Preganglionic parasympathectomy decreases salivary SIgA secretion rates from the rat submandibular gland.

Immunoglobulin A (IgA) is transported into saliva by salivary cells expressing the polymeric immunoglobulin receptor (pIgR). In rat salivary glands, autonomic nerves stimulate this process. To examine how nerves affect pIgR-mediated IgA secretion, the chorda-lingual nerve was sectioned. One week after preganglionic parasympathectomy, both the stimulated and unstimulated rates of salivary IgA secretion were reduced, despite similar glandular amounts of IgA. Biochemical analysis of cells from parasympathectomised and control glands indicated reduced membrane expression of pIgR. It appears the removal of long-term parasympathetic input has affected the routing of pIgR within salivary cells and reduced the SIgA transport into saliva.

Salivary secretion of immunoglobulin A by submandibular glands in response to autonomimetic infusions in anaesthetised rats.

Salivary secretion of immunoglobulin A (lgA) by submandibular glands is increased by stimuli from autonomic nerves. Since it is unclear which specific autonomic receptors transduce such stimuli, we have infused autonomimetics intravenously and compared secretion of fluid, IgA and stored proteins (peroxidase and total protein) with secretory responses during electrical stimulation of the parasympathetic nerve supply in anaesthetized rats. The greatest secretion of IgA was evoked by the alpha-adrenoceptor agonist phenylephrine and this was reduced by the beta-adrenoceptor blocking drug propranolol. The secretion of fluid or proteins but not IgA was increased with frequency of nerve stimulation and dose of methacholine (cholinergic), isoprenaline (beta-adrenergic) or phenylephrine (alpha-adrenergic).

Constitutive secretion of immunoglobulin A and other proteins into lumina of unstimulated submandibular glands in anaesthetised rats.

Salivary fluid secretion is dependent upon reflex stimuli mediated by autonomic nerves. In order to determine if immunoglobulin A (IgA) and salivary proteins are secreted in the absence of nerve stimulation, small volumes (< 2 microl) of saliva were consecutively collected from the submandibular duct of anaesthetised rats following rest pauses in order to sample the protein contents of the ductal system. Within the first 5 microl of such saliva collected by parasympathetic nerve stimulation, IgA and other salivary proteins reached peak concentrations that were over 20-fold greater than levels in parasympathetically stimulated saliva subsequently collected during a 5 min period of stimulation. Confocal microscopy of TRITC-labelled IgA added to live, acutely isolated submandibular acini indicated that it did not enter the lumina by paracellular leakage. IgG is thought to enter saliva by paracellular leakage but did not accumulate in luminal saliva in the present study. Electrophoresis suggested that the major proteins secreted in the absence of stimulation were the same as those present in subsequently stimulated saliva. It can be concluded that IgA and other major submandibular proteins are secreted into glandular lumina in the absence of nerve stimulation. The functional significance of such unstimulated protein secretion is at present unclear.

Ultrastructural histochemical studies of secretory granule replenishment in rat submandibular granular tubules after cyclocytidine-induced secretion.

Rat submandibular glands have been examined electron microscopically at various times after degranulating the granular tubules by injecting cyclocytidine (75 mg/kg i.p.), to study events in the reformation of secretory granules in these cells. The changes were progressive but not synchronous in the cells. The first evidence of recovery was the re-appearance of glycogen particles 6 h after injection. Residual secretory granules were small and located periluminally at that time. More granules were present at 15 h after injection but they were still small and placed periluminally. There was more glycogen in the cells and some was present in aggregates. At 1 day after injection there were more secretory granules and they tended to be larger than previously. The secretory granules increased in size and number progressively thereafter and the cells appeared like normal controls by day 7. During the recovery, fusion profiles were seen between granules from 2 days onwards. Throughout, few Golgi complexes were detected and this may be related with the low glycosylation of the secretory proteins in these cells. The results confirm that the reformation of the secretory granules in granular tubule cells is a slow process that involves fusions of smaller granules.

Variation in the response to ductal obstruction of feline submandibular and sublingual salivary glands and the importance of the innervation.

