Influence of Desalivation on Acid Secretory Output and Gastric Mucosal Integrity in the Rat

Transcription

1 GASTROENTEROLOGY 1981;81:335-9 Influence of Desalivation on Acid Secretory Output and Gastric Mucosal Integrity in the Rat KAROLINA A. SKINNER (nee MENKAL) and BARRY L. TEPPERMAN Department of Physiology, University of Western Ontario, London, Canada Exogenous administration of substances extracted from rodent salivary glands may increase the resistance of the gastric mucosal barrier to the disruptive actions of ulcerogenic agents and/or reduce acid secretory output. In the present study the influence of desalivation in the rat on acid secretory function and mucosal integrity has been investigated. Experiments were performed on rats 4 wk after removal of the major salivary glands and ligation of the salivary ducts. Intravenous infusion of pentagastrin ( p.g/kg/min) resulted in an increase in acid output in desalivated rats although the response was less than that observed in sham-operated controls. Intraluminal instillation of a bile salt solution (5 mm in 150 mm Hel) in control animals resulted in a significant loss of H+ and the luminal appearance of Na+ and K+. The net fluxes of Na+ and K+ were exacerbated in the desalivated group. Furthermore, the mean area of bile salt-induced ulceration was significantly greater in desalivated rats than in the control group. In the absence of bile salt treatment there was no difference in either the transmural ionic fluxes or area of ulceration between the two groups. These results suggest that removal of the major salivary glands in rats decreases the resistance of the gastric mucosa to bile salt-induced damage. The existence of a substance, or substances with antisecretory and antiulcer activities originating from the salivary glands has been suggested for the past 30 yr. Evidence for the existence of these fac- Received December 10, Accepted March 9, Address requests for reprints to: Dr. Barry L. Tepperman, Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada NCA 5Cl. This work was supported by a grant from the Medical Research Council of Canada (Grant No. MA6426). A preliminary account of this work was presented at the 31st Annual American Physiological Society Fall Meeting, October 12-17, 1980, Toronto by the American Gastroenterological Association /81/ $02.50 tors includes an inhibition of basal or stimulated gastric acid secretion after intravenous injection of extracts of canine or human salivary glands or juice (1-4). Furthermore, similar treatment has been shown to reduce or abolish stress or steroid-induced ulcers in rats (3). Possible mediators of these effects may be anyone of a number of peptide or glycopeptide substances which have been extracted from both salivary glands and saliva (5,6). Although the physiologic significance of these purified peptide substances is unknown, they produce many of the same biologic effects as do the crude salivary extracts including inhibition of acid secretion (7,8) and protection against ethanol-induced gastric mucosal damage (9). However, it is not known whether these peptides or other salivary factors exert similar actions physiologically on the gastric mucosa. Some epidemiologic evidence exists linking a decrease in ingestion of saliva to an increase in the incidence of gastric ulceration (10), and alterations in salivary flow rate and composition have been noted in duodenal ulcer patients (11,12). Furthermore, in preliminary studies Barker et a1. (13) have found that removal of the salivary glands in rats increased the incidence of severe gastric ulceration after vagotomy. However, the possibility that the salivary glands physiologically influence gastric secretory activity and/or mucosal integrity has not been extensively investigated. In the present study we have examined the effects of removal of the submaxillary and sublingual glands and ligation of the parotid ducts on acid output and bile salt-induced ulceration and the permeability characteristics of rat gastric mucosa. Materials and Methods Animal Preparations Male Sprague-Dawley rats ( g) were used in these studies. Under pentobarbital anesthesia the sub-

