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1 J. Phyil. (1984), 351, pp With 5 text-figures Printed in Great Britain EFFECT OF SIALOADENECTOMY AND SALIVARY GLAND EXTRACTS ON GASTROINTESTINAL MUCOSAL GROWTH AND GASTRIN LEVELS IN THE RAT BY K. A. SKINNER, B. D. SOPER AND B. L. TEPPERMAN From the Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5C1 (Received 18 August 1983) SUMMARY 1. We have examined gastrointestinal mucosal growth 30 days after surgical removal of the submandibular-sublingual salivary glands and ligation of the parotid ducts of rats. The rate of [3H]thymidine uptake in vitro as an estimation of DNA synthesis and the content of DNA and RNA were examined in the oxyntic, duodenal and proximal colonic mucosa. 2. DNA synthesis, DNA and RNA content of oxyntic mucosa were reduced in sialoadenectomized rats when compared to sham-sialoadenectomized control animals. There was no change in the degree of [3H]thymidine incorporation or DNA content of the duodenal or colonic mucosa. 3. Intraperitoneal injection ofan aqueous extract ofthe submandibular-sublingual salivary glands of rats (4-0 mg tissue protein in 0-1 M-sodium phosphate buffer administered twice a day for 15 days) increased the rate of DNA synthesis and the total mucosal DNA and RNA content in the oxyntic mucosa. Injections of extracts of spleen or muscle did not produce consistent results. 4. Administration of epidermal growth factor (10 jug/kg) or pentagastrin (250 psg/kg) resulted in an increase of the level of DNA synthesis observed in the oxyntic mucosa of sialoadenectomized rats. 5. Plasma and antral tissue levels of the trophic hormone, gastrin, were not significantly decreased in sialoadenectomized rats treated with 041 M-sodium phosphate-buffered saline. However, treatment with the salivary tissue extract did result in significant reductions in both plasma and tissue levels of gastrin. 6. We conclude that elimination of the major salivary glands in the rat results in a decrease in [3H]thymidine uptake and DNA content of the gastric oxyntic mucosa. These effects are not mediated via a reduction in endogenous levels of the trophic hormone, gastrin. Administration of an aqueous extract of salivary tissue exerted a small but significant trophic influence on the oxyntic mucosa of the rat. INTRODUCTION Stimulation of salivation and systemic administration of some peptides extracted from saliva or salivary gland tissue have been shown to exert trophic effects on the 1 PHY 351

2 2 K. A. SKINNER AND OTHERS mucosa of the gastrointestinal tract. Exogenous administration of epidermal growth factor (EGF), a polypeptide extracted from rodent salivary glands, has been shown to produce keratinization of gastric mucosa, with a thicker cellular nucleated epithelial layer and an increase in the mitotic index in neonatal mouse forestomach tissue (Cohen & Elliot, 1963; Steidler & Houghton, 1980). Studies by Feldman, Aures & Grossman (1978) and Johnson & Guthrie (1980) demonstrated that exogenous administration of EGF stimulated ornithine decarboxylase activity, and increased the content of DNA and RNA and the rate of DNA synthesis in rat oxyntic mucosa. EGF also appears to stimulate DNA synthesis or mitosis in rodent intestinal mucosa (Al-Nafussi & Wright, 1982; Chabot & Hugon, 1980). Removal of the salivary glands in rodents has been shown to result in a marked decline in the degree and rate of whole body growth (Narashiman & Gangla, 1966) and a reduction in the weight of the digestive tract (Bueker & Schenkein, 1964). Sialoadenectomy has also been associated with a reduction in the metabolic activity and growth of a variety of tissues including adrenals, testes, brain (Narashiman & Gangla, 1966; Velasco-Plaza, Menendez-Patterson & Marin, 1979), thymus and spleen (Martinez-Hernandez, Nakane & Pierce, 1973). Recently, it has been shown (Li, Schattenkerk, Huffman, Ross & Malt, 