G. B. Proctor

Secretory responses in granular ducts and acini of submandibular glands in vivo to parasympathetic or sympathetic nerve stimulation in rats.

SummaryThe roles of sympathetic and parasympathetic nerves in the secretion of saliva from submandibular glands of rats have been tested by electrical stimulation of either nerve for 1 h unilaterally in separate animals. The flows of saliva thereby induced and their protein content were monitored. Structural changes in each gland were assessed by light- and electron microscopy and compared with the unstimulated contralateral control gland, and the extent of the changes was determined morphometrically. Sympathetic nerve stimulation induced a relatively low flow of saliva that was rich in protein and was accompanied by extensive degranulation from both acinar and granular duct cells. In contrast parasympathetic nerve stimulation induced a considerable flow of saliva that had a low protein content and no detectable degranulation occurred from the secretory cells. It is possible, therefore, that some protein in parasympathetic saliva may have arisen from a non-granular pathway.

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.

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.

New specific assays for tonin and tissue kallikrein activities in rat submandibular glands assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva

At least fourteen separate bands of proteinase activity, labelled A-N, were identified by an enzyme overlay membrane technique, using oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) substrates in rat submandibular gland extracts fractionated on pH 4-6.5 isoelectric focusing gels. The proteinases were eluted into an ammonium bicarbonate buffer pH 9.8 containing 0.1% Triton X-100 and the relative contribution of each band to total activity evaluated using D-Val-Leu-Arg-AFC (DVLR-AFC) and Z-Val-Lys-Lys-Arg-AFC (ZVKKR-AFC) as substrates. Immunoblotting of band eluants run on sodium dodecyl sulphate gels with antibodies showed that band A was identical with tonin and bands K-N contained tissue kallikrein. Tonin was found to hydrolyse ZVKKR-AFC but not DVLR-AFC. Estimates of the Km values of tissue kallikrein for DVLR-AFC and tonin for ZVKKR-AFC were found to be similar (approx. 20 microM) yet the former enzyme hydrolysed its substrate five times faster. Tonin was inhibited by soybean trypsin inhibitor (SBTI) but not by aprotinin. Tissue kallikrein, on the other hand, was inhibited by aprotinin but was considerably more resistant to inhibition by SBTI. In tissue extracts 95% of the ZVKKR-AFC lytic activity in the presence of 1 microM aprotinin is due to tonin and a similar percentage of the DVLR-AFC hydrolysing activity in the presence of 10 microM SBTI is due to tissue kallikrein. These findings were used for the specific measurement of these two proteinases in submandibular gland extracts and in saliva without prior purification. Using these inhibitor based assays we revealed qualitative differences in the composition of proteinases secreted into saliva during parasympathetic versus sympathetic stimulation of the submandibular gland. The distribution of proteinases in sympathetic saliva is very similar to that found in submandibular extracts but on parasympathetic stimulation, although much less proteinase is released, the contributions of the more acidic isomers of tissue kallikrein are increased and that of tonin and other proteinases dramatically decreased. The data suggest that parasympathetic and sympathetic nerves induce proteinase secretion via different pathways.

Influences of secretory activities in rat submandibular glands on tissue kallikrein circulating in the blood

