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MS 0707 Received 15 February 2000; accepted after revision 18 May 2000.
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ABSTRACT |
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INTRODUCTION |
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Previous studies on reflex secretion from submandibular glands in conscious rats have shown that outputs of saliva differ with different afferent stimulations (Matsuo et al. 1994), being great with grooming and eating dry food pellets, but less during rejection behaviour when a bitter fluid is placed in the mouth. However, little is known about the relative contributions of the parasympathetic and sympathetic nerves during such reflex activities.
Experimental studies using nerve stimulations indicate that, in isolation, parasympathetic nerves exercise different effects on the secretory cells in rat submandibular glands from those of the sympathetic nerves. Parasympathetic stimulation causes a copious flow of saliva with low outputs of protein and no detectable degranulation of acinar or granular tubule cells (Garrett et al. 1991). Nevertheless, both peroxidase from acini and tissue kallikrein from granular tubules continue to be secreted in low amounts during parasympathetic stimulation (Anderson et al. 1995). Subsequent work has shown that the parasympathetic secretion of kallikreins occurs via the so-called 'constitutive' vesicular route (Garrett et al. 1996) and not from preformed granules. On the other hand, sympathetic nerve stimulation per se causes less fluid secretion, but exocytosis of secretory granules occurs from both acini and granular tubules (Garrett et al. 1991). Dual nerve stimulation experiments have shown that when very low frequency sympathetic stimulation is added to parasympathetic stimulation the output of acinar peroxidase is greatly increased, but soon reaches a plateau as the sympathetic frequency increases (Anderson et al. 1995). The same study also showed that the secretion of ductal tissue kallikrein from storage granules in granular tubules required a much higher frequency of sympathetic drive. This and other work (Garrett et al. 1997) confirms that the dependence on sympathetic impulses for granule exocytosis is different with respect to frequency for the two secretory cell types in rat submandibular glands. Recent investigations have also revealed that stimulation of either autonomic input can upgrade the basal rate of salivary secretion of secretory IgA (SIgA), which is derived from IgA released by interstitial plasma cells (Carpenter et al. 1998) and moves to lumina by transcytosis. However, with dual nerve stimulation the effects were never more than additive for SIgA (Carpenter et al. 2000), whereas for tissue kallikrein and peroxidase they were synergistic (Anderson et al. 1995).
The purpose of the present study was to analyse different proteins secreted into submandibular saliva under similar reflex conditions to those used by Matsuo et al. (1994), and also during heat-induced secretion (Hainsworth & Stricker, 1970; Elmér & Ohlin, 1971), in order to differentiate activities being induced in the separate cell types within the glands. It was anticipated that this would shed light on the types of autonomic drive occurring reflexly in response to different afferent stimuli. Furthermore, the same reflex stimulations were repeated in the animals after eliminating sympathetic impulse traffic to the gland by preganglionic sympathectomy (decentralization), thereby providing supplementary information about the natural contribution of sympathetic impulses to the saliva normally formed during the reflex conditions used.
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METHODS |
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Eleven normal adult male Wistar rats weighing 242-300 g (Charles River Breeders, Osaka, Japan) were used. All animal protocols were performed in accordance with The Guiding Principles for the Care and use of Animals approved by the Council of The Physiological Society of Japan.
Surgical procedures and collection of saliva
The rats were fasted overnight but given free access to water. They were deeply anaesthetized with an I.P. injection of sodium pentobarbitone (50-75 mg kg-1) and prophylactically treated with a penicillin suspension (30000 units I.M.). The duct of the left submandibular gland was exposed near its orifice at the floor of the mouth through an incision made at the corner of the mouth. The duct was cannulated with polyethylene tubing (3 mm long) of the largest possible bore. The tubing was fixed to the duct and the dorsal edge of the mandibular bone between incisor and molar teeth using a butyl-2-cyanoacrylate adhesive and dental acrylic resin. The free end of the tubing was connected to a second piece of polyethylene tubing (0·8 mm i.d., 1·1 mm o.d., 
7 cm long) that was bent subcutaneously backwards over the masseter and temporalis muscles and led out through an incision made on the top of the skull. For oral injection of the bitter tasting solution, an intraoral fistula was made by a method similar to that described by Phillips & Norgren (1970). Briefly, a curved surgical needle was inserted in the buccal mucosa lateral to the upper molar teeth and out through the incision over the skull to guide the polyethylene tubing (0·8 mm i.d., 1·1 mm o.d., 7 cm long) into place. The salivary and intraoral tubes were cemented to the top of the skull with dental acrylic resin.