2 336 SKINNER AND TEPPERMAN GASTROENTEROLOGY Vol. 81, No.2 mandibular/sublingual gland complexes were removed after the ducts were ligated. Parotid gland ducts were ligated and severed. This process has been shown to result in atrophy of the parotid tissue within 1 wk of ligation (14). Effective salivectomy was confirmed by the observation of an increase in prandial drinking by approximately 200% compared with control animals (15). Desalivated animals were used for experiments 4 wk postoperatively and only after they had regained their presurgical weight and continued to gain weight at a rate similar to that observed in sham-operated control rats. Acid Secretory Studies Animals were fasted for 20 h before anyone experiment but were allowed tap water ad libitum. Under urethane anesthesia (0.6 ml/1oo g body wt of a 25% solution) a silastic catheter (ID in., Dow Corning Corp., Midland, Mich.) was inserted into the jugular vein, and another catheter (ID in.) was inserted into the gastric lumen via the duodenum. The duodenum was ligated so that fluid was restricted to the gastric lumen. The volume instilled remained in the stomach as evidenced by the ability to recover all the fluid placed in the stomach. Two milliliters of a 0.9% saline solution was instilled into the stomach and replaced at 10-min intervals with fresh solution. The sample volumes were measured, and the acid concentration was determined by titration to ph 7.0 with 0.02 N NaOH using an Autoburet and ph meter (Radiometer, Copenhagen). Acid output was calculated and expressed as microequivalents per hour. After a 1-h basal period in which 0.9% saline was infused intravenously, secretion was stimulated by infusion of pentagastrin (Peptavlon, Ayerst) into the jugular vein in the dose range 0.1 flg/kg/min to 0.4 flg/kg/min. Infusions were performed using a Harvard infusion pump (model VDC; Harvard Apparatus, S. Natick, Mass.) at an infusion rate of 3 ml/h. Each dose was administered over a period of 60 min. The mean secretory response to each dose was calculated from the average output of the last three 10-min collection periods. Bile Salt Studies To test the relationship between salivary gland removal and gastric mucosal barrier integrity we have studied the effect of topical application of the ulcerogenic agent sodium taurocholate (bile salt) (16), to the gastric mucosa of another group of sham-operated and des alivated rats. Solutions were instilled into the stomach via the duodenal catheter. At the end of each 10-min period the solution was drained from the stomach and replaced by fresh solution. A control solution consisting of 150 mm HCI was initially instilled into the gastric lumen. The stomach was irrigated with the HCI solution for four periods. After this the stomachs were irrigated with a solution of 5 mm sodium taurocholate in 150 mm HCI for six periods, followed by a final four periods of HCI alone. After each 10-min collection the volume was measured, and the H+ concentration was determined by electrometric titration as described previously. The Na+ and K+ concentration of each sample was determined by flame photometry (Radiometer, Copenhagen, model FLM3). Total amounts of Na+, K+, and H+ in the recovered fluid were calculated for each period. The net flux of each ion was determined by subtracting the amount put into the gastric lumen from the amount recovered. To study the effect of desalivation on mucosal permeability in the absence of a pharmacologic damaging agent, similar experiments were performed in a separate group of sham-operated and des ali va ted animals. In this study the stomach was irrigated for 14 periods only with the 150 mm Hel solution. Ulcer Index After the bile salt and HCl irrigation experiments the rats were killed by cervical fracture, the stomachs were removed, opened along the greater curvature, and rinsed with cold saline. The stomach was spread and pinned on a flat surface and examined with a binocular dissecting microscope. Since ulcers were not always straight but often were observed to be "L" shaped, both the length and width of each lesion was measured, and the total area of ulceration was recorded as the ulcer index for each rat. Small petechial hemorrhages were not included in these calculations. Measurement of ulcers was performed without knowledge of whether the animal had been sham-operated or desalivated. The presence or absence of salivary tissue was determined by postmortem examination only after measurement of mucosal ulceration. Statistics In acid secretory and permeability studies each response was dependent to a certain extent upon the preceding response level. Therefore, these data were analyzed using a nested analysis of variance (17). To ensure homogeneity of variance the test was performed after log transformation of the data. Ulcer index data were analyzed using Student's t-test for unpaired values. All data were analyzed using the Hewlett-Packard 9845 computer. Results Gastric Acid Secretion Infusion of increasing doses of pentagastrin resulted in an incremental increase in acid secretion in both desalivated and sham-operated control rats (Figure 1). Basal acid outputs in both groups (n = 10 for both desalivated and control groups) were not statistically different. However, the mean secretory response to intravenous pentagastrin of the desalivated groups was diminished (p = 0.054) when compared with the acid output from control animals.