1983) that increased secretion of submandibular saliva in the mouse promoted proliferation of the intestinal mucosa. Stimulants of salivary secretion did not increase intestinal growth when administered to sialoadenectomized animals. In the present study we have examined the in vitro incorporation of [3H]thymidine, as well as the DNA and RNA content in the oxyntic, duodenal and colonic mucosa of the rat 30 days after surgical removal of the submaxillary and sublingual salivary glands and ligation of the parotid ducts. We have also studied the ability of a parenterally administered aqueous extract of submandibular and sublingual glands of Sprague-Dawley rats to influence mucosal growth. The effects of sialoadenectomy and the salivary extract on plasma and tissue levels of the trophic antral hormone, gastrin, have been investigated as a possible mode of influence of the salivary glands on gastrointestinal mucosal growth. METHODS Animal preparation. Male Sprague-Dawley rats ( g) were used in these studies. Under pentobarbitone anaesthesia (50 mg/kg I.P.) the submandibular-sublingual gland complexes were removed bilaterally after their ducts were ligated. These glands were rinsed in ice-cold saline and stored at -20 C until used for the preparation of the salivary gland extract. The parotid gland ducts were ligated bilaterally and severed. This process has been shown to result in degranulation of both acinar and ductal cells within 1 week of ductal ligation and total atrophy of the parotid tissue within 3 weeks of the time of surgery (Tamarin, 1967). At the same time a group of rats was sham sialoadenectomized by exposure of the glands and ducts to serve as controls in these studies. Animals were maintained in a room at 22 C under constant day-night rhythm and given food and tap water ad libitum. The weight of each animal was recorded daily. The rats were killed 30 days after surgery. The rate of DNA synthesis was estimated by the in vitro incorporation of [3H]thymidine. DNA synthesis and mucosal DNA and RNA levels were measured in the oxyntic portion of the stomach, the duodenum and colon. Estimation of [8H]thymidine incorporation and muco8al DNA and RNA content. In the first study, the stomach, duodenum and proximal colon were removed after the animals were killed by cervical fracture. The mucosa was scraped quickly from the oxyntic gland area of the stomach, the duodenum and the proximal colon and weighed.

3 SIALOADENECTOMY AND THE GUT The rate of DNA synthesis in the scraped mucosal samples was measured by incubating the tissue at 37 0 for 30 min in medium 199 (Gibco Laboratories, Grand Island, NY, U.S.A.) containing 2 #Ci/ml of ['H]thymidine (5 Ci/mmol, Amersham, Canada). The flasks were continuously gassed with 95% O,-5 % CO during the incubation. The reaction was stopped with 0-4 N-perchloric acid containing carrier thymidine at a 5 mm concentration. Samples were washed with 0-2 N-perchloric acid and RNA was removed by hydrolysing in 0-3 N-KOH for 90 min at 37 0C. After standing on ice for 10 min the tubes were centrifuged and the RNA content of the supernatant was determined using the orcinol reaction (Ceriotti, 1955). The DNA in the residual pellet was solubilized by added 10% perchloric acid and heated to 700C for 20 min. The solution was centrifuged at 500 g for 10 min. The DNA content of the supernatant was determined by the diphenylamine method described by Giles & Myers (1965) using 2-deoxy-D-ribose (Sigma, St Louis) as a standard. The DNA and RNA content of the tissue were expressed as micrograms per 100 milligrams of the wet weight of mucosa. The incorporation of [3H]thymidine into DNA was determined by adding 1 ml of supernatant to 10 ml of scintillation fluid (ACS, Amersham, Canada) and counting for 10 min in an LKB Rackbeta scintillation counter. DNA synthesis was expressed as disintegrations per minute of [3H]thymidine incorporated