Is there a fundic factor which regulates G cells in the antrum?

JOURNAL OF SURGICAL RESEARCH 4 7 , 8-11 (1989)

Is There a Fundic Factor Which Regulates G Cells in the Antrum? 1 PETER J. FABRI, M.D., 2 MICHAEL A. FABIAN, M.D., WILLIAM R. GOWER, JR., PH.D., AND TIMOTHY H. KNIERIM Department of Surgery, University of South Florida College of Medicine, and Surgical Service, James A. Haley Veterans' Hospital, Tampa, Florida 33612

Both surgical fundusectomy and pharmacologic acid i n h i b i t i o n h a v e b e e n s h o w n to p r o d u c e a n t r a l G c e l l h y p e r p l a s i a i n rats. F u n d u s e c t o m y h a s a l s o b e e n s h o w n to b e a r e l i a b l e , r e p r o d u c i b l e m e a n s to p r o d u c e h y p e r g a s trinemia. In contrast, treatment of animals with famot i d i n e , a h i s t a m i n e H2 r e c e p t o r a n t a g o n i s t , or o m e p r a z o l e , a H + - K + - A T P a s e e n z y m e i n h i b i t o r , f a i l e d to p r o d u c e h y pergastrinemia. This suggests that elimination of acid inhibition may not be the sole mechanism of hypergast r i n e m i a a f t e r f u n d u s e c t o m y . W e h y p o t h e s i z e d t h a t removal of the gastric fundus may remove a factor which c o n t r o l s G c e l l a c t i v i t y . © 1989 AcademicPress, Inc.

INTRODUCTION In 1905, Edkins [1] discovered that extracts from hog antrum stimulated gastric acid secretory activity. In 1964, Gregory and Tracy [2] isolated pure gastrin from hog antral mucosa and demonstrated this substance as the causative agent in the stimulation of gastric acid secretion. With the development of a radioimmunoassay for gastrin in 1968 by McGuigan and Trudeau [3], sensitive studies on the various actions of this recently purified hormone were possible. Gastrin secretion in vivo has been found to be regulated by a complex set of mechanisms involving endocrine [4], paracrine [5], and neurocrine [6, 7] features. Antral gastrin release can be stimulated by alkaline pH, proteins, amino acids, calcium, vagal stimulation, and luminal stimulation by food [8]. Intravenous substances that stimulate gastrin release include calcium, epinephrine, gastrin releasing peptide, and bombesin [9-12]. Experimentally, hypergastrinemia together with antral G cell hyperplasia has been induced in the rat by vagotomy [13-15], fundusectomy [13], or antrocolonic transposition [15]. Previous work from this laboratory documented consistent, significant G cell hyperplasia and hypergastrinemia after fundusectomy at 21 days. However, various investigators have suggested that a nonacid luminal en-

1 Presented at the A n n u a l Meeting of the Association for Academic Surgery, Salt Lake City, Utah, N o v e m b e r 16-19, 1988 2 To w h o m reprint requests should be addressed. 0022-4804/89 $1.50 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

vironment, by itself, is not sufficient to cause G cell hyperplasia [16, 17]. In the present study, to further elucidate the mechanisms of G cell hyperplasia, we compared serum immunoreactive gastrin (IRG), G cell number (GCN), and antral gastrin content (AG) in rats with either surgical (fundusectomy) or pharmacologic (famotidine or omeprazole) reduction of gastric acid secretion. MATERIALS AND METHODS Forty male Sprague-Dawley rats (200-250 g) were allocated into two groups: surgical manipulation (fundusectomy or sham) or pharmacologic manipulation (treatment or control). Animals undergoing similar manipulation were then kept in common cages. Animals undergoing surgery were deprived of food for 24 hr prior to the procedure, but were allowed water ad libitum. These animals were then separated into two groups: surgical fundusectomy or sham surgery with laparotomy only. Animals undergoing operation were anesthetized with intraperitoneal pentobarbital (35 mg/kg), and a sterile surgical procedure was performed in the method described by Gower et al. [18]. Animals were allowed access to water upon recovery and access to rat chow 24 hr after surgery. Pharmacologically manipulated animals were separated into subgroups as follows: T en animals received famotidine (a gift from Merck, Sharp, and Dohme), 2 mg/kg! day, three animals received omeprazole (a gift from AB H~issle, Sweden), 140 mg/kg/day, and three animals received polyethylene glycol (vehicle). All substances were administered every 12 hr by gavage. At 3 weeks, five fundusectomized animals, five famotidine-treated animals, and all omeprazole-treated animals were fasted for 24 hr, anesthetized with intraperitoneal pentobarbital, and exsanguinated by inferior vena cava puncture. T he stomachs were then immediately harvested. Serum was stored at - 2 0 ° C and assayed for gastrin immunoreactivity by radioimmunoassay as previously described [19]. Gastrin antibody 3-02-11-78, which recognizes gastrin G-34 and G-17 in equivalent concentrations, was used throughout this study. Synthetic G-17 (Sigma Chemical Co.) was used as both standard and label. Tissue gastrin was then determined as follows: Excised stomachs were opened along the greater curvature, rinsed in saline, stretched, and pinned mucosa side up in a wax-