A variable response following ductal ligation of feline salivary glands corresponds to the human condition but contrasts with a predictable atrophy in obstructed salivary glands of rodents popularly used as a model for human salivary problems. The present investigation is concerned with a possible reason for the variable response, namely the preservation of the innervation. Ducts of feline submandibular and sublingual salivary glands were ligated with or without the inclusion of the chorda tympani. Inclusion led to a delayed initial response followed by progressive atrophy until the parenchyma was extremely atrophic, whereas avoidance of the chorda led to the variable response in which variable numbers of acini of a similar form to normal persisted. The results establish the atrophic effect of inclusion of the chorda tympani in ductal ligation and indicate the caution that should be exercised in the extrapolation of the rodent model to the human condition.

Nerve-evoked secretion of immunoglobulin A in relation to other proteins by parotid glands in anaesthetized rat.

Secretion of fluid and proteins by salivary cells is under the control of parasympathetic and sympathetic autonomic nerves. In a recent study we have shown that, in the rat submandibular gland, autonomic nerves can also increase the secretion of IgA, a product of plasma cells secreted into saliva as SIgA (IgA bound to Secretory Component, the cleaved poly-immunoglobulin receptor). The present study aimed to determine if parotid secretion of SIgA is increased by autonomic nerves and to compare SIgA secretion with other parotid proteins stored and secreted by acinar and ductal cells. Assay of IgA in saliva evoked by parasympathetic nerve stimulation immediately following an extended rest period under anaesthesia indicated that it had been secreted into intraductal saliva in the absence of stimulation during the rest period. The mean rate of unstimulated IgA secretion (2.77+/-0.28 microg min(-1) g(-1)) and the 2.5-fold increase in IgA secretion evoked by parasympathetic stimulation were similar to results found previously in the rat submandibular gland. Sympathetic nerve stimulation increased SIgA secretion 2.7-fold, much less than in the submandibular gland. SDS-PAGE and Western blot analysis with anti-IgA and anti-Secretory Component antibodies confirmed that SIgA was the predominant form of IgA in saliva. Acinar-derived amylase and ductal-derived tissue kallikrein were more profoundly increased by parasympathetic and particularly sympathetic stimulation than SIgA. Overall, the results of the present study indicate that SIgA forms a prominent component of unstimulated parotid salivary protein secretion and that its secretion is similarly increased by stimulation of either autonomic nerve supply. The secretion of other parotid salivary proteins that are synthesized and stored by acinar or ductal cells is upregulated to a much greater extent by parasympathetic and particularly sympathetic stimulation.

The effects of ductal obstruction on the acinar cells of the parotid of cat.

Twenty-nine parotids ligated for between 1 and 365 days were examined by light and electron microscopy. Major changes in the acini were seen at 4 days and included vacuolation, disintegration, extravasation, apoptosis, phagy and a reduction in number and size of secretory granules. There was a further reduction in secretory granules from 7 to 12 days, but acinar cells persisted even up to 365 days, some contained a luminal concentration of small secretory granules and occasionally acinar cells of a similar appearance to normal were found. These findings contrast with a reported absence of acinar cells from the obstructed parotid of rat and show that parotid acinar cells are able to persist and retain an appearance indicative of secretory activity.

Sympathetic decentralization abolishes increased secretion of immunoglobulin A evoked by parasympathetic stimulation of rat submandibular glands.

Salivary secretion of immunoglobulin A (IgA) in response to electrical stimulation of the parasympathetic nerve supply was assessed bilaterally in the submandibular glands of anaesthetized rats 1 week following unilateral pre-ganglionic sympathectomy (decentralization). Nerve-mediated stimulation on the non-denervated side increased IgA secretion several fold above an unstimulated rate of secretion whereas sympathetic decentralization reduced the parasympathetically stimulated secretion of IgA without affecting the basal rate. Glandular levels of IgA were increased following decentralization compared to the control glands. Salivary levels of free secretory component (FSC), the cleaved polymeric immunoglobulin receptor (plgR), were increased by parasympathetic stimulation and reduced by sympathectomy, though not as much as IgA. The decreased secretion of FSC suggests a reduced production of plgR and may account in part, for reduced IgA secretion following long-term removal of sympathetic nerve impulses.

Reflex secretion of proteins into submandibular saliva in conscious rats, before and after preganglionic sympathectomy.