per milligram of tissue wet weight. Preparation and injection of salivary gland tissue extract. The extract was prepared by a modification of the procedure described by Martinez-Hernandez et al. (1973). The glands which had been stored at -20 'C were thawed, adherent adipose tissue was dissected away and the remaining salivary tissue was weighed. The glands were minced with scissors and suspended in an iced phosphate-buffered saline solution consisting of 0'1 M-sodium phosphate buffer in 0-15 M-NaCl. Soybean trypsin inhibitor (Sigma, St Louis; 0-3 ml of a 0-2 % solution per gram wet weight of tissue) was added to the buffer to reduce proteolytic activity. The homogenizing buffer was adjusted to 8 ml/g tissue. After emulsification for 2 min in a high-speed VirTis blender (VirTis, Gardiner, New York), the tissue was reblended with twenty complete strokes in a Broeck homogenizer. The suspension was centrifuged at g for 30 min at 40C. The supernatant was collected. The pellet was resuspended in phosphate-buffered saline and centrifuged again at g for 30 min. After centrifugation the first and second supernatants were pooled and used as the salivary gland extract. The protein concentration of final extract was measured by the method of Bradford (1976). As a control for these studies, extracts of rat spleen and skeletal muscle were prepared in an identical manner to that described for salivary tissue. Each of the extracts was administered to sialoadenectomized rats by intraperitoneal injection for a period of 15 days. Injections were commenced 15 days after the surgery for removal of the salivary glands and ligation of the parotid ducts and were given twice a day at 12 h intervals. The amount of extract given was equivalent to 4 mg of extract protein per injection. The volume of each injection was 0 4 ml. This dose of salivary gland extract was found to depress secretagoguestimulated acid output by approximately 30% and to increase the integrity of the rat gastric mucosa to pharmacological damage (Tepperman & Soper, 1983). As a control, comparable volumes of 0-1 M-phosphate-buffered saline were administered by a similar regimen to sialoadenectomized animals. The weight of each animal was recorded daily. At the end of the 15-day treatment period, animals were killed by cervical fracture, the stomach, duodenum and colon were removed, the mucosa was scraped and the rate of in vitro [3H]thymidine incorporation and the mucosal DNA and RNA content were determined as previously described. Injection of EGF and pentagastrin. In a separate group of sialoadenectomized rats, EGF (10,ug/kg culture grade; a gift from Dr H. Gregory, ICI Ltd., Macclesfield), or pentagastrin (250,ug/kg; Ayerst, Montreal) were administered by intraperitoneal injection. Injections followed the same regimen as described above for tissue extracts. The doses of EGF and pentagastrin had previously been shown to stimulate growth in rat gastric mucosa (Johnson & Guthrie, 1980). lastrin determinations in plasma and antral tissue. At the end of the experimental period, rats were stunned by a blow to the head and blood was withdrawn by cardiac puncture. Approximately 3 ml of blood was mixed with 10 u. of sodium heparin, centrifuged at low speed and the plasma collected. Extraction of gastrin from antral tissue was performed as previously described by Lichtenberger, Welsh & Johnson (1976). The antrum was removed, trimmed and weighed. The tissue was homogenized in 1 ml of ice-cold de-ionized water for 30 s using a Broeck homogenizer. An aliquot of the homogenate mixture (0-5 ml) was added to 2 ml of boiling de-ionized water and the extract was boiled for 20 min. The sample was then centrifuged at g for 30 min and the gastrin 1-2 3

