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Secretion from minor salivary glands following ablation of the major salivary glands in rats
ARCHIVES OF ORAL BIOLOGY
PERGAMON
Archives
of Oral Biology
44 (1999)
S45-S48
Secretion Corn minor salivary glands following ablation of the major salivary glands in rats J. Blazsek’l, of Oral Biology, lDepartment
G. Varga2
Semmelweis University of Medicine; *Ir&ute bpesf
of Experimental Medicine, Hungarian
Academy of Sciences;Bu-
HQWY
Since minorsalivary glands are tiny and dispersed, ductal cammlation cannot be used when studying their function. The present study was devised to develop a method of measuring minor salivary gland function by excision of the major glands. Female rats (230-280 g) were anaesthetized with sodium pentobarbital. Ablation of the submandibular, sublingual and parotid glands was performed through a sag&al neck incision. Sham-operated rats served as controls. Croups of sialadenectomized animals were investigated immediately and after 1 week, 2 weeks and 3 months. To study secretory function, the mouth was rinsed with 250 pl water in every 5 min and protein and amylase concentrations were measured. After an initial 50 min of basal secretion pilocarpine (1 mg/kg, i.p.) was given. Bilateral ablation of both submandibular, sublingual and parotid glands led to a moderate loss of body weight and a considerable increase in water intake. No other obvious abnormality was observed for periods up to 90 days following surgery. We deduce that the minor glands secrete approximately 14 % of protein and 1 % of amylase in whole saliva. Secretion is maintained even after 90 days following removal of the major glands. Surgical removal of the major salivary glands allows the secretory function of the minor glands in rats to be studied in vivo. Key words: minor salivcury glands, ablation, secretion, protein, amylase., rat 1. Introduction
2. Materials and methods
Investigation of minor salivary timction in rodents is technically rather difficult. The presence of minor salivary glands has been described by histochemical, cytochemical and biochemical methods in rats (Green and Embery, 1984; Nicolas and Redman, 1985; Roberts and JatTe, 1986; Nagato et al., 1!997; Hand, this issue). Up till now, however, no attempts have been made to evaluate the secretion of these glands in vivo. Secretion of the major salivary glands can be measured by direct cannulation of then ducts. However, this method cannot be used when studying minor glands, especially in small rodents since the glands are tiny and dispersed throughout the mouth. The vast majority of mixed saliva is derived from the major glands. Therefore, it is not possible to measure minor salivary secretion while the function of the major glands remains intact. For this reason, our aim was to establish whether in vivo excision of the major salivary glands allows the me-ement of minor salivary gland function.
Female Wistar Crl.(Wi)Br Charles River rats (230-280 g) from the breeding colony of Semmelweis University of Medicine were kept in light controlled, air-conditioned rooms and given food and water was given ad libitum. Rats were anaesthetized with sodium pentobarbital (40 mg/kg, i.p.). Through a sagittal neck incision bilateral submandibular and sublingual sialoadenectomy (ablation) was per+ormed together with excision of both parotid glands. Care was taken to preserve both nerves and blood vessels in the neck area during the surgical procedure. One group of rats served as sham-operated controls. After surgery, animals were allocated to one of five groups. Two groups and the sham-operated controls were studied immediately after surgery, while secretory function in the other groups was investigated 1 week, 2 weeks or 3 months later. In chronic experiments body weight gain food intake and water intake was measured for the first three weeks after surgery.
*Correspondiag author. Tel: +36-l-210415
Fax: +36-l-210-4421
0003-9969/99/$-see front matter 0 1999 Published PII: SOOO3-9969(99)00049-7
by Elsevier
Both acute and chronically ablated rats were tracheotomized before starting secretory experiments under pentoScience
Ltd. All rights reserved
S46
.I. Blazsek, G. Varga /Archives
of Oral Biology 44 (1999) S45-S48
barbital anaesthesia. To evaluate secretory function of the minor glands, the mouth was rinsed with 250 pl water/5 mm for 150 mm. Control animals received no stimulation during this period; experimental groups received pilocarpine-HCl (1 mg/kg, i.p:; Sigma, St. Louis, MO) after 50 min. The mouth rinse samples were put into Eppendorf tubes and centrifuged at 5500 g for 10 min to sediment the debris. Protein [Bradford, 197611 and amylase [phadebas test (Ceska et al., 1969)] concentrations were measured in the snpernatant using a Bio-Rad micro-plate reader.