After recovery from anaesthesia (on the same day at least 4 h after injection of pentobarbitone), the animal was moved to a test box (a clear plastic cylinder 30 cm in diameter and 20 cm high). Reflex saliva was collected in polyethylene tubing (1·1 mm i.d., 1·6 mm o.d., 20-30 cm long) that was connected to the implanted salivary tubing close to the skull. After reflex stimulation, the collecting tubing was removed and the saliva was transferred to a preweighed culture tube. The saliva sample was weighed and immediately stored at -20°C until analysed. The volumes of saliva secreted were determined assuming a specific gravity of 1·0 g ml-1. The four kinds of reflex stimuli, feeding, rejection of a bitter tasting solution, exposure to heat and grooming (see below for details), were repeated twice. The order of the stimuli was varied each time and in different animals. The animals were allowed access to water between reflex stimuli for 15-30 min. After collection of saliva animals were returned to their home cage and given laboratory food pellets and water. Food, but not water, was withheld 3 h later.
The next day the rats were deeply anaesthetized as previously, and the left cervical sympathetic trunk was sectioned via an incision made along the mid-line of the ventral surface of the neck. After the rats had recovered from anaesthesia, saliva was collected in the same way as before sympathetic decentralization. After the last saliva samples had been collected, the animals were killed with an overdose of sodium pentobarbitone (> 100 mg kg-1 I.P.), and the submandibular glands were excised bilaterally (7-11 h after preganglionic sympathectomy). Each gland was immediately weighed and a thin transverse wedge was fixed for 1 week at 4°C in 4 % paraformaldehyde in 0·5 M cacodylate buffer, pH 7·2, containing 7·5 % sucrose.
Reflex stimulation
Food given in the test box was a mash consisting of 0·9 parts distilled water to one part powder diet (NMF powder, Oriental Co., Japan) by weight. Rats chewed the mash continuously for a few minutes at a time. For evoking the bitter taste rejection behaviour an aliquot (0·1 ml) of 0·02 M quinine hydrochloride was injected into the mouth through the intraoral fistula. When a single injection did not induce a sufficient amount of saliva for chemical analyses, two to four aliquots of quinine were injected. For heat exposure, the test box was moved to an incubator with a glass window which permitted continuous observation of the animals. Vigorous salivation occurred after about 20 min at a temperature of 40 ± 1°C and saliva was collected for 5-7 min; during this period the rats extended their bodies on the floor without grooming. Saliva during grooming was collected at room temperature (24-26°C). Grooming often occurred after drinking of water, but drinking water by itself did not induce secretion of saliva.
Assays of salivary proteins
The levels of SIgA present in each saliva sample were quantified by enzyme-linked immunosorbent assay (ELISA). This was performed on 96-well microtitre plates (Griffiths & Neilson, Billingshurst, UK) which were coated overnight at 4°C with rabbit anti-rat IgA (Serotec Ltd, Oxford, UK) diluted 1 in 2000 with 0·1 M sodium carbonate buffer (pH 9·6). Plates were washed 3 times in 0·1 M phosphate-buffered saline (pH 7·0) containing 0·15 M sodium chloride and 0·1 % Tween 20 (PBS-T) followed by water and then samples were placed on the coated plates and serially diluted in PBS-T. Samples were incubated on the plates for 2 h at 37°C then the plates were washed as above. SIgA purified from rat bile (Carpenter et al. 1998) was quantified by a modified Lowry protein assay (Petersen, 1977) and was used as a standard for quantifying the SIgA content of samples. Horseradish peroxidase-labelled rabbit anti-rat IgA antibodies (Serotec Ltd), diluted 1 in 2000 in PBS-T, were incubated on the plate for 1 h at 37°C. Following incubation, plates were washed with PBS-T then incubated at room temperature in the peroxidase substrate tetramethyl benzidine (Sigma, Poole, UK) at a concentration of 3 mg ml-1 in DMSO and diluted 1:20 in 0·1 M sodium acetate buffer, pH 5·5. The reaction was terminated by the addition of 50 µl of 2 M H2SO4 and absorbance was measured at 450 nm in an automated microplate reader (BioRad Labs Ltd, Hemel Hempstead, UK).