3 August 1981 DESALIV ATION AND GASTRIC MUCOSAL FUNCTION 337 Figure 1. Mean acid output (± SEM) from the rat stomach in response to intravenous infusion of pentagastrin. Each dose of pentagastrin was infused for 60 min. The mean secretory response to each dose was calculated from the average output of the last three to-min periods. The acid response from desalivated rats (e e, n = 10) and from sham control rats (0--0, n = 10) is displayed here. Indicates that p = by the nested analysis of variance. O~---''------' ' ' r Pentagastnn dose (.u9/kg/mln) Gastric Mucosal Permeability and Ulceration The change in the net transmucosal fluxes of H+, Na+, and K+ in response to bile salt treatment are displayed in Figure 2. There was no significant difference between the two groups (n = 13 for desalivate and control) in the rate of disappearance of H+ from the gastric lumen. The appearance of Na+ in the gastric lumen was significantly greater (p ~ 0.05) in the desalivated group (n = 7) than in sham-operated animals (n = 7). Furthermore, desalivated rats (n = 13) displayed a significant (p ~ 0.01) increase in net K+ flux when compared with the control group (n = 13). This greater increase in Na+ and K+ fluxes in the desalivated group was apparent within 10 min of application of the sodium taurocholate solution. Ionic fluxes remained elevated for the duration of bile application and had not returned to control levels even after removal of the bile and a 40-min exposure to the control Hel solution. In the absence of sodium taurocholate the net fluxes of H+, Na+, and K+ in the desalivate (n = 8) and control (n = 8) groups were not different (Figure 3). A small degree of ulceration was apparent in both sham-operated and desalivated rats exposed to 150 mm Hel alone (Table 1). However, addition of sodium taurocholate (5 mm) to the Hel solution resulted in a significant (p ~ 0.05) increase in the area of ulceration only in the des ali va ted group. Ulcers were confined exclusively to the glandular portion of the stomach. Discussion Extirpation of the submandibular-sublingual salivary gland complexes and ligation of the parotid ducts in rats resulted in a decreased acid output in response to intravenous secretagogue infusion. An incremental increase in acid output was observed both in control and desalivated animals. Desalivated 5mM No-taurocholate Time (lomln periods post control) Figure 2. Effect of 5 mm sodium taurocholate in 150 mm HCI on the change (ll) from control in net fluxes of H+, Na+, and K+ across the rat stomach. The mean response (± SEM) from desalivated (e e) and control (0--0) rats is displayed. The flux for each period was calculated as the change in net flux from the average of the last two control periods before bile salt instillation. After period 6 the stomach was bathed with a solution of 150 mm HC!. Data were analyzed as described in Figure 1. p ::s 0.05; p ::s 0.01.

4 338 SKINNER AND TEPPERMAN GASTROENTEROLOGY Vol. 81, No.2 150mM Hel 6 8 1'0 1'2 Time (10 min penods) Figure 3. Effect of 150 mm HCl on the net fluxes of H+, Na+, and K+ across the rat stomach. The mean response (± SEM) from desalivated (.e, n = 8) and sham control (0--0, n = 8) rats is displayed. Data were analyzed as described in Figure 1. animals displayed an attenuation of the acid secretory response to pentagastrin infusion when compared with sham controls. Salivary extracts and peptides of salivary origin have been found to produce antisecretory effects after parenteral administration (1-4,7). If these agents function physiologically as secretory inhibitors, then their removal might be expected to augment acid output. In contrast an attenuation of the response to secretagogue administration has been observed. Therefore, it is not likely that the endogenous salivary factors function physiologically to depress gastric acid secretion. In the present study it was observed that desalivation in rats resulted in an exacerbation of the net transmural ionic fluxes and the degree of mucosal ulceration associated with bile salt-induced damage to the gastric mucosal barrier. These results confirm and extend the preliminary findings of Barker et al. (13) in which de salivation produced a greater predisposition to ulceration in rats. These data suggest the existence of some protective factor in saliva and/or salivary glands. The existence of such a factor is further supported by the demonstration that salivary extracts and certain salivary peptides exert protective actions on gastric mucosa (3,9). Since removal of the salivary glands resulted in a depression of the acid response to a secretagogue, it is likely, 14 that the protective action of the salivary factor(s) on the gastric mucosa may be independent of an inhibition of acid secretion. It has recently been demonstrated that the protective influence of saliva may not be unique to the gastric mucosa. Hutson et al. (18) have demonstrated that topical application of saliva to skin wounds by licking significantly accelerated the rate of wound healing in rats. After desalivation the rate of healing was significantly depressed. In spite of an increase in the incidence of ulceration and an increase in Na+ and K+ appearance: H+ back-diffusion did not appear significantly greater in the desalivated group than in sham controls after bile salt administration. However, other studies have revealed that gastric mucosal barrier damage can occur in the absence of an increase in the rate of luminal acid loss (19). It is possible that H+ back-diffusion is masked by an increase in acid secretion, which is known to occur in damaged mucosa (20,21), and possibly is mediated by an increase in mucosal histamine release. To investigate whether salivectomy itself resulted in gastric mucosal damage and ulceration, the permeability characteristics of the gastric mucosa were studied in desalivated rats in the absence of bile salt application. Since the permeability characteristics and ulcer index of the desalivated group were not greater than that of the sham-operated counterpart, this result suggests that desalivation per se does not damage the gastric mucosal barrier. It is possible, however, that in desalivated animals the barrier becomes "weakened" and therefore is less resistant to treatment with an ulcerogenic drug. It is concluded that removal of the salivary glands and ligation of the salivary ducts results in an attenuation of the acid response to intravenous secretagogue administration and a decrease in the ability of the gastric mucosal barrier to withstand a phar- Table 1. Effect of Desalivation on the Mean Area of Ulceration in Rats Receiving Topical Applications of HCI Alone or Taurocholate + HCI Mean area of ulceration Treatment n (mm2±sem) Sham control group 150 mm HCl alone ± mm sodium taurocholate ± 3.3 in 150mMHCl Desalivate group 150 mm HCl alone ± mm sodium taurocholate ± 6.3 a in 150mMHCl a Indicates a significant (p ~ 0.05) difference from the bile-treated sham control group by the t-test for unpaired data.