Changes in serum levels of rat tissue kallikrein (rK1) in venous blood were measured, using a newly developed radioimmunoassay, before and after autonomic nerve stimulations of submandibular salivary secretion. rK1 secreted into saliva under these conditions was measured by radioimmunoassay and by enzymic activity assay, using the fluorogenic peptide substrate D‐Val‐Leu‐Arg‐7‐amino‐4‐trifluoromethylcoumarin (AFC). Following an overnight fast, serum rK1 concentration was 30‐40 ng ml‐1. Unilateral electrical stimulation of the submandibular sympathetic nerve supply (at 50 Hz in bursts of 1 s every 10 s for 60 min) evoked a small flow of saliva with a very high rK1 concentration, resulting in a large output of rK1 of 2104.4 +/− 603.5 micrograms (n = 6). Such stimulation caused a large degranulation of granular duct cells and a corresponding reduction in glandular rK1 content. Unilateral electrical stimulation of the parasympathetic nerve supply (at 5 Hz continuously for 60 min) evoked a copious flow of saliva with a very low rK1 concentration, resulting in a low output of rK1 (18.1 +/− 4.9 micrograms; n = 6). Despite these large differences in salivary outputs of rK1, serum concentrations of rK1 were increased similarly following either sympathetic or parasympathetic stimulation by 48 and 46%, respectively. If the submandibular duct was briefly obstructed during sympathetic stimulation, inducing leakage and glandular oedema, then serum rK1 increased greatly (40‐fold); a similar increase to that seen by others in previous studies without deliberate obstruction. Four days after bilateral submandibular‐sublingual sialadenectomy serum rK1 concentration was reduced by approximately 50%. The results indicate that submandibular glands normally contribute to circulating levels of rK1 in rats, but this contribution is independent of the amounts of rK1 secreted into saliva by sympathetically induced exocytosis, and is likely to arise from basal vesicular transport. However, if glandular leakage occurs during sympathetic stimulation of submandibular secretion this then causes increases in the circulating levels of rK1 that correlate with the large amounts being secreted into saliva.

In vivo secretory responses of submandibular glands in streptozotocin-diabetic rats to sympathetic and parasympathetic nerve stimulation.

Submandibular gland responses to sympathetic and parasympathetic nerve stimulation were studied in streptozotocin-diabetic rats. Morphologically, the acinar cells in control glands were relatively uniform in size and contained electron-lucent granules. The granular ducts were distinguished by the presence of electron-dense granules. With the exception of intracellular lipid droplets and the presence of a few autophagosomes in diabetic glands, no consistent differences in acinar cell structure were observed. In contrast, the diameter of the granular ducts and the granule content of their cells were less in diabetic glands. At 3 weeks sympathetic flow rate, salivary protein concentration, and total protein output were unaffected by diabetes. Sympathetic flow rate was greater at 3 months, and the concentration of protein in the saliva was lower. In 6-month diabetic rats flow rate remained increased, but protein concentration and total protein output were reduced. The decrease in salivary protein concentration at 3 and 6 months was accompanied by a reduction in secretory granule release from acinar and granular duct cells. No consistent differences in flow rate, protein concentration, protein output, or secretory granule release were observed following parasympathetic stimulation. We conclude that the effects of diabetes on nerve-stimulated flow rate and protein release depend on the duration of diabetes and the type of stimulation, and are independent of one another.

Lipid analysis of the major salivary glands in streptozotocin-diabetic rats and the effects of insulin treatment

Two separate sets of experiments were performed on female Wistar rats made diabetic with streptozotocin: (1) a time-course study where groups of three animals were removed at weekly intervals, up to 4 weeks after induction of diabetes, with an age-matched group of control (normal) animals kept for 4 weeks; (2) six further animals were made diabetic and kept for 7 weeks; three of these were given insulin in the final week. At the required time the animals were anaesthetized and the salivary glands removed and preserved by fixation or freezing. The frozen tissues were later homogenized and the protein and lipid content analysed. Histologically, intracellular lipid droplets had accumulated in the majority of the diabetic salivary glands. In the time-course experiment, the visible amount of intracellular lipid reached a maximum after 2 weeks and then decreased, with a concomitant disappearance of interstitial lipid. The increased lipid content was not attributable to any one class. The fatty acid profiles of the glands showed an increase in the percentages of C18:0 (stearic acid) and C18:2w6 (linoleic acid) and a decrease in the percentages of C18:1w9 (oleic acid) and C20:4w6 (arachidonic acid). After 1 week of insulin treatment the lipid content and the fatty acid profiles returned to normal. Thus the effect of insulin on salivary gland lipid metabolism is rapid both in its occurrence and reversibility. The effects seen in the diabetic rats are considered to be due to a lack of insulin and not to the presence of streptozotocin.