Total protein content of saliva samples was assayed by absorbance at 215 nm (Arneberg, 1971) using a human serum albumin/gamma globulin standard (Sigma). Peroxidase was assayed using the fluorogenic substrate dichlorofluorescin (Molecular Probes Europe, Leiden) which is converted to dichlorofluorescein (DCF) in the presence of hydrogen peroxide and peroxidase (Proctor & Chan, 1994). A standard curve of DCF (Sigma) was prepared and peroxidase activity was expressed in micromoles of DCF per minute (DCF units).
Tissue kallikrein was assayed using the fluorogenic substrate D-valyl-leucyl-arginyl-7-amino-4-trifluoromethylcoumarin (dVLR-AFC; Enzyme Systems Products, USA) in the presence of soya bean trypsin inhibitor (Sigma) as previously described (Shori et al. 1992). A standard curve of the product 7-amino-4-trifluoromethylcoumarin (AFC; Enzymes Systems Products) was prepared and tissue kallikrein activity was expressed in micromoles of AFC per minute (AFC units).
Electrophoresis of salivary proteins
Saliva samples (equivalent to 40 µg total protein) were reduced using dithiothreitol and loaded onto 4-20 % SDS-PAGE separating gels containing Tris and glycine (Novex, Frankfurt, Germany) and electrophoresis was performed according to the manufacturer's instructions. Resolved proteins were stained with a solution of 0·2 % Coomassie Brilliant Blue R-250 (Sigma) in 25 % methanol and 10 % acetic acid and gels were destained with 10 % acetic acid. In order to confirm that IgA was secreted in the form SIgA, that is IgA bound to secretory component (SC), electrophoretically resolved proteins were electroblotted onto 0·45 µm nitrocellulose membranes (Anderman & Co., Kingston Upon Thames, UK) and probed with peroxidase-labelled rabbit anti-rat IgA (Serotec) or with unlabelled rabbit anti-rat SC (Universal Biologicals Ltd, Stroud, UK) followed by biotinylated goat anti-rabbit antibody (Sigma Ltd, Poole, UK) and avidin-biotin complex (Vector laboratories Ltd, Peterborough, UK). Binding was detected using enhanced chemiluminescence (ECL; Amersham, Pharmacia, Biotech, Aylesbury, UK) and photographic film as previously described (Carpenter et al. 1998).
Results are expressed as means ± S.E.M. Student's paired or unpaired t tests with the Bonferroni correction, where appropriate, were used to compare means ± S.E.M. of particular groups. Means for different groups were compared by one-way ANOVA and P < 0·05 or the equivalent corrected value was considered significant. Flow rates and outputs for each animal were related to the wet weight in grams of the control gland (g-1).
Histology
The fixed slices of the glands were embedded in paraffin wax. Sections were cut at 5 µm, mounted on glass slides, the left (test) and right (control) sides from each animal were mounted on the same slide. Sections from the glands were stained conventionally with haematoxylin and eosin (H & E), for mucosubstances with Alcian Blue-periodic acid-Schiff's reagent (AB/PAS) or with p-dimethylaminobenzaldehyde (DMAB) to show the granular tubules.
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RESULTS |
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Macroscopic features
All glands appeared to be normal and showed no evidence of oedema. Salivary gland weights were: left sympathetically decentralized test glands 172·0 ± 6·4 mg and right control glands 155·1 ± 4·6 mg. The difference between the weights was highly significant (P < 0·001).
Salivary flow and protein secretion
Results are based on 22 observations for each reflex stimulation before and also after sympathetic decentralization.