150
im 50 0
1.2 Statistical analysis Values are given as mean + standard deviation. Experiments were performed using 3-5 parallel samples, except in the ablated 90&y group where we had only two animals. Cumulative protein and amylase secretions over 100 min were calculated assuming that all secreted protein and amylase was recovered in the rinsing fluid. This is not a precise measure but, as experimental conditions in all experiments were identical, it allowed us to compare secretory activity between different groups over long time periods. Croups were compared by analysis of variance (ANOVA). Calculations were performed using the InStat program (GraphPad Software, Inc., San Diego, CA). 3. Results No animals died during or after surgical ablation of the submandibular, sublingual and parotid glands and no obvious sign of abnormality was seen up to 90 days after surgery. However, ablation of major glands led to a decrease in body weight during the first three weeks after surgery when compared to sham-operated controls (Fig. 1A). Despite this, weekly food intake either as an absolute value (g) or normal&d to body weight (g/lOOg) was not significantly different (Fig. lB-C). In contrast, water intake increased by 71,72 and 53 %, respectively, during the first, second and third weeks after surgery (Fig. 1D). In part this was due to a change in feeding behaviour of ablated animals: they consumed chow in small portions, with &qmnt interruptions to drink water to help moisten the food. Acute ablation of major salivary glands led to a dramatic decrease in protein and amylase concentration in the mouth with values reaching .new steady-state levels within 30 min after surgery. Protein concentration dropped from around 400 &ml to 100 (y/ml in the rinsing solution (Fig. 2A). The decrease in amylase concentration was even more pronounced (Fig 3A). The basal secretions in ablated animals correspond to outputs of about 300 pg/lOO min protein and 3 U/100 min amylase outputs. Pilocarpine stimulation induced a four-fold increase in protein output (Fig. 2B), and a ten-fold increase in amylase output (Fig. 3B). These values represent 14 % of the protein and 1 % of the amylase secreted by intact rats receiving the same
140 120 100 50 50 40 20 0 1-w
144w 21-w
nwbyfoH
-.w.
7-w
14-wZ~QI
wmiy w&l -.w.
Figure 1. Effect of bilateral removal of submandibular, subliigual and parotid glands m, body weight (A), food intake (IS), food intake ormalizedtobodyweight(C)andwaterintakenamalizedtobody k3ight @) iu fats. Open columns representsham-operatedcontrol,
ffied columns indicate ablated animals, O-day = day of surgery, 7-day, Wday and Zlday are 7, 14, and 21 days after sqeq, respectively.Values are mean fl S.D.; 11=3-l?,wO.05 vs shamcperatedomnoh stimulation. In response to pilocarpine stimulation protein outputs in the sialadenectomized rats were 1.10 * 0.05 mg/lOO min (sham-operated 7.67 f 1.4 mg/lOO mm) and amylase outputs were 25 f 5 U/100 min (sham-operated 2116 f 937 U/l00 mm). In chronically ablated rats, initial concentrations of protein and amylase were lower than in rats immediately after surgical removal of the major salivary glands. Steady levels of protein and amylase concentrations were reached inthethirdorfourth5minrinse.Thesesteadybasal secretory values were not significantly different from those observed after acute extirpation of the major salivary glands (Fig. 2A-3A). Protein and amylase outputs in response to pilocarpme stimulation in 7day and 14day ablated rats were again not signiticantly diffemnt from those observed immediately .after surgery (Fig. 2B and 3B). As there were only two animals in the 9Oday ablated group all we can state is that oral protein and amylase secretion is still maintained three months after extirpation of the major salivary glands (Fig. 2B-3B). 4. Discussion There is a large number of studies involving ablation of submandibular and sublingual glands (Sarosiek J et al., 1988; Konturek et al., 1990; Z&es et al., 1995; Lambotte et al., 1997) because the submandibular gland in rats is
.I. Blazsek, G. Varga /Archives
qf Oral Biology 44 (1999) S4S-S48
s47
.