5 August 1981 DESALIV A TION AND GASTRIC MUCOSAL FUNCTION 339 macologic insult. The mediator of this effect is unknown, although a number of possibilities may be suggested. Desalivation may result in the removal of a trophic substance which may influence the gastric mucosa. Removal of the salivary glands has been shown to result in a marked decline in the rate and degree of growth of a number of species (22) and a reduction in the metabolic activity of tissues including adrenal gland, testes, and brain (23). Exogenous administration of some purified salivary peptides, including epidermal growth factor, exert potent trophic influences on the gastrointestinal tract (24). In addition, chronic stimulation of salivary secretion has been shown to promote mucosal cell proliferation in the small intestine (25). Therefore the decreased acid output and/or the decreased resistance of the gastric mucosal barrier to a pharmacologic damaging agent might be explained by a hypotrophy of the gastric mucosa. The mode of the action of the salivary glands on gastric mucosal function and integrity is currently under investigation. References 1. Code CF. Ratke HV. Livermore GR. Lunberg W. Occurrence of gastric secretory inhibitor activity in fresh gastric and salivary mucin. Fed Proc 1949;8: Menguy R. Masters YF. Manzi J. Characterization and partial purification of sialogastrone. Surgery 1967;62: Miyoshi A. Moriga M. Ohbayashi M. et al. Studies on a gastric secretion inhibitory substance in human saliva (Salivogastrone). Jpn Arch Intern Med 1969;16: Kobayaski M. Morimoto T. Yamamoto M. Studies on the inhibitory substances of gastric secretion in salivary glands. I. The inhibitory activity of gastric secretion in mouse submaxillary glands. and its specificity. J Pharm Soc Jpn 1972;92: Taylor JM. Mitchell WM. Cohen S. Epidermal growth factor. Physical and chemical properties. J Bioi Chern 1972;247: Heitz PU. Kasper M. Van Noorden S. et al. Immunohistochemical localisation of urogastrone to human duodenal and submandibular glands. Gut 1978;19: Bower JM. Gamble R. Gregory H. et al. The inhibition of gastric acid secretion by epidermal growth factor. Experientia 1975;31: Gregory H. Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature (Lond) 1975; 257: Pilot MA. Deregnaucourt J. Code CF. Epidermal growth factor increases resistance of the gastric mucosal barrier to ethanol in rats. Gastroenterology 1979;76: Malhotra SL. Protective action of saliva in peptic ulceration. Scand J Gastroenterol 1967;2: Blum AL. Salivary secretion in duodenal ulcer disease. Gut 1972;13: Nagwani PL. Naik SR. Sachdev S. et al. Correlation of salivary and gastric acid secretions in duodenal ulcer patients in tropics. Gut 1970;20: Barker JH. Lund JP. Dellow PG. Desalivation and the incidence of experimental gastric ulceration. Proc Can Fed BioI Soc 1969;12: Tamarin A. Secretory cell alterations associated with submaxillary gland duct ligation. In: Schneyer LH. Schneyer CA. eds. Secretory mechanisms of salivary glands. New York: Academic Press. 1967: Epstein AN. Spector D. Samman A. Goldblum C. Exaggerated prandial drinking in the rat without salivary glands. Nature (Lond) 1964;201: Davenport HW. Destruction of the gastric mucosal barrier by detergents and urea. Gastroenterology 1968;54: Sokal RR. Rohlf FJ. Biometry: The principles and practice of statistics in biological research. San Francisco: WH Freeman Hutson JM. Niall M. Evans D. Fowler R. Effect of salivary glands on wound contraction in mice. Nature (Lond) 1979; 279: Davenport HW. Protein-losing gastropathy produced by sulfhydryl reagents. Gastroenterology 1971;60: Davenport HW. Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 1966;50: Thompson MR. Studies on the acid secretion that occurs during injury to the gastric mucosa. Gastroenterology 1976; 71: Narashiman MS. Gangla VG. Influencia regulatoria de la gumdula submaxilar sobre el crecimiento. Curr Sci 1966;35: Velasco Plaza A. Menendez-Patterson A. Marin B. Effects of the extirpation of submandibular salivary glands on the oxidative activity of nervous and glandular structures in the male rat. Arch Oral Bioi 1979;24: Feldman EJ. Aures D. Grossman MI. Epidermal growth factor stimulates ornithine decarboxylase activity in the digestive tract of the mouse. Proc Soc Exp Bioi Med 1978;15: Li AKC. Schattenkerk ME. Huffman RG. Submandibular saliva promotes small bowel intestinal proliferation. Clin Res 1980;28:483A.