The salivary flow rates during the different reflex conditions, feeding, heat, rejection and grooming, in normal animals showed consistent differences between each; grooming caused a significantly greater flow of saliva than heat exposure, which produced the second highest flow of saliva (Fig. 1A). Rejection- and feeding-evoked salivary flow rates were significantly lower than during heat exposure or grooming. After sympathetic decentralization the salivary flow rates were not significantly reduced and with each of the stimuli the relative order of salivary flow was maintained.
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A, salivary flow rate. Grooming (g) evoked a significantly higher flow than heat (h, P < 0·01), rejection (r, P < 0·001) or feeding (f, P < 0·001), and in turn heat evoked a higher flow rate than rejection (P < 0·001). Sympathetic decentralization did not significantly reduce fluid secretion and the difference between grooming and heat was no longer significant. B, protein concentration. Heat-evoked saliva had a significantly lower protein concentration (P < 0·05) than the other salivas. Sympathetic decentralization significantly reduced protein concentrations (P < 0·01) in all salivas except heat-evoked saliva. C, protein output. Grooming-evoked secretion of salivary protein was significantly greater than with the other stimuli (P < 0·001) and output on feeding was in turn greater than that on heat or rejection (P < 0·001). Following sympathetic decentralization the outputs of protein into saliva from grooming, rejection and feeding were significantly reduced (P < 0·01). n = 22. | ||
Total salivary protein concentration (Fig. 1B) was similarly high in grooming- (5·64 ± 1·06 mg ml-1), feeding- (6·95 ± 0·66 mg ml-1) and rejection-evoked saliva (5·53 ± 0·72 mg ml-1) but was significantly much lower (P < 0·01) in heat-evoked saliva (1·59 ± 0·36 mg ml-1). Salivary protein concentration was greatly reduced after sympathetic decentralization, except during heat exposure. Output (concentration × flow rate) of total salivary protein was highest for grooming- and lowest for heat-evoked saliva (Fig. 1C). The reduced protein output during grooming, feeding or rejection behaviour after sympathetic decentralization reflected reductions in protein concentration. Although there was a slight reduction in the output of protein during heat exposure, it was not significant. However, following preganglionic sympathectomy the output of protein into rejection-evoked saliva (64·4 ± 14·2 µg g-1 min-1) was significantly less (P < 0·01) than that into heat- (165·4 ± 30·6 µg g-1 min-1) or grooming-evoked saliva (183·1 ± 59·5 µg g-1 min-1).
Secretion of peroxidase
Peroxidase secretion from acinar cells showed similar patterns to total protein secretion, and heat-evoked saliva had a significantly lower concentration of peroxidase activity (P < 0·01) than the other stimuli (Fig. 2A). Following sympathetic decentralization salivary peroxidase activity, like protein concentration, decreased to similar levels for each reflex stimulation with significant (P < 0·01) reductions in peroxidase activity for all stimuli except heat (Fig. 2A). Output of peroxidase from glands with an intact innervation was highest on feeding or grooming and lowest with heat or rejection behaviour (Fig. 2B). The reductions in peroxidase outputs following the sympathetic decentralization were marked except for heat, being only 10 % of the pre-sympathectomy activity for grooming- and feeding-evoked salivas (Fig. 2B).