4 4 K. A. SKINNER AND OTHERS concentration in the supernatant determined. Both the plasma samples and the tissue extract were stored at -20 'C. Gastrin concentration in both plasma and tissue extracts was determined radio-immunochemically as described by Stadil & Rehfeld (1973), with the exception that the charcoal dextran method was used to separate bound and free gastrin. Synthetic human gastrin was used as a standard and for the preparation of '261-labelled gastrin (Stadil & Rehfeld, 1972). The antiserum used was number 2604 (a generous gift to Dr T. J. MacDonald from Professor J. Rehfeld). The characteristics of this antibody have been described previously (Rehfeld, Stadil & Rubin, 1972). Plasma gastrin concentration was expressed as femtomoles per millilitre and antral gastrin was expressed as picomoles per 100 milligrams of tissue wet weight. Radioreceptor a88ay for EGF-like activity. Determination of EGF activity in extracts of salivary glands, spleen and skeletal muscle was done using a radioreceptor assay described by Imai, Tsushima, Sasaki & Matsuzaki (1979). The assay is based on the finding that mouse liver is a rich source of specific EGF binding sites. The livers of male mice were homogenized in 10 vol. of ice-cold 50 mm-tris HCl (ph 7 4) containing 10 mm-mgcl2. 6H20. The homogenate was centrifuged at g for 20 min and the precipitate was resuspended in an equal volume of Tris buffer containing 01 % bovine serum albumin (BSA) labelled EGF (Collaborative Research; 150 psci/g) was incubated with 200 g tissue homogenate protein. The total volume in each assay tube was 2-0 ml. The assays were conducted at 22 'C for 120 min. The final concentration of 126I-labelled EGF in each tube was 1-2 x M. A displacement curve was generated by simultaneous incubation with serial dilutions ofunlabelled EGF (receptorgrade; Collaborative Research) in the concentration range ng/ml. In addition, the displacement activity of mouse EGF (culture grade) and pentagastrin were also investigated. These peptides were added in the concentration range ng/ml. Competition in the binding of 1251-labelled EGF to the mouse liver particulate fraction by serial dilutions of extracts of salivary tissue, spleen and muscle was also investigated. The approximate amount of EGF-like activity in the tissue extracts was determined from the standard curve of 125I-labelled EGF displacement by receptor grade EGF. Results were calculated using a least-squares fit to a log-logit plot. Analy8i8 of data. All data are represented as means and S.E. of means. Means were compared using Student's t test for unpaired data or a many-sample, one-way analysis of variance and Duncan's multiple range test. The differences between means were considered significant at P < RESULTS Post-mortem inspection 30 days after ligation of the parotid gland ducts did not reveal the presence of any remaining parotid salivary tissue in sialoadenectomized rats. Excision of the submandibular-sublingual gland complexes and ligation of the parotid ducts resulted in a transient but significant decrease of g in mean body weight of rats treated with phosphate-buffered saline. The decrease in body weight generally occurred within the first two post-operative days. After the initial period of weight loss, the sialoadenectomized rats injected with phosphate buffer increased in weight by g over the remainder of the post-operative period with a mean weight increase of g/day. The weight of sham-operated rats increased by g in the 30-day post-operative period, with a mean increase of g/day. After the initial period of weight loss the rates of weight gain between sham-operated animals and sialoadenectomized rats were not significantly different. Intraperitoneal injections of the extracts of salivary glands, spleen or skeletal muscle did not change the course of weight gain as observed in the phosphatebuffer-treated sialoadenectomized group of rats. Furthermore, injection of EGF or pentagastrin did not significantly influence the increase in body weight over the 30-day post-operative period.