Enzyme Histochemistry of Tryptase in Stomach Mucosal Mast Cells of the Mouse

We investigated the histochemical characteristics of mast cell tryptase in different mouse tissues. By use of peptide substrates, tryptase activity could be demonstrated in unfixed connective tissue mast cells in different tissues, including the stomach. Tryptase activity was better localized after aldehyde fixation and frozen sectioning, and under such conditions was also demonstrated in mucosal mast cells of the stomach but not in those of the gut mucosa. Double staining by enzyme histochemistry followed by toluidine blue indicated that the tryptase activity was present only in mast cells and that all mast cells in the stomach mucosa contained the enzyme. The peptide substrates z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthlyamide, which are substrates of choice for demonstrating tryptase in other species, were most effective for demonstrating mouse tryptase. The use of protease inhibitors further indicated that activity present in all mast cells was tryptase. Safranin O did not stain stomach mucosal mast cells, suggesting that the tryptase present in these cells was active in the absence of heparin sulfate proteoglycan.

Nerve-induced secretion of glycoconjugates from cat submandibular glands: a correlative study with lectin probes on tissues and saliva.

Horseradish peroxidase-conjugated lectins were used on tissue sections to localize the main secretory glycoproteins in cat submandibular glands and on Western blots to evaluate their movement into saliva with selective nerve stimulation. Central acinar cells bound lectins from Arachis hypogaea (PNA) specific for the terminal disaccharide Gal beta 1, 3GalNac, Griffonia simplifolia (GSA I-B4) specific for terminal alpha Gal, and Lotus tetragonolobus (LTA) specific for fucose. Lectins from Limax flavus (LFA) specific for sialic acid and Dolichos biflorus (DBA) specific for terminal alpha GalNac reacted preferentially with demilunar cells, whereas apical granules in striated ducts were recognized principally by LTA. Parasympathetic stimulation promoted the release of lectin-reactive glycoconjugates from both central and demilunar cells. In contrast, sympathetic stimulation caused almost complete release of LTA-reactive granules in striated ducts and only moderate secretion from demilunar cells. Lectin blots of stimulated saliva discriminated many of the constituent bands, providing information about their glycosylation. Several bands were common to both parasympathetic and sympathetic saliva, and many bands gave wider ranges of lectin binding than anticipated from the histochemistry. The major component in parasympathetic saliva was a glycoconjugate of less than 12 KD which reacted with every lectin tested. Lectin blots of sympathetic saliva showed a prominent diffuse LTA-reactive band around 33 KD, which was attributed to tissue kallikrein. The identity and cellular origin of most bands in stimulated submandibular saliva are still unclear but the technique shows considerable promise for improving the recognition and characterization of individual glycoconjugates.

Proteinase activities in bovine atrium and the possible role of mast cell tryptase in the processing of atrial natriuretic factor (ANF)

1. Using low salt, Triton X-100 and high salt extracts of bovine atria, two main proteinases were identified by means of fluorogenic oligopeptide substrates. 2. An acidic proteinase, extracted in low salt and Triton X-100 was identified as cathepsin B, but it caused little hydrolysis of the Z-Gly-Pro-Arg- containing substrate that resembles the cleavage site for activation of pro-ANF. 3. An alkaline proteinase was extracted only with high salt and had characteristics of the serine proteinase tryptase. It cleaved Z-Gly-Pro-Arg- containing substrates more efficiently than others tested and was localized in and around mast cells histochemically. Previously, Imada et al., 1988 (J. biol. Chem. 263, 9515-9519) found an identical enzyme would cleave ANF from pro-ANF. 4. These results suggest therefore that mast cell tryptase may be involved in the activation of ANF from pro-ANF.