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A, peroxidase activity. Heat (h)-evoked saliva had a significantly lower peroxidase activity (P < 0·01) than the other salivas. Following sympathetic decentralization peroxidase activity was markedly consistent between the different salivas and there were significant (P < 0·01) reductions in the activities in grooming (g)-, rejection (r)- and feeding (f)-evoked salivas though there was no significant change in heat-evoked saliva. B, peroxidase output. Grooming produced a greater output of peroxidase than heat (P < 0·001) and rejection (P < 0·001). Similarly, feeding produced a greater output than during heat (P < 0·01) or rejection (P < 0·01). After sympathetic decentralization, significant reductions in salivary peroxidase output occurred during grooming (P < 0·05), feeding (P < 0·001) and rejection (P < 0·001). C, tissue kallikrein activity. Feeding produced a greater tissue kallikrein activity in saliva than the other stimuli though the differences were not significant. Following sympathetic decentralization tissue kallikrein activity was reduced in feeding- and grooming-evoked salivas but this was only significant (P < 0·05) for the latter. D, tissue kallikrein output. Feeding evoked a significantly higher output (P < 0·05) than rejection. After sympathetic decentralization tissue kallikrein output was reduced significantly during grooming (P < 0·05), feeding (P < 0·02) and rejection (P < 0·02) but not in heat-induced saliva. E, IgA concentration. Heat evoked a lower concentration of IgA than feeding (P < 0·01) or grooming (P < 0·001). Sympathetic decentralization caused no significant changes in the concentrations. F, IgA output. Grooming produced a greater output (P < 0·01) than the other stimuli and sympathetic decentralization caused a decreased output with grooming (P < 0·05). n = 22. | ||
Secretion of tissue kallikrein
Tissue kallikrein secretion by ductal cells showed a different pattern (Fig. 2C) from that for total protein or acinar peroxidase. Kallikrein activity was highest in feeding-evoked saliva and lower in saliva samples evoked by the other stimuli but the differences were not statistically significant. Sympathetic decentralization decreased tissue kallikrein activity significantly in grooming-evoked saliva (P < 0·05); it also reduced tissue kallikrein activity evoked by feeding, but this difference was not statistically significant. However, the outputs of tissue kallikrein activity were reduced significantly by sympathetic decentralization during feeding, grooming and rejection but not with heat exposure (Fig. 2D).
Salivary IgA secretion
The concentration of salivary IgA in heat-evoked saliva was less than that in feeding- (P < 0·01) or grooming-evoked saliva (P < 0·001) and no changes were seen following sympathetic decentralization (Fig. 2E). When expressed as outputs, IgA secretion was greater on grooming than during feeding (P < 0·003), heat (P < 0·001) or rejection (P < 0·001) and the output on grooming was reduced by sympathetic decentralization (P < 0·05) (Fig. 2F).
Composition of salivary protein resolved by electrophoresis
Equal amounts of total protein from salivas were electrophoresed and this revealed almost identical compositions of different proteins in samples evoked by feeding, grooming and rejection behaviour (Fig. 3). However, the protein composition of saliva evoked by heat showed major differences from those of the other stimulations. Proteins which were provisionally identified as the acinar cell products mucin and glutamine-rich protein on the basis of their metachromatic staining with Coomassie Brilliant Blue R-250 and their electrophoretic mobility (Moreira et al. 1989) were present in proportionately lower amounts in heat-evoked saliva than in that from the other stimulations (Fig. 3). Following sympathetic decentralization the protein composition of feeding-, grooming- and rejection-evoked salivas changed and took on an electrophoretic banding pattern resembling that of heat-evoked saliva with subtle changes in the intensities of different bands (Fig. 3). In particular the relative amounts of glutamine-rich protein and mucin appeared to be reduced by preganglionic sympathectomy. Western blotting using antibodies directed against IgA and SC indicated that IgA was almost exclusively secreted in the form of SIgA for all types of stimulation, before and after sympathetic decentralization (data not shown).
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Resolved proteins were stained with Coomassie Brilliant Blue R-250, and compared with molecular weight standards (MW). Feeding (f)-, rejection (r)- and grooming (g)-evoked salivas showed similar patterns and intensities of protein bands, and were different from heat (h)-induced saliva. Numbers 1-5 indicate the position of proteins which show changes following sympathetic decentralization. Two particular proteins - mucin (1) and glutamine-rich protein (2) (identified on the basis of mobility and metachromatic staining) - in saliva from normally innervated glands were proportionately less in heat-induced saliva. Following Sx banding patterns in feeding, rejection and grooming changed to resemble those of heat-induced saliva which showed little change following sympathetic decentralization. | ||
Histology
All glands appeared histologically normal. The control glands always appeared as if some acinar protein secretion had been occurring (see Fig. 4B), as is usual even in 'resting' glands (Garrett & Harrop, 1976). However, on the sympathetically decentralized side the acinar cells were loaded with mucosubstance (see Fig. 4A) as had previously been found 24 h after sympathetic decentralization (Garrett & Harrop, 1976). No readily discernible differences were found between the granular tubules in the control and test glands.