5 SIALOADENECTOMY AND THE GUT Thirty days after surgery the in vitro incorporation of [3H]thymidine by the oxyntic mucosa of sialoadenectomized animals was decreased by approximately 40 % (P < 0-05) when compared to sham-operated control rats (Fig. 1). Administration of salivary gland extract resulted in statistically lower levels of DNA synthesis from the levels observed in the phosphate-buffer-treated group ofsham-sialoadenectomized animals. However, the rate of [3H]thymidine incorporation in the salivary extract 5 'A Oxyntic gland -E 40 ** M nz4u h i~ 400 T a)." 300 T <0CD z E 200 cr8 I 100 0). A.Tt. 4sbS' CO 4' 4(b c? 4 4JZ Fig. 1. Mean (+S.E. of mean) in vitro incorporation of [3H]thymidine, DNA and RNA content of the mucosa of the oxyntic gland of the stomach in sialoadenectomized animals (salx.) treated with either salivary tissue extract (SGE, n = 14) or phosphate-buffered saline (PBS, n = 15) and sham-sialoadenectomized rats (n = 11). * indicates significant differences (P < 0-05) from the sham-operated group by analysis of variance and Duncan's multiple range test. ** indicates a significant increase in DNA levels from the sham control group. t indicates a significant increase from the salx. control group. group was greater than that observed in sialoadenectomized rats treated with phosphate buffer. Similarly, treatment with spleen or muscle extracts did not restore the rate of [3H]thymidine incorporation to that observed in sham-sialoadenectomized rats. Salivary gland extract treatment did result in a slightly greater incorporation of [3H]thymidine into oxyntic mucosa than was observed with spleen or muscle extracts. Similarly, the amount of DNA in the mucosa of the oxyntic gland area was significantly decreased in the buffer-treated sialoadenectomized group of animals when compared to sham-operated rats. Administration of the salivary gland tissue I I

6 6 K. A. SKINNER AND OTHERS extract increased gastric DNA and RNA content to a level which was significantly different from those observed in sialoadenectomized rats treated with phosphatebuffered saline. Administration of spleen or muscle resulted in a greater oxyntic gland DNA concentration than was evident in sialoadenectomized rats treated with buffered saline. However, neither treatment produced a similar increase in tissue RNA levels. In duodenal and colonic mucosa neither sialoadenectomy nor administration of salivary gland extract to sialoadenectomized rats influenced the rate of [3H]thymidine incorporation, or DNA and RNA content (Fig. 2). Duodenum 0), X 40 Z-' 30 T C 20 Colon CL 10 0).~120 E80, 0) 30 -;500 *2 400 ze? <300T Cc 200 ~z100 3L0 Fig. 2. Mean (+.E. of mean) in vitro incorporation of [3H]thymidine, DNA and RNA content of the mucosa of the duodenum and proximal colon in sialoadenectomized rats (salx.) treated with either salivary tissue extract (SGE, n = 14) or phosphate-buffered saline (PBS, n = 15) and sham-sialoadenectomized rats (n = 11). Administration of culture grade EGF to sialoadenectomized rats resulted in an increase in the rate of DNA synthesis and DNA content in the oxyntic mucosa (Fig. 3). The level of RNA in the gastric mucosa, while greater than that in sialoadenectomized rats treated with buffered saline, was not significantly different. EGF treatment had a negligible effect on the duodenal and colonic mucosa, with only a significant increase in the duodenal content of DNA. The sialoadenectomized group ofrats treated with pentagastrin displayed a significant increase only in [3H]thymidine incorporation into oxyntic mucosa. Pentagastrin treatment did produce a slight increase in the DNA and RNA levels of oxyntic mucosa, but these increases were not statistically significant. There was no significant influence exerted on duodenal or colonic mucosa by pentagastrin treatment.

7 SIALOADENECTOMY AND THE GUT Oxyntic gland Duodenu m F 1' Colion 7 0X a) O A cm a) Co EL-0 ze EE C -6 cr e cn 0, tap xq 4( x 0 N+b ELLI t a I txt Sae t 4xi- I C qpe %+ C" 41 C+ Fig. 3. Mean ( +s.e. of mean) in vitro incorporation of [3H]thymidine and the content of DNA and RNA in the oxyntic gland of the stomach, duodenum and proximal colon in sialoadenectomized rats (salx.) treated with either 10,ug/kg epidermal growth factor (EGF, n = 7), 250 ug/kg pentagastrin (PG, n = 7) or phosphate-buffered saline (PBS, n = 15). * indicates significant differences (P < 0 05) from the PBS group by analysis of variance and Duncan's multiple range test., E E x 'a 1U.0 (D w 'a Spleen Muscle SGE Concentration of peptide/tube I II~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 0 10 x 10lx 102x 103x 104x 105x 106X Dilution factor of tissue extracts Fig. 4. Competition in binding of '251-labelled EGF to the mouse liver particulate fraction by receptor grade EGF (RG-EGF), culture grade EGF (CG-EGF), salivary gland extract (SGE), pentagastrin, spleen extract and skeletal muscle extract. Each point is the mean (±+.E. of mean) of four determinations.

