarely seen in normal unoperated tissue. Myoepithelial cells were also investigated since changes in their morphology reflected changes in the acini morphology not readily detected by conventional staining. Actin staining revealed the presence of some shrunken acini in the atrophic tissue, whereas they had regained their normal morphology in the deligated gland suggesting that the acini were recovering. Some acini during deligation regained aquaporin 5 expression, which had decreased during atrophy. SMG-B protein, located in the pro-acinar cell during gland development and usually found in the intercalated duct cells in the adult, was detected in the newly formed acini of the deligated gland. This study suggests that morphological markers of regeneration appear as early as 3 days following ligation removal.

Innervation and secretory function of transplanted human submandibular salivary glands.

Free submandibular gland autotransplantation is used to treat absolute tear deficiency. Although disconnected from any peripheral innervation, most transplants show increasing secretion for years. We have evaluated the secretory activity and autonomic innervation of such transplants. Secretory activity of glands in response to parasympatholytics and parasympathomimetics was evaluated by Schirmer's test and Technetium scintigraphy. Submandibular gland tissue specimens taken before and after transplantation were examined histologically. Relative hypersecretion during the first postoperative week suddenly decreased but then slowly increased during the first postoperative year. Hypersecretion was significantly reduced by parasympatholytics while carbachol rapidly increased secretion. Histology of transplanted glandular tissue showed parenchymal atrophy. Cholinesterase-positive nerves were abundant and in a similar distribution to normal with scattered positive ganglion cells. Adrenergic axons were fewer than normal and irregularly distributed. Early hypersecretion may be due to release of neurotransmitters from degenerating terminal axons. This is followed by a period of minimal secretion during which hypersensitivity of acinar cells develops. With spontaneous reinnervation, secretion is accentuated by external sympathetic vasomotor adrenergic drive. This shows that submandibular glands can remain viable despite complete separation from their normal nerve supply and are capable of regaining a substantial secretory activity for years.

Intraoral duct ligation without inclusion of the parasympathetic nerve supply induces rat submandibular gland atrophy.

The atrophic effect of ligating the main duct of the right submandibular gland was examined in rat using a novel intraoral approach that did not include the chorda lingual (CL) nerve. Comparison was made with the effect of duct ligation including the attached CL nerve as carried out in previous studies. In all animals, the contralateral, unligated left submandibular gland was used as a control. At different times (1, 2, 7, 14 and 21 days) after ligation, glands were removed and weighed. Tissue was fixed for morphological analysis and homogenized for biochemical assay of secretory proteins. After 21 days, ligated glands showed a significant decrease in wet weight compared with unligated glands. Weight loss was the greatest (P < 0.05) in glands ligated with the CL nerve included. Light microscopy revealed that following ligation, an initial inflammatory reaction was followed by severe atrophy of acini and granular ducts. The atrophy was less severe when the CL nerve was not ligated. Secretory proteins were decreased from day 1 onwards following duct ligation in both groups. It can be concluded that most of the atrophy induced by duct ligation is independent of damage caused to the parasympathetic nerve supply, although the latter causes a greater atrophy presumably due to denervation.

Recovery of rat submandibular salivary gland function following removal of obstruction: a sialometrical and sialochemical study.

Functional recovery of the rat submandibular gland following ligation of the main excretory duct was examined. Rat submandibular glands were ligated for 1, 4 and 8 weeks using a micro-clip with a plastic tube. Micro-clips were removed and glands were allowed to recover for periods of 8, 16 and 24 weeks. Submandibular glands were stimulated with autonomimetic drugs (methacholine and isoprenaline) and salivas were collected from atrophic or de-ligated and contralateral control glands. Glands recovered almost full size (92% of control gland) following 24 weeks of de-ligation. Saliva volume secreted by ligated/de-ligated (RSM) and control (LSM) glands were similar with different doses of agonists. Protein output expressed per gram of tissue wet weight was similar from both ligated/de-ligated and control glands with all doses of agonist. Sodium and chloride levels were higher from de-ligated glands than contralateral control glands. Protein electrophoresis showed similar profiles of salivary proteins in all samples with some minor differences. Acinar cells in de-ligated glands showed a normal morphology, as indicated by light microscopy, whilst granular ductal cells were fewer and contained fewer secretory granules. Sodium potassium ATPase staining of striated ducts in de-ligated glands was similar to that of control glands. It can be concluded that rat submandibular glands can regenerate following severe atrophy and secrete normal amounts of saliva containing broadly a full profile of secretory proteins. In contrast to acinar cells, ductal cells appear not to recover full function.