Figure 2. Basal and pilooarpine-stimulated protein secretion of minor salivary glands fc~llowing a&e or chronic removal of the major salivary glands in rata. (A) Protein conoentration in the 250 ml mouth rinses that were used to recover minor salivary secretion periodically every 5 min. Groups: acute, umtimulated ( 0 ); pilocarpine-stimulation alter aoute ablation ( R ), 7 days ( * X 14 days(+)and90daya(.D)afkr.surgery.(B)Totalproteinoutput in response to pilooarpine stimulation (i.e. 100 min cumulative value). Open column represents unatimulated, acutely ablated rats. Filled oolums show values obtained a&r pilocarpiue stimulation following aoute ablatim, or 7 days or 14 days subsequent to surgery. Closed circles represent individual values of the two animals in which secretion was measured 90 days atIer removal of tbe major salivary glands. Values are mean it S.D.; n=3-5 except for go-day values where II=?, *p
the primary source of epidermal growth factor (EGF), a factor
involved in the maintenance
of mucosal integrity in
the oral cavity and upper gastrointestinal tract. Such studies are made relatively easy by the compact and capsulated nature of the gland. Conversely, rat parotid glands have scattered lobules, making it more difficult to remove them without damaging surrounding tissues. Therefore, there have been very few attempts to eliminate parotid function in rats, and these have made use of duct ligation or denervation (Ekstrom et al., 1988; Schneyer et al., 1991, 1992). In this present study we simultaneously removed bilateral sub.mandibular, sublingual and parotid glands. This led to only a moderate loss of body weight, and induced no other obvious abnormalities. Since food intake did not change considerably, the primaty cause of
Figure 3. Baaal and pilocarpine&imulated amylase oooomtration (A) and output (B) Tom minor salivary glands following aoute or chronic removal of the major salivary glanda in rata. Symbols as in legend to Fk.2.
body weight loss is probably trauma due to surgery. As expected, removal of the major salivary glands considerably increased water intake as the rats learned to moisten food by drinking excess water in substitution for the missing salivary lubrication. Several different methods are available to study secretory function of the minor salivary glands in humans, both in vivo (Ferguson, this issue) and in vitro (Turner, this issue), but very little is known about their function in rats. Our data indicate that their secretory function can be studied in rats in vivo by surgically removing the major glands. Futher, our data show that these glands contribute significantly to salivary function: following pilocarpine stimulation they secrete approximately 14 % of salivary protein and 1 % of salivary amylase. The contribution to whole saliva protein is greater than the contribution to whole saliva amylase in accord with morphological observations that the majority of minor salivary glands are mucous while serous cells represent only a small proportion (Hand, this issue). We have assumed that the protein and amylase recovered by mouthrinsing is secreted mainly by the minor salivary glands and that any surface cells detached by the rinsing procedure and any residual secretory proteins mmaining in
S48
J. Blazsek, G. Varga /Archives
incompletely removed ductal elements of the major glands would not represent a significant contribution to the rinse. In fact, any celhtlar debris was removed by centriftrgation before the assays. Moreover, pilocarpine stimulated both protein and amylase secretion, in a similar manner to its action on the major salivary glands in rats (Schueyer and Hall, 1965) or minor glands in humans (Ferguson, this issue). This suggests that proteins in our samples are derived from sources under complex parasympatheticsympathetic control. The magnitude of increase in amylase concentration, a protein specific to saliva, in response to pilocarpme was similar to that of increase in protein concentration indicating a certain degree of parallelism in the secretion of proteins, as in major salivary glands (Lerner et al., 1991, Cook et al., 1994). Finally, comparing data obtained in acute and chronic experiments, both basal and pilocarpine-stimulated protein secretion were maintained at a similar level even after 90 days of ablation, suggesting a stable and inducible source of proteins in the mouth. Nevertheless,we are now analyzing the protein constituents of our samples to confhm the correctness of our assumptions.. In conclusion, our results suggest that after surgical removal of the major salivary glands the secretory function of the minor glands can be evaluated in vivo in rats. The only changes we observed up to 90 days after ablation of the submandibular, sublingual and parotid glands were a moderate loss of body weight and a considerably increased water intake. Our data indicate that approximately 14% of the protein and 1% of the amylase in whole saliva stimulated by pilocarpine arises from minor glands.