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A, left sympathetically decentralized gland after reflex stimulations, showing that the plump acinar cells had retained mucin, which appears diffuse within the cells. B, right control gland with an intact innervation from the same animal after reflex stimulations, showing that a depletion of acinar mucin has occurred, and the remaining secretory granules are smaller than in the left gland and concentrated mainly around lumina. Scale bar, 100 µm. | ||
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DISCUSSION |
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The results from the different animals were remarkably consistent for each type of stimulation, irrespective of their order or the time after anaesthesia. Therefore it is considered that neither the preceding anaesthesia nor the preceding stimulus had much effect on the secretory responses.
Ohlin (1968) showed that sympathetic impulses make a small contribution to gustatory reflex flows of fluid from rat submandibular glands and also on exposure to heat (Elmér & Ohlin, 1971). However, under the present reflex conditions any contribution by these nerves to the flow of saliva must have been minimal because it was not reduced significantly by sympathetic decentralization. Therefore, the present results indicate that the widely different reflex conditions used must have induced different patterns of parasympathetic drive centrally, since the flow rates differed so much.
However, sympathetic decentralization reduced the amounts of acinar peroxidase secreted reflexly into submandibular saliva to very low levels. Electrophoresis also showed that glutamine-rich protein and mucin, both acinar cell products (Moreira et al. 1989), were decreased relative to the other proteins. These reductions indicate that sympathetic drive plays an important part in the normal secretion of protein by the acinar cells. This had been suggested functionally by our dual nerve stimulation experiments (Anderson et al. 1995), and histologically by the retention of acinar mucin 24 h after sympathetic decentralization (Garrett & Harrop, 1976). The fact that this histological change was already manifest within 7-11 h after sympathetic decentralization in the present study, with a corresponding increase in gland weight, indicates that normally these cells in conscious rats receive a substantial input of sympathetic drive during normal reflex activity. As indicated in the Introduction, very low frequency sympathetic stimulation can increase peroxidase outputs considerably during dual nerve stimulations (Anderson et al. 1995), which must mean that the acinar cells are relatively sensitive to adrenergic influences, so far as protein secretion is concerned. This makes it unlikely that circulating catecholamines had been making much of a contribution during the reflex conditions used, otherwise the decentralization would not have had so great an effect on the acinar secretion of protein. The high peroxidase results during feeding suggest that a fair degree of sympathetic drive must have been occurring at such times. Salivary protein secretion ran similarly to that of acinar peroxidase and was likely therefore to reflect mainly acinar secretion of proteins. During parasympathetic stimulation per se proteins are also secreted from both submandibular acini and ducts, but in low amounts (Anderson et al. 1995).
Our results indicate that the least sympathetic drive occurred during the early phases of heat exposure, since the saliva it produced from glands with an intact innervation contained the lowest concentrations of proteins and peroxidase activity, and also showed different electrophoretic patterns compared to the other stimuli, with relatively less of some proteins. Furthermore, during heat exposure none of the proteins secreted into the saliva was changed substantially by sympathetic decentralization. However, this interpretation appears to be in conflict with opinions expressed by others about salivary events during heat exposure. Based on the outputs of salivary kallikrein into both saliva and blood during prolonged exposure to heat, Berg et al. (1990) inferred that this had been caused by sympathetic drive but conceded that it may have represented 'a developing pathological condition associated with heat shock'. Damas (1996) similarly considered that during prolonged heat exposure, 'rat submaxillary glands are subject to considerable sympathetic stimulation' but his glands showed oedema - not seen in the present work with shorter exposure to heat and free access to water between episodes. Thus, when our results are taken into account it may be considered that, during the initial less stressful more physiological periods of heat exposure, while there is still an absence of grooming, the drive for the secretion of submandibular saliva in rats comes largely from the parasympathetic input, whereas with prolonged more stressful heat exposure sympathetic and adrenal mechanisms may eventually become involved.