F A B R I E T AL.: F U N D I C F A C T O R R E G U L A T E S G C E L L S

lined dental tray. A 1- to 2 - m m longitudinal strip was excised f r o m the esophagogastric junction to the pylorus, weighed, minced, a n d placed into a 13 X 100-mm P y r e x tube with 0.5 cc deionized water. T h e strip was boiled for 10 min, homogenized in a P o t t e r - E l v e h j e m tissue homogenizer, t h e n centrifuged for 30 m i n at 2000g. T h e sup e r n a t a n t was r e m o v e d for gastrin r a d i o i m m u n o a s s a y . G cell n u m b e r was d e t e r m i n e d by a modification of the m e t h o d originally described by L e h y et al. [20]. After rem o v a l of a longitudinal strip for d e t e r m i n a t i o n of tissue gastrin, the r e m a i n i n g p o r t i o n of s t o m a c h was rinsed in phosphate-buffered saline solution, stretched, a n d pinned m u c o s a side up to dental wax-lined dishes. A n t r a were fixed in freshly m a d e B o u i n ' s solution for 24 hr, t h e n each a n t r u m was cut into five parallel strips p e r p e n d i c u l a r to the lesser curvature f r o m the esophageal j u n c t i o n to the pylorus. Strips were w a s h e d in 80% ethanol overnight, dehydrated, a n d e m b e d d e d in paraffin. F i v e - m i c r o m e t e r sections were cut f r o m the distal side of each strip, deparaffinized, a n d stained for gastrin b y indirect immunofluorescence [18]. G a s t r i n - c o n t a i n i n g cells were counted at 200× magnification along the entire length of each section a n d scored as G cells per high p o w e r field. F o r each rat, a m e a n cell c o u n t per visual field was obtained b y averaging the m e a n result of all five sections. At 6 weeks, the r e m a i n i n g f u n d u s e c t o m i z e d a n d famo t i d i n e - t r e a t e d a n i m a l s were sacrificed in t h e s a m e m a n ner, with identical s e r u m a n d tissue processing as before. T o evaluate the effect of dose of H2 antagonists, a separate series of animals were administered famotidine twice daily by gavage in a dose of 2, 4, or 8 m g / k g / d a y for 21 days. On D a y 21, gastric c o n t e n t s were a s p i r a t e d percut a n e o u s l y a n d p H was measured. Blood s a m p l e s were t a k e n for gastrin r a d i o i m m u n o a s s a y . P e n t a g a s t r i n in a dose of 6 # g / k g was a d m i n i s t e r e d i n t r a v e n o u s l y and, 15 m i n later, the s t o m a c h harvested, gastric p H measured, a n d a n t r u m h a r v e s t e d for gastrin content. Statistical Analysis M e a n a n d s t a n d a r d e r r o r of the m e a n were t a b u l a t e d for all groups of d a t a a n d results c o m p a r e d b y u n p a i r e d t test. Significant difference was accepted if P < 0.05. M e a n squares were c o m p a r e d by A N O V A as well. TABLE 1

Procedure Fundusectomy--sham F u n d u s e c t o m y - - 3 weeks F u n d u s e c t o m y - - 6 weeks PEG control F a m o t i d i n e - - 3 weeks F a m o t i d i n e - - 6 weeks O m e p r a z o l e - - 3 week s * P < 0.05.