Acknowledgements The authors thank Andrea Csapo, Lilian Dahnodi and Orsolya Szucs for their excellent technical assistance. This work was supported by grants from the Hungarian National Research Fund (T-022401) and from the Research Fund of the Hungarian Ministry for Social Welfare @IT 02 193/96). References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72,248-254. Ceska, M., Brown, B., Biiath, K., 1969. A rapid method for the clinical determination of alpha-amylase activities in human serum and urine. Gptimal conditions. Clin Chim Acta 26,437-444. Cook, D.L, Van Lennep, E.W., Roberts, ML., Young, J.k, 1994. Secretion by the major salivary glands. In: Physiology of the gastrointes&al tract, Third edition @d&d by Johnson L.R et al.) pp. 1061-l 117, Raven Press, New York_ Ekstrom, J., Gamett, J.R, Mansson, B., Tobin, G., 1988. The e&&s of atropine and chronic sympathe&omy on maximal parasympathetic stimulation of parotid saliva. J. Physiol. (London) 403, 105-l 16
of Oral Biology 44 (1999) S~_T~-S@ Konturek, P.K., Brzozowski, T., Konturek, S.J., Dembinski, A., 1990. Role of epidermal growth factor, pnx@landin, and sulfhydryls in stress-induced gastric lesions. Gastmmterology 99, 1607-161 5. Green, D.RJ., Embery, G.. 1984. Incorporation of inorganic [35S]-sulphate iuto glycoproteins of rat buocal and palatal minor salivary glands in vivo and in vitro. Arch. oral Biol. 29,335-341. J_ambotte, L., Slia, A., Triest, S., Maitner, D., Bamnski, A, Barker, A, Li, B., 1997. Effect of sialoadenectomy and epidermal growth factor administration on liver regeneration after partial hepa&&my. Hepatology 25,607-612. Lemer, A, Rosenthal, MA, Liebow, C., L&e&xl, E., 1991. Salivary secretion. In: Textbook of Gastroenterology (Edited by Yamada T. et al.) pp.218-233, J.B. Lippincott Company, PhiladelPhia. Nagato, T., Ren, X.Z., Toh, H., Tandler, B. (1997) Ultmshucture of Weber’s salivary glands of the root of the tongue in the fat (1997) Anat. Rec. 249,435-440. Nicolas, S., Redman RS.. 1985. Uhrastmcture of the anterior buccal gland ofthe rat. Anat. Rec. 213, 140-149. Roberts, LM., Jaffe, R (1986) LinSual lipase: immuno@xhemical localiion in the rat von Ebner gland Gastroenterology 90, 1170-l 175. Ssrosiek, J, Bilski, J., Mmty, V.L., Slomiany, A, Slomiany, B.L., 1988. Role of salivary epidennal growth factor in the maintenauce of physicochemical characteristics of oral and gastric mucosal mucus coat. Biochem Biophys. Res. Commun. 152, 1421-1427. Schneyer, CA, Hall, H.D. (1965) Comparison of rat salivas by auriculo-temporal and pilocarpine stimulation. Am. J. Physiol. 209, 484488. Schneyer, CA, Humphreys-Beher, M.G., Hall, H.D., 1991. Inhibition of activity-induced parotid growth by submaadiiular gland ablation or autonomic denervation. Am. J. Physiol. 261, G723G727. Schueyer, CA, Hall, H.D., Humphreys-Beher, M.G., 1992. Effects of nerve growth factor on cyclocytidine-induced gowth responses of sympathecomized parotid or parotid of partially desalivated rat. J Auton Nerv Syst 38,29-35. Zelles, T., Purushotham, K.R., Macauley, S.P., Gxfbrd G.E., Humphreys-Beher, M.G., 1995. Saliva and growth factorx the fountain of youth resides in us all. J. Dent. Res. 74, 18263182.