8 8 K. A. SKINNER AND OTHERS 1251-labelled EGF bound specifically to a g homogenate preparation of male mouse liver. Addition of increasing amounts of a receptor-grade quality EGF preparation resulted in good displacement in the range between 0-25 and 2500 ng (Fig. 4). A less pure preparation of EGF (culture grade) was a less potent inhibitor of 1251-labelled EGF binding. Increasing the amount of salivary gland extract in the incubation medium also produced a dose-dependent inhibition of 1251-labelled EGF binding. Extracts of spleen and muscle and increasing amounts of pentagastrin did not displace labelled EGF bound to the liver particulate fraction. E (50 E C: n G) E a. co Fig. 5. Mean (+ s.e. of mean) plasma and antral mucosal levels of gastrin of sialoadenectomized rats (salx.) treated with either salivary tissue extract (SGE, 0) or phosphatebuffered saline (PBS, *) or sham-sialoadenectomized rats (El). The numbers of animals in each group are: sham, antrum n = 12, plasma n = 11; salx.+sge, antrum n = 13, plasma n = 12; salx. + PBS, antrum n = 14, plasma n = 15. * indicates P < 005 compared to the sham control group by analysis of variance and Duncan's multiple range test. Analysis of the standard curve of 1251-labelled EGF displacement by receptor-grade quality EGF was used to determine the EGF-like activity in the dose of salivary gland extract administered to each rat (1-4 jug/day). Sialoadenectomy resulted in a small, statistically insignificant reduction in both plasma and antral mucosal levels of gastrin. However, administration of salivary tissue extract resulted in a further and significant reduction in plasma and tissue gastrin levels (Fig. 5). DISCUSSION The methodology employed in this study has been used widely for the measurement of cellular multiplication in the gastrointestinal tract, and the reliability of the assays

9 SIALOADENECTOMY AND THE GUT 9 has been tested extensively (Willems, Van Steenkiste & Limbosch, 1972; Johnson & Guthrie, 1974). [3H]thymidine has been observed to be selectively incorporated into DNA during the pre-mitotic DNA synthesis phase (Amano, Messier & Leblond, 1959). Measurement of tissue DNA content in conjunction with the determination of tissue DNA synthesis establishes whether increases in synthesis are matched by increases in cell turnover. However, it has been acknowledged that the method used in the present study to assess DNA synthesis has some limitations (Maurer, 1981). Specifically it has been pointed out that the use of thymidine incorporation to assess the degree of cellular proliferation depends upon a variety of factors including changes in lymphoid tissue and bacterial flora, and variations in the size of the endogenous thymidine pool. There is no evidence to suggest that any of the above-mentioned parameters are altered by removal of the salivary glands in the present investigation. In the present study it was not possible to distinguish between bound thymidine and thymidine incorporated into DNA. However, in these experiments a decrease in [3H]thymidine incorporation into rat oxyntic mucosa in vitro was associated with a decrease in tissue DNA content. In other studies stimulation of DNA synthesis as measured by thymidine incorporation was followed by an increase in cell division and increased DNA content (Willems et al. 1972; Lichtenberger, Miller, Erwin & Johnson, 1973). Therefore it appears that the methods used in this study allow at least qualitative assessment of changes in DNA synthesis and growth in gastrointestinal mucosa. The results reported here demonstrate that surgical removal of the submandibular and sublingual salivary glands and atrophy of the parotid tissue by ductal ligation were associated with a decline in the in vitro rate of [3H]thymidine incorporation into the oxyntic mucosa of mature Sprague-Dawley rats. These data confirm and extend the results of previous studies. Sialoadenectomy in rats was followed by atrophy of a number of tissues including the gastrointestinal tract (Bueker & Schemkein, 1964; Martinez-Hernandez et al. 1973; Narashiman & Gangla, 1966; Velasco-Plaza et al. 1979). The depressed rate of [3H]thymidine incorporation observed in the stomach does not appear to