The potential for rat submandibular glands to secrete kallikreins in large amounts during high frequency sympathetic nerve stimulation is well documented (see Anderson et al. 1995). However, such mechanisms seem to have been little in evidence during the reflex stimulations we studied and the activities of tissue kallikrein in the saliva remained relatively low throughout. The greatest extra secretion of tissue kallikrein occurred during the eating of soft chow. The outputs of tissue kallikrein in saliva during feeding, grooming and rejection were reduced by sympathetic decentralization, which suggests that a small amount of high frequency sympathetic stimulation of the granular tubules may have been occurring during eating, grooming and rejection but not during heat exposure. Nevertheless, any secretion of kallikrein was not extensive and no obvious differences were seen in the granular tubules between normal and decentralized glands. So it remains an open question when the potential for a large sympathetically induced secretion of kallikreins is fulfilled in life and under what natural reflex conditions it may occur or for what purposes. The glands certainly contain large reserves of these secretory proteinases.
The secretion of IgA presents a different picture from that for the other proteins. Although the concentration of IgA in heat-evoked saliva was less than with the other stimuli, sympathectomy did not cause any decreases in the concentrations. The concentrations were about twice those for parasympathetic stimulation per se at 5 Hz in a different strain of rat (Carpenter et al. 1998) but considerably less than with high frequency sympathetic stimulation at 50 Hz in bursts. Experimentally, the outputs of IgA increased as a result of increasing the frequency of parasympathetic nerve stimulation up to but not above 5 Hz (Carpenter et al. 2000). The present results probably reflect the parasympathetic drive that had been occurring reflexly and, under these conditions, any additional sympathetic drive had little or no further influence on the IgA secreted into the saliva. It is unlikely, therefore, that there had been widespread sympathetic activity at high frequency in the glands, as was also suggested from our results for kallikrein secretion.
Experimental studies by Anderson et al. (1995) show that increasing the frequency of continuous parasympathetic nerve stimulation causes increases in flow and limited increases in total protein secreted, including peroxidase and tissue kallikrein. Simplistically therefore it might be considered that after sympathetic decentralization all the differences are attributable to frequency differences in the parasympathetic drive. Nevertheless, the secretion of the different proteins after sympathectomy did not fully accord with the differences in flow rates. However, in life, it is improbable that all nerves ever fire together or at synchronous rates under reflex conditions. Therefore some of the differences may relate to variations in the glandular distribution of the reflex parasympathetic impulses and their extent, thereby affecting acini and tubules unequally, as well as to differences in their firing rates. Furthermore, if there are any differential increases in neuropeptide release from the nerves under reflex conditions then they are likely to cause increases in the secretion of acinar proteins but not of kallikrein (see Anderson et al. 1998). So there are many possible explanations as to why the reflex secretion of protein is not uniform after sympathetic decentralization.
The present results suggest that the reflex stimuli used activated the sympathetic and parasympathetic drives differentially to the submandibular gland. Neuroanatomical studies in rats using the transneuronal transport of a virus reveal the forebrain centres that can influence the sympathetic and parasympathetic drives for salivation (Jansen et al. 1992; Hübschle et al. 1998). Thus the submandibular parasympathetic nerve has polysynaptic connections with the hypothalamus controlling feeding (the lateral hypothalamic area), heat-loss (the preoptic and anterior hypothalamic area) and grooming (the paraventricular nucleus and dorsal hypothalamic area; Roeling et al. 1993). On the other hand the superior cervical ganglion, which contains the postganglionic sympathetic neurones innervating the salivary glands, has connections with the feeding centre and a part of the grooming centre (only the paraventricular nucleus), but not with the heat-loss centre (Strack et al. 1989). Moreover, electrophysiological studies (Yamamoto, 1989; Matsuo, 1999) showed that both sympathetic and parasympathetic nerves of rat submandibular glands responded to electrical stimulation applied to the feeding centre and insular cerebral cortex corresponding to the masticatory and gustatory areas. These findings suggest that the heat-loss centre exclusively activates the parasympathetic nerve (as in the early stages of heat exposure used in the present experiments), whereas the forebrain centres for feeding and mastication activate both sympathetic and parasympathetic nerves of the salivary glands. The present study indirectly demonstrates that such differential activation of the salivary autonomic nerves occurs during natural reflex conditions. Under our conditions, heat exposure did not evoke any obvious movement of the jaw, so oral sensory inputs were likely to make little contribution to the salivation. However, under the other reflex conditions, oral sensory inputs also participated in both sympathetic and parasympathetic drives (Matsuo & Yamamoto, 1989). In particular, salivation during taste rejection may be induced by oral sensory inputs without forebrain descending influences, since the rejection behaviour occurred in both normal and chronic decerebrate rats (Grill & Norgren, 1978).