PERGAMON
Archives
of Oral Biology
44 (1999)
S45-S48
Secretion Corn minor salivary glands following ablation of the major salivary glands in rats J. Blazsek’l, of Oral Biology, lDepartment
G. Varga2
Semmelweis University of Medicine; *Ir&ute bpesf
of Experimental Medicine, Hungarian
Academy of Sciences;Bu-
HQWY
Since minorsalivary glands are tiny and dispersed, ductal cammlation cannot be used when studying their function. The present study was devised to develop a method of measuring minor salivary gland function by excision of the major glands. Female rats (230-280 g) were anaesthetized with sodium pentobarbital. Ablation of the submandibular, sublingual and parotid glands was performed through a sag&al neck incision. Sham-operated rats served as controls. Croups of sialadenectomized animals were investigated immediately and after 1 week, 2 weeks and 3 months. To study secretory function, the mouth was rinsed with 250 pl water in every 5 min and protein and amylase concentrations were measured. After an initial 50 min of basal secretion pilocarpine (1 mg/kg, i.p.) was given. Bilateral ablation of both submandibular, sublingual and parotid glands led to a moderate loss of body weight and a considerable increase in water intake. No other obvious abnormality was observed for periods up to 90 days following surgery. We deduce that the minor glands secrete approximately 14 % of protein and 1 % of amylase in whole saliva. Secretion is maintained even after 90 days following removal of the major glands. Surgical removal of the major salivary glands allows the secretory function of the minor glands in rats to be studied in vivo. Key words: minor salivcury glands, ablation, secretion, protein, amylase., rat 1. Introduction
2. Materials and methods
Investigation of minor salivary timction in rodents is technically rather difficult. The presence of minor salivary glands has been described by histochemical, cytochemical and biochemical methods in rats (Green and Embery, 1984; Nicolas and Redman, 1985; Roberts and JatTe, 1986; Nagato et al., 1!997; Hand, this issue). Up till now, however, no attempts have been made to evaluate the secretion of these glands in vivo. Secretion of the major salivary glands can be measured by direct cannulation of then ducts. However, this method cannot be used when studying minor glands, especially in small rodents since the glands are tiny and dispersed throughout the mouth. The vast majority of mixed saliva is derived from the major glands. Therefore, it is not possible to measure minor salivary secretion while the function of the major glands remains intact. For this reason, our aim was to establish whether in vivo excision of the major salivary glands allows the me-ement of minor salivary gland function.
Female Wistar Crl.(Wi)Br Charles River rats (230-280 g) from the breeding colony of Semmelweis University of Medicine were kept in light controlled, air-conditioned rooms and given food and water was given ad libitum. Rats were anaesthetized with sodium pentobarbital (40 mg/kg, i.p.). Through a sagittal neck incision bilateral submandibular and sublingual sialoadenectomy (ablation) was per+ormed together with excision of both parotid glands. Care was taken to preserve both nerves and blood vessels in the neck area during the surgical procedure. One group of rats served as sham-operated controls. After surgery, animals were allocated to one of five groups. Two groups and the sham-operated controls were studied immediately after surgery, while secretory function in the other groups was investigated 1 week, 2 weeks or 3 months later. In chronic experiments body weight gain food intake and water intake was measured for the first three weeks after surgery.
*Correspondiag author. Tel: +36-l-210415
Fax: +36-l-210-4421
0003-9969/99/$-see front matter 0 1999 Published PII: SOOO3-9969(99)00049-7
by Elsevier
Both acute and chronically ablated rats were tracheotomized before starting secretory experiments under pentoScience
Ltd. All rights reserved
S46
.I. Blazsek, G. Varga /Archives
of Oral Biology 44 (1999) S45-S48
barbital anaesthesia. To evaluate secretory function of the minor glands, the mouth was rinsed with 250 pl water/5 mm for 150 mm. Control animals received no stimulation during this period; experimental groups received pilocarpine-HCl (1 mg/kg, i.p:; Sigma, St. Louis, MO) after 50 min. The mouth rinse samples were put into Eppendorf tubes and centrifuged at 5500 g for 10 min to sediment the debris. Protein [Bradford, 197611 and amylase [phadebas test (Ceska et al., 1969)] concentrations were measured in the snpernatant using a Bio-Rad micro-plate reader.