1. An indwelling catheter was placed in the left submandibular duct of rats, under pentobarbitone anaesthesia, and connected to an outflow cannula that emerged above the skull. 2. Saliva was collected from the outflow cannula in conscious rats, the same day after recovery from anaesthesia, under four different reflex conditions: grooming, heat exposure, rejection of a bitter tasting substance and feeding on softened chow, repeated in different orders. 3. Saliva flow was greatest for grooming and least for rejection. Protein concentrations were least with heat but much greater and similar for the other stimulations. Acinar peroxidase activity was high for feeding, intermediate for grooming and rejection, and again lowest with heat. Tubular tissue kallikrein activities were moderately low, being greatest with feeding and least with grooming. Secretory immunoglobulin A (SIgA) concentration was least with heat and similar for the other stimulations. 4. The next day, under pentobarbitone anaesthesia, the left preganglionic sympathetic trunk was sectioned (sympathetic decentralization) and, after recovery, the preceding stimulations were repeated. Flow of saliva showed little change, but protein and peroxidase concentrations and outputs decreased dramatically with grooming, rejection and feeding to levels similar to those with heat, which showed little change. Tissue kallikrein was lowered less dramatically, but the reductions in output were significant except with heat. Patterns of proteins resolved by electrophoresis changed for grooming, rejection and feeding and became similar to saliva from heat, which showed little change. No significant effects on SIgA concentrations occurred. 5. Gland weights from the sympathetically decentralized side were greater than from the intact side at the end of the experiments and histologically showed retention of acinar mucin. 6. Thus reflex sympathetic drive varied with the different stimulations; it was least during heat, but it had pronounced effects on acinar secretion of proteins during the other stimulations. At the same time this sympathetic drive had less impact on tissue kallikrein secretion from tubules and had little influence on flow or the concentration of SIgA secreted.

Immunoglobulin A secretion into saliva during dual sympathetic and parasympathetic nerve stimulation of rat submandibular glands.

Salivary secretion of proteins from rat submandibular glands was studied using graded stimulation of the parasympathetic nerve in isolation, and then at a fixed rate in combination with graded sympathetic nerve stimulation. Increasing the frequency of parasympathetic nerve stimulation per se caused a gradual increase in the secretion of peroxidase (from acini) but only small increases in proteinase (from ductal cells) and IgA outputs. Dual stimulations, with an increasing frequency of sympathetic nerve stimulation on a background of low frequency parasympathetic nerve stimulation, showed that maximal acinar secretion of peroxidase required only a low frequency of additional sympathetic stimulation, whereas ductal secretion of kallikrein was greatest with the highest frequency of additional sympathetic stimulation (20 Hz in bursts). IgA secretion also required high frequency additional sympathetic stimulation in bursts for greatest output. Although a synergism occurred with parasympathetic plus sympathetic nerve stimulation for the secretion of both peroxidase and kallikrein it was not evident for the secretion of IgA. This presumably reflects a difference for exocytosis of proteins stored in granules (e.g. peroxidase and kallikrein) compared to those proteins continuously transported across the plasma membrane in vesicles by transcytosis. This work confirms that vesicular movement of secretory IgA can be increased by both parasympathetic and sympathetic nerve stimulation, but the frequency parameters differ for each nerve.

Ultrastructural histochemical studies of secretory processes in rat submandibular granular tubules during intermittent sympathetic nerve stimulation.

Secretory changes in the cells of granular tubules in rat submandibular glands have been studied sequentially during electrical stimulation of their sympathetic nerves. Results were assessed in a series of biopsied lobes from the same gland, taken at different times during the sympathetic stimulation. Changes were not synchronous between adjacent cells and it appeared that the time for the onset of secretory events differed between cells but, once set in action, a chain of similar events occurred. Nevertheless, some cells appeared to remain refractory throughout. Initially, some alignment of granules to the adjacent plasma membrane occurred and occasional evidence for classical exocytosis was seen. However, from early on microvesicles appeared in more luminally located granule membranes and were associated with granule fusions, that became common and led to the formation of large irregular aggregates. Most of the secretion of granule contents appeared to be through openings of aggregates into lumina. With granule fusions the intra-membrane microvesicles became internalised and tended to increase in size with time; they were commonly expelled with the contents of the aggregates. Fragments of cytoplasm also became incorporated in aggregate formation. Cytoplasm, often containing glycogen, also formed luminal blebs over some granular tubule cells and appeared to pass into the secretion by an apocrine process. At the end of stimulation multivesicular bodies were seen in association with redundant aggregates.