Results after preganglionic sympathectomy suggest that the reflex stimuli evoked different patterns of parasympathetic drive which may unequally stimulate acini and granular tubules. Such parasympathetic drives may be attributed to activation of different kinds of parasympathetic nerves. This speculation is supported by the following findings. Electrophysiological studies in anaesthetized rats showed that taste and mechanical stimuli of the oral region activate different populations of the preganglionic parasympathetic fibres innervating the submandibular gland, and that mechanical stimulation produced higher impulse frequency than taste stimulation (Matsuo & Kusano, 1984; Matsuo et al. 1989). When electrical stimulation was applied to the feeding centre, the taste-related neurones were more often facilitated than the mechano-related neurones (Matsuo & Kusano, 1984). However, when the body temperature increased from 39 to 40°C in the anaesthetized rats, both salivary flows induced by taste and mechanical stimulation were similarly increased, before starting heat-evoked salivation (Kanosue et al. 1986), suggesting that heat exposure activates many parasympathetic neurones irrespective of their relation to taste or mechanical stimulation. These electrophysiological findings imply that there are differences in recruitment of parasympathetic neurones and in their impulse frequency under different reflex conditions. It is likely that such a variability in the parasympathetic drive is reflected in the differences in flow rate and composition of saliva observed after sympathectomy in the present study.
Conclusion
Any sympathetic drive during these reflex stimulations had little effect on the flow of saliva. Sympathetic responses were mainly reflected in the secretion of acinar proteins, which indicated that sympathetic drive varied with the reflex stimulations tested and was least during heat exposure. The reflex sympathetic impulses had less effect on the secretion of kallikrein from the tubules and little or no effect on the concentrations of IgA secreted. Both these features suggest it was unlikely that there had been widespread sympathetic activity at high frequency.
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REFERENCES |
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| Anderson, L. C., Garrett, J. R., Zhang, X. & Proctor, G. B. (1998). Protein secretion from rat submandibular acini and granular ducts: effects of exogenous VIP and substance P during parasympathetic nerve stimulation. Comparative Biochemistry and Physiology A 119, 327-331. | |
| Anderson, L. C., Garrett, J. R., Zhang, X., Proctor, G. B. & Shori, D. K. (1995). Differential secretion of proteins by rat submandibular acini and granular ducts on graded autonomic nerve stimulations. The Journal of Physiology 485, 503-511 | [Abstract] |
| Arneberg, P. (1971). Quantitative determination of protein in saliva. A comparison of analytical methods. Scandinavian Journal of Dental Research 79, 60-64. | [Medline] |
| Berg, T., Johansen, L. & Poulsen, K. (1990). Exocrine and endocrine release of kallikrein after reflex-induced salivary secretion. Acta Physiologica Scandinavica 139, 29-37 | [Medline] |
| Carpenter, G. H., Garrett, J. R., Hartley, R. H. & Proctor, G. B. (1998). The influence of nerves on the secretion of immunoglobulin A into submandibular saliva in rats. The Journal of Physiology 512, 567-573 | [Abstract/Full Text] |
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This work was supported by a Wellcome research travel grant to R.M., a Leverhulme Emeritus award to J.R.G. and a Wellcome Trust project grant to G.B.P.
Corresponding author
J. R. Garrett: Department of Oral Pathology, KCSMD, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.
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