150
im 50 0
1.2 Statistical analysis Values are given as mean + standard deviation. Experiments were performed using 3-5 parallel samples, except in the ablated 90&y group where we had only two animals. Cumulative protein and amylase secretions over 100 min were calculated assuming that all secreted protein and amylase was recovered in the rinsing fluid. This is not a precise measure but, as experimental conditions in all experiments were identical, it allowed us to compare secretory activity between different groups over long time periods. Croups were compared by analysis of variance (ANOVA). Calculations were performed using the InStat program (GraphPad Software, Inc., San Diego, CA). 3. Results No animals died during or after surgical ablation of the submandibular, sublingual and parotid glands and no obvious sign of abnormality was seen up to 90 days after surgery. However, ablation of major glands led to a decrease in body weight during the first three weeks after surgery when compared to sham-operated controls (Fig. 1A). Despite this, weekly food intake either as an absolute value (g) or normal&d to body weight (g/lOOg) was not significantly different (Fig. lB-C). In contrast, water intake increased by 71,72 and 53 %, respectively, during the first, second and third weeks after surgery (Fig. 1D). In part this was due to a change in feeding behaviour of ablated animals: they consumed chow in small portions, with &qmnt interruptions to drink water to help moisten the food. Acute ablation of major salivary glands led to a dramatic decrease in protein and amylase concentration in the mouth with values reaching .new steady-state levels within 30 min after surgery. Protein concentration dropped from around 400 &ml to 100 (y/ml in the rinsing solution (Fig. 2A). The decrease in amylase concentration was even more pronounced (Fig 3A). The basal secretions in ablated animals correspond to outputs of about 300 pg/lOO min protein and 3 U/100 min amylase outputs. Pilocarpine stimulation induced a four-fold increase in protein output (Fig. 2B), and a ten-fold increase in amylase output (Fig. 3B). These values represent 14 % of the protein and 1 % of the amylase secreted by intact rats receiving the same
140 120 100 50 50 40 20 0 1-w
144w 21-w
nwbyfoH
-.w.
7-w
14-wZ~QI
wmiy w&l -.w.
Figure 1. Effect of bilateral removal of submandibular, subliigual and parotid glands m, body weight (A), food intake (IS), food intake ormalizedtobodyweight(C)andwaterintakenamalizedtobody k3ight @) iu fats. Open columns representsham-operatedcontrol,
ffied columns indicate ablated animals, O-day = day of surgery, 7-day, Wday and Zlday are 7, 14, and 21 days after sqeq, respectively.Values are mean fl S.D.; 11=3-l?,wO.05 vs shamcperatedomnoh stimulation. In response to pilocarpine stimulation protein outputs in the sialadenectomized rats were 1.10 * 0.05 mg/lOO min (sham-operated 7.67 f 1.4 mg/lOO mm) and amylase outputs were 25 f 5 U/100 min (sham-operated 2116 f 937 U/l00 mm). In chronically ablated rats, initial concentrations of protein and amylase were lower than in rats immediately after surgical removal of the major salivary glands. Steady levels of protein and amylase concentrations were reached inthethirdorfourth5minrinse.Thesesteadybasal secretory values were not significantly different from those observed after acute extirpation of the major salivary glands (Fig. 2A-3A). Protein and amylase outputs in response to pilocarpme stimulation in 7day and 14day ablated rats were again not signiticantly diffemnt from those observed immediately .after surgery (Fig. 2B and 3B). As there were only two animals in the 9Oday ablated group all we can state is that oral protein and amylase secretion is still maintained three months after extirpation of the major salivary glands (Fig. 2B-3B). 4. Discussion There is a large number of studies involving ablation of submandibular and sublingual glands (Sarosiek J et al., 1988; Konturek et al., 1990; Z&es et al., 1995; Lambotte et al., 1997) because the submandibular gland in rats is
.I. Blazsek, G. Varga /Archives
qf Oral Biology 44 (1999) S4S-S48
s47
.
Figure 2. Basal and pilooarpine-stimulated protein secretion of minor salivary glands fc~llowing a&e or chronic removal of the major salivary glands in rata. (A) Protein conoentration in the 250 ml mouth rinses that were used to recover minor salivary secretion periodically every 5 min. Groups: acute, umtimulated ( 0 ); pilocarpine-stimulation alter aoute ablation ( R ), 7 days ( * X 14 days(+)and90daya(.D)afkr.surgery.(B)Totalproteinoutput in response to pilooarpine stimulation (i.e. 100 min cumulative value). Open column represents unatimulated, acutely ablated rats. Filled oolums show values obtained a&r pilocarpiue stimulation following aoute ablatim, or 7 days or 14 days subsequent to surgery. Closed circles represent individual values of the two animals in which secretion was measured 90 days atIer removal of tbe major salivary glands. Values are mean it S.D.; n=3-5 except for go-day values where II=?, *p
the primary source of epidermal growth factor (EGF), a factor
involved in the maintenance
of mucosal integrity in
the oral cavity and upper gastrointestinal tract. Such studies are made relatively easy by the compact and capsulated nature of the gland. Conversely, rat parotid glands have scattered lobules, making it more difficult to remove them without damaging surrounding tissues. Therefore, there have been very few attempts to eliminate parotid function in rats, and these have made use of duct ligation or denervation (Ekstrom et al., 1988; Schneyer et al., 1991, 1992). In this present study we simultaneously removed bilateral sub.mandibular, sublingual and parotid glands. This led to only a moderate loss of body weight, and induced no other obvious abnormalities. Since food intake did not change considerably, the primaty cause of
Figure 3. Baaal and pilocarpine&imulated amylase oooomtration (A) and output (B) Tom minor salivary glands following aoute or chronic removal of the major salivary glanda in rata. Symbols as in legend to Fk.2.