Constitutive secretion of kallikreins in vivo from rat submandibular glands.

In parasympathetic saliva from rat submandibular glands the relative proportions of the various tissue kallikreins differ from those in sympathetic saliva. Kallikreins in sympathetic saliva arise from exocytosis of prepackaged granules in granular tubules, so the kallikreins in parasympathetic saliva must come from a non-granular pool, and are likely to be secreted through a constitutive vesicular route. During periods devoid of stimulation in anaesthetised rats, the kallikreins have been found to accumulate progressively in glandular lumina in parasympathetic-like proportions. As this transport of kallikrein into lumina occurs continuously in vivo, independently of any stimulation or any secretion of fluid, it must arise by constitutive vesicular secretion. During parasympathetic stimulation, the kallikreins are secreted into the saliva at a greater rate than in the resting state but their proportions remain the same and the means by which this increase occurs is open to debate. Constitutively secreted true tissue kallikrein (rK1) has been found to have a different molecular form from that in secretory granules. The submandibular glands also contribute to the kallikreins normally circulating in the blood. Serum levels of kallikrein increased equally during either parasympathetic or sympathetic stimulation and were independent of the amounts secreted into the saliva, so are likely to have arisen from constitutive secretion via the basal sides of the cells, morphological evidence for which has been found in the mouse (Penschow & Coghlan, 1993).

Neural regulation of blood flow in the rat submandibular gland.

Blood flow in salivary glands is regulated mainly by sympathetic and parasympathetic nerve activity. This study was carried out to determine the relative contributions of cholinergic, adrenergic and peptidergic neurotransmitters to the control of submandibular blood flow in the rat using laser-Doppler flowmetry. Parasympathetic impulses caused a rapid atropine-sensitive vasodilation followed by a maintained increase in blood flow, a portion of which remained in the presence of both atropine and L-NAME. In contrast, continuous sympathetic stimulation caused an intense vasoconstriction that was followed by a prolonged after-vasodilation. The same number of impulses delivered in bursts resulted in a cyclic vasoconstriction followed by a rapid vasodilation. Alpha-adrenoceptor blockade largely abolished the vasoconstriction, and the duration and magnitude of the after-vasodilation were reduced. Inhibition of nitric oxide (NO) synthase by L-NAME reduced the vasodilation. The addition of a beta-adrenoceptor antagonist eliminated the sympathetic vasodilator response, but in the presence of complete alpha- and beta-adrenoceptor blockade and L-NAME a small vasoconstriction remained. We conclude that the vasoconstrictor effects of sympathetic stimulation of the rat submandibular gland are due to alpha-adrenergic receptor activation and probably also NPY, and the vasodilator effects are due to NO and beta-adrenergic activity. Parasympathetic vasodilation was due to NO-independent mechanisms mediated by acetylcholine and substance P, and NO-dependent mechanisms mediated by VIP.

The influence of nerves on the secretion of immunoglobulin A into submandibular saliva in rats.

1. The influence of sympathetic and parasympathetic nerve stimulations on salivary secretion of immunoglobulin A (IgA) was studied in the submandibular glands of anaesthetized rats by stimulating the nerve supplies with bipolar electrodes. 2. Although the flow of saliva from sympathetically stimulated glands was only 23% of that from parasympathetically stimulated glands the output of IgA was over 2-fold greater. This difference was attributable to influences of the nerves on IgA secretion through the epithelial cell polymeric immunoglobulin receptor-mediated pathway, as Western blotting with specific antibodies to IgA and secretory component revealed that secretory IgA (SIgA) dominated in all saliva samples. 3. Study of saliva secreted in sequential periods of nerve stimulation or following rest pauses suggested that SIgA secretion occurred in the absence of stimulation but this was upregulated 2.6- and 6-fold by parasympathetic and sympathetic nerve stimulations, respectively, compared with the calculated unstimulated rate. 4. The IgA content of extensively stimulated glands was 77% of levels in unstimulated contralateral control glands despite a secretion into saliva equivalent to almost 90% of the glandular IgA content. The IgA may be synthesized and secreted by glandular plasma cells at a rate which exceeds demand and/or such synthesis may be upregulated by nerve impulses. 5. The results indicate that salivary secretion of SIgA is upregulated by nerve impulses and that sympathetic nerves induce a greater effect than parasympathetic nerves.