be related to a general decline in body weight or alterations in food intake. After a small initial weight loss in the first two post-operative days, sialoadenectomized rats treated with either saline or tissue extracts gained weight at a rate similar to that of sham-operated animals, suggesting that food intake had returned to normal. This suggestion is strengthened by previous demonstrations that food intake in rats returns to normal within 6 days of removal of the salivary glands (Epstein, Spector, Samman & Goldblum, 1964; Kissileff & Epstein, 1969). Furthermore, Martinez-Hernandez et al. (1973) observed that atrophy of certain tissues following salivary gland extirpation was evident even if compared to pair-fed control animals. Therefore the results of the present study do not appear to be due non-specifically to a decline in nutrient intake. Administration of the salivary tissue extract to sialoadenectomized rats resulted in a significantly greater rate of [3H]thymidine incorporation and DNA content of the oxyntic mucosa than was observed in the sialoadenectomized group treated with phosphate buffer. These levels were however significantly less than those observed in the sham control group. Salivary gland extract was able to restore RNA to levels

10 10 K. A. SKINNER AND OTHERS observed in the sham-sialoadenectomized group of rats. These results suggest that administration of an extract of salivary gland tissue exerted a slight trophic influence on growth of oxyntic mucosa in the sialoadenectomized rat. This trophic effect in sialoadenectomized animals appears specific for homogenates of salivary tissue. In the present study, administration of comparable amounts of the extracts of rat spleen and skeletal muscle did not significantly alter the rate of DNA synthesis of the oxyntic mucosa of sialoadenectomized rats. This observation is similar to that reported by Martinez-Hernandez et al. (1973), in which extracts of submandibular gland tissue but not liver could influence tissue growth. Furthermore, while treatment with salivary gland extract was able to restore tissue RNA to the levels observed in shamoperated rats, RNA levels in the spleen- and muscle-extract-treated groups were significantly lower. In contrast, the DNA content was either not affected or higher in these two groups. These data may be interpreted as a reduction in cellular exfoliation as a result of injection of these extracts. This latter possibility has not been examined in the present study. The results of the present study also indicate that plasma and antral levels of gastrin are slightly depressed by sialoadenectomy. Gastrin is a trophic hormone for the gastrointestinal mucosa (Johnson, 1976). It is possible that the association between a reduction in oxyntic mucosal DNA synthesis and sialoadenectomy is related to the reduced circulating and tissue levels of gastrin. Injection of the gastrin analogue pentagastrin did result in an increase in the rate of DNA synthesis in the oxyntic mucosa of sialoadenectomized rats. However, there is evidence that the atrophic effect of sialoadenectomy in the present study was not mediated exclusively by low levels of circulating or tissue gastrin: the reduction in gastrin levels was not statistically significant. Administration of pentagastrin did not increase the DNA or RNA content of the gastric mucosa, while sialoadenectomy was able to reduce these parameters. Furthermore, treatment with the salivary tissue extract was able to partially reverse the atrophic effect of sialoadenectomy on the gastric mucosa. However, the gastrin levels in sialoadenectomized rats were even further decreased when the salivary tissue extract was administered to these animals. Another likely mechanism through which the salivary glands might affect gastric mucosal growth is via the endogenous trophic substance, EGF (Feldman et al. 1978; Johnson & Guthrie, 1980). Administration of a culture-grade preparation of EGF resulted in an increase in DNA synthesis in the oxyntic mucosa of sialoadenectomized rats. Salivary gland extracts which exhibited a slight trophic influence on the oxyntic mucosa of sialoadenectomized rats did contain some EGF-like activity, as determined by their