body weight loss is probably trauma due to surgery. As expected, removal of the major salivary glands considerably increased water intake as the rats learned to moisten food by drinking excess water in substitution for the missing salivary lubrication. Several different methods are available to study secretory function of the minor salivary glands in humans, both in vivo (Ferguson, this issue) and in vitro (Turner, this issue), but very little is known about their function in rats. Our data indicate that their secretory function can be studied in rats in vivo by surgically removing the major glands. Futher, our data show that these glands contribute significantly to salivary function: following pilocarpine stimulation they secrete approximately 14 % of salivary protein and 1 % of salivary amylase. The contribution to whole saliva protein is greater than the contribution to whole saliva amylase in accord with morphological observations that the majority of minor salivary glands are mucous while serous cells represent only a small proportion (Hand, this issue). We have assumed that the protein and amylase recovered by mouthrinsing is secreted mainly by the minor salivary glands and that any surface cells detached by the rinsing procedure and any residual secretory proteins mmaining in
S48
J. Blazsek, G. Varga /Archives
incompletely removed ductal elements of the major glands would not represent a significant contribution to the rinse. In fact, any celhtlar debris was removed by centriftrgation before the assays. Moreover, pilocarpine stimulated both protein and amylase secretion, in a similar manner to its action on the major salivary glands in rats (Schueyer and Hall, 1965) or minor glands in humans (Ferguson, this issue). This suggests that proteins in our samples are derived from sources under complex parasympatheticsympathetic control. The magnitude of increase in amylase concentration, a protein specific to saliva, in response to pilocarpme was similar to that of increase in protein concentration indicating a certain degree of parallelism in the secretion of proteins, as in major salivary glands (Lerner et al., 1991, Cook et al., 1994). Finally, comparing data obtained in acute and chronic experiments, both basal and pilocarpine-stimulated protein secretion were maintained at a similar level even after 90 days of ablation, suggesting a stable and inducible source of proteins in the mouth. Nevertheless,we are now analyzing the protein constituents of our samples to confhm the correctness of our assumptions.. In conclusion, our results suggest that after surgical removal of the major salivary glands the secretory function of the minor glands can be evaluated in vivo in rats. The only changes we observed up to 90 days after ablation of the submandibular, sublingual and parotid glands were a moderate loss of body weight and a considerably increased water intake. Our data indicate that approximately 14% of the protein and 1% of the amylase in whole saliva stimulated by pilocarpine arises from minor glands.
Acknowledgements The authors thank Andrea Csapo, Lilian Dahnodi and Orsolya Szucs for their excellent technical assistance. This work was supported by grants from the Hungarian National Research Fund (T-022401) and from the Research Fund of the Hungarian Ministry for Social Welfare @IT 02 193/96). References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72,248-254. Ceska, M., Brown, B., Biiath, K., 1969. A rapid method for the clinical determination of alpha-amylase activities in human serum and urine. Gptimal conditions. Clin Chim Acta 26,437-444. Cook, D.L, Van Lennep, E.W., Roberts, ML., Young, J.k, 1994. Secretion by the major salivary glands. In: Physiology of the gastrointes&al tract, Third edition @d&d by Johnson L.R et al.) pp. 1061-l 117, Raven Press, New York_ Ekstrom, J., Gamett, J.R, Mansson, B., Tobin, G., 1988. The e&&s of atropine and chronic sympathe&omy on maximal parasympathetic stimulation of parotid saliva. J. Physiol. (London) 403, 105-l 16
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