Protein secretion from rat submandibular acini and granular ducts: effects of exogenous VIP and substance P during parasympathetic nerve stimulation.

The influences of exogenous vasoactive intestinal peptide (VIP) and substance P on the release of peroxidase from acini and true tissue kallikrein (rK1) from granular ducts of the rat submandibular gland were studied during continuous parasympathetic stimulation. Parasympathetic nerve impulses caused a moderate flow of saliva (mean +/- SD, 108+/-26 microl/g tissue/min) that had a low protein concentration (174+/-88 microg/ml). The outputs of peroxidase and rK1 were minimal (14.3+/-11.8 pmol DCF/g tissue/min and 6.5+/-3.4 nmol AFC/g tissue/min, respectively). When administered intravenously, VIP had no apparent effect on the overall flow rate, but caused a significant increase in the output of peroxidase; 450% at 1 microg/kg and a further 10-fold increase at 10 microg/kg. In contrast, substance P (1 microg/kg) evoked a marked increase in flow rate (68%), and peroxidase secretion increased only 3-fold. The output of rK1 was unaffected by either VIP or substance P. Our results support the hypothesis that acinar, but not granular duct, protein secretion is evoked by non-adrenergic, non-cholinergic peptides released from parasympathetic nerve terminals.

The enzymic potential of tissue kallikrein (rK1) in rat submandibular saliva depends on whether it was secreted via constitutive or regulated pathways.

The enzymic activity and immunoreactivity of rat tissue kallikrein (rK1) secreted at rest by granular duct cells of unstimulated submandibular glands has been compared with that secreted on autonomic nerve stimulation. Although a direct vesicular, constitutive secretory pathway operates for rK1 secretion from granular duct cells of unstimulated and parasympathetically stimulated glands the rK1 was not present in a pro-form and actually showed a greater enzymic activity per unit immunoreactive protein than the granule-derived rK1 in sympathetically evoked saliva. Constitutively secreted rK1 was found to be in a single chain molecular form by reducing SDS gel electrophoresis. In contrast rK1 secreted from the storage granule pool of granular duct cells on sympathetic nerve stimulation was present in much higher amounts and occurred in both one-chain and two-chain forms as revealed by SDS gel electrophoresis under reducing conditions. The lower enzymic potential of rK1 in sympathetically evoked saliva might be accounted for by its conversion to a two-chain form.

Use of a cast spreader during reoperative sternotomy.

We report a modified use for an old tool: separation of the sternal edges during repeat sternotomy with a cast spreader. We have found that this technique improves our exposure, causes no trauma to bone, and minimizes the risk of damage to underlying mediastinal structures.

Secretory interactions between the sympathetic and parasympathetic innervations of the submandibular gland in the anaesthetized cat.

Interactions between the sympathetic and parasympathetic innervations of the submandibular gland have been investigated in the anaesthetized cat. At low frequencies of chorda lingual (parasympathetic) stimulation, simultaneous stimulation of the ascending cervical sympathetic nerve in bursts (20 Hz for 1 s at 10 s intervals) increased the flow of submandibular saliva, but the effect was never more than additive. The output of protein was consistently reduced by simultaneous stimulation of both the sympathetic and parasympathetic innervations, below that evoked by stimulation of either alone. Sympathetic stimulation was more effective than parasympathetic stimulation in promoting the secretion of tissue kallikrein and peroxidase in the submandibular saliva. The output of the latter enzyme, in response to sympathetic stimulation, was significantly reduced by simultaneous stimulation of the parasympathetic innervation at frequencies greater than 1 Hz, but nevertheless exceeded the amount secreted during chorda stimulation alone. Thus, this protocol provided no evidence of synergy between the two divisions of the autonomic nervous system with respect to any submandibular secretory function that was recorded. However, following the administration of a small dose of atropine (2-15 micrograms kg-1 i.v.), sufficient to block secretion during chorda stimulation alone, the flow of saliva, in response to sympathetic stimulation, was potentiated when superimposed on a background of parasympathetic stimulation at all frequencies that were employed. This effect was abolished by larger doses of atropine, indicating that it was dependent upon activation of muscarinic receptors, only some of which could have been blocked by the initial dose.