ability to displace labelled EGF bound to a mouse liver homogenate. This activity was not evident in other tissue extract preparations or by pentagastrin. The low level of EGF-like activity calculated to exist in the salivary tissue homogenate may account for the small trophic influence this extract exerted on gastric mucosal growth in the sialoadenectomized rat. The effects of sialoadenectomy and administration of exogenous EGF, pentagastrin and salivary tissue extract on the sialoadenectomized animal appeared specific for oxyntic mucosa. Changes in the growth of either duodenal or colonic mucosa were inconsistent. Whereas pentagastrin has been shown to influence growth throughout the gastrointestinal tract (Johnson, 1976) there is some doubt as to whether EGF

11 SIALOADENECTOMY AND THE GUT has the same widespread stimulatory capabilities for the gut (Chabot & Hugon, 1980; Johnson & Guthrie, 1980; Malo & Menard, 1982). Nevertheless, the inability of elimination of salivary tissue or administration of some trophic substances in the present study to alter growth in the small intestine or colon, as has been shown in previous studies (Johnson, 1976), may reflect a situation unique to the sialoadenectomized rat. These present data are consistent with previously published reports from our laboratory. Sialoadenectomy in the rat has been shown to increase the susceptibility of the gastric mucosa to bile salt-induced damage (Skinner & Tepperman, 1981). Furthermore, the degree of mucosal damage could be reduced or abolished by administration of an extract of salivary tissue (Tepperman & Soper, 1983). It is possible that the influence of sialoadenectomy on mucosal integrity may be mediated through a reduction in tissue growth which increased the susceptibility of the mucosa to bile salt-induced damage. Similarly, Takeuchi & Johnson (1979) have shown that a decrease in gastric mucosal DNA synthesis increased the susceptibility of the stomach to stress ulceration in rats. Furthermore, these effects could be reversed by pentagastrin administration. Konturek and co-workers (Konturek, Radecki, Brzozowski, Piastucki, Dembinski, Dembinska-Kiec, Zmuda, Gryglewski & Gregory, 1981) have shown that treatment of rats with EGF effectively protects them from aspirin-induced ulcers. As was the case for pentagastrin, the protective effect may be attributable to the ability of EGF to stimulate mucosal DNA synthesis. In conclusion, a reduction in oxyntic mucosal growth was observed in rats in which the submandibular-sublingual salivary glands were removed and the parotid ducts ligated. The effect may be mediated via a salivary tissue trophic factor. A possible candidate for this trophic factor is EGF. We are grateful to the MRC of Canada for financial support. We thank Dr T. J. MacDonald, Department of Medicine for performance of the radio-immunoassay for gastrin and Ms M. Allen and Mrs N. Wilson for typing the manuscript. 11 REFERENCES AL-NAFUsSI, A. I. & WRIGHT, N. A. (1982). The effect of epidermal growth factor (EGF) on cell proliferation of the gastrointestinal mucosa in rodents. Virchow8 Arch. B. Cell. Pathol. 40, AMANO, M., MESSIER, B. & LEBLOND, C. P. (1959). Specificity of labelled thymidine as a DNA precursor in autoradiography. J. Histochem. Cytochem. 7, BRADFORD, M. M. (1976). A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Aaldyt. Biochem. 72, BUEKER, E. D. & SCHEMKEIN, I. (1964). Effects of daily subcutaneous injections of nerve growth stimulating protein fractions on mice during postnatal to adult stage. Ann. N. Y. Acad. Sci. 118, CERIOTrrI, G. (1955). Determination of nucleic acids in animal tissues. J. biol. Chem. 214, CHABOT, J. G. & HUGON, J. S. (1980). Stimulation of DNA synthesis in mouse intestinal mucosa by epidermal growth factor (EGF). J. Cell Biol. 87, 155a. COHEN, S. & ELLIOT, G. A. (1963). The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J. invest. Derm. 40, 1-5. EPSTEIN, A. N., SPECTOR, D., SAMMAN, A. & GOLDBLUM, C. (1964). Exaggerated prandial drinking in the rat without salivary glands. Nature, Lond. 201, FELDMAN, E. J., AURES, D. & GROSSMAN, M. I. (1978). Epidermal growth factor stimulates

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