Serum IRG (pg/ml) 62 402 409 73 74 71 98

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RESULTS Results are s u m m a r i z e d in T a b l e 1. Figure 1 shows s e r u m I R G for each group. At b o t h 3 a n d 6 weeks, fund u s e c t o m i z e d a n i m a l s experienced very significant increases in I R G c o m p a r e d to those in control a n i m a l s a n d p h a r m a c o l o g i c a l l y m a n i p u l a t e d animals. Omeprazolet r e a t e d a n i m a l s d e m o n s t r a t e d a small increase in I R G t h a t was n o t statistically significant. As seen in Fig. 2, all f u n d u s e c t o m i z e d a n d famotidinet r e a t e d a n i m a l s experienced a significant increase in G C N . O m e p r a z o l e - t r e a t e d a n i m a l s did not. Fundusect o m i z e d a n i m a l s experienced a significantly greater increase in G C N m a r k e d l y earlier t h a n t h a t in famotidinet r e a t e d animals. A n t r a l gastrin c o n t e n t was e x a m i n e d in f u n d u s e c t o m ized a n i m a l s at 6 weeks a n d in f a m o t i d i n e - a n d o m e p r a zole-treated a n i m a l s at 3 a n d 6 weeks. As can be appreciated in T a b l e 1, fundusectomized a n d f a m o t i d i n e - t r e a t e d a n i m a l s d e m o n s t r a t e d statistically significant increases in a n t r a l gastrin c o n t e n t c o m p a r e d to those in control animals. W h i l e o m e p r a z o l e - t r e a t e d a n i m a l s showed an increase in a n t r a l gastrin c o n t e n t as well, the change was n o t statistically significant. A m o n g the d o s e - r e s p o n s e animals, a c h l o r h y d r i a was p r e s e n t in essentially all a n i m a l s a f t e r 21 days regardless of dose. N o significant change in gastric acid c o n t e n t or p H was n o t e d following the a d m i n i s t r a t i o n of p e n t a g a s trin, suggesting adequate acid blockade. S e r u m gastrin i r n m u n o r e a c t i v i t y was not significantly different a m o n g the t h r e e dosage groups (2 m g / k g / d a y , 88 _ 26 pg/ml; 4 m g / k g / d a y , 78 __ 12 pg/ml; 8 m g / k g / d a y , 176 -+ 73 p g /

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JOURNAL OF SURGICAL RESEARCH: VOL. 47, NO. 1, JULY 1989 dose we u s e d d i d n o t e l e v a t e a n t r a l g a s t r i n c o n t e n t , although they did d e m o n s t r a t e a significant increase in serum IRG.

3.0q

CONCLUSION 2.5C

F u n d u s e c t o m y p r o d u c e s b o t h G cell h y p e r p l a s i a a n d h y p e r g a s t r i n e m i a i n m a l e S p r a g u e - D a w l e y rats. F a m o t i d i n e , 2 m g / k g / d a y , p r o d u c e s a s i g n i f i c a n t G cell h y p e r p l a s i a b u t n o t h y p e r g a s t r i n e m i a , e v e n after 6 weeks of t r e a t m e n t . O m e p r a z o l e , 140 m g / k g / d a y , p r o d u c e s a n i n crease i n G C N a n d s e r u m I R G , t h o u g h n o t s t a t i s t i c a l l y s i g n i f i c a n t a f t e r o n l y 3 weeks of t r e a t m e n t . T h u s a f u n d i c f a c t o r m a y be p r e s e n t t h a t e x e r t s c o n t r o l over G cell activity.

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ACKNOWLEDGMENTS SHAM

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Gastrin cell number. Abbreviations same as for Fig. 1.

ml). A n t r a l g a s t r i n c o n t e n t was likewise n o t d i f f e r e n t a m o n g t h e t h r e e g r o u p s s u g g e s t i n g t h a t a d e q u a t e acid i n h i b i t i o n h a d b e e n a c c o m p l i s h e d (2 m g / k g / d a y , 2844 + 509 p g / m g ; 4 m g / k g / d a y , 3014 _ 296 p g / m g ; 8 m g / k g / d a y , 2705 _ 294 p g / m g ) . DISCUSSION F u n d u s e c t o m y p r o d u c e d a m a r k e d a n d s i g n i f i c a n t increase i n s e r u m I R G , G C N , a n d A G as expected. I n cont r a s t , f a m o t i d i n e - a n d o m e p r a z o l e - t r e a t e d a n i m a l s failed to s h o w a s i g n i f i c a n t i n c r e a s e i n s e r u m I R G , b u t did show a n i n c r e a s e i n G C N a n d AG. T h e r e are a n u m b e r of possible e x p l a n a t i o n s for o u r f i n d i n g s . O n e is t h a t t h e f u n d u s m a y c o n t a i n a f a c t o r w h i c h r e g u l a t e s G cell a c t i v i t y a n d f u n c t i o n , w h i c h is r e m o v e d d u r i n g f u n d u s e c t o m y . T h i s w o u l d also h e l p t o e x p l a i n t h e m a r k e d a n d r a p i d i n c r e a s e i n s e r u m I R G , G C N , a n d A G s e e n as e a r l y as 3 w e e k s after fundusectomy in our animals. A l t e r n a t i v e l y , g a s t r i n p r o d u c t i o n m a y lag b e h i n d G cell p r o l i f e r a t i o n i n t h i s s e t t i n g a n d m o r e t i m e m u s t elapse b e f o r e a rise i n s e r u m I R G is s e e n i n p h a r m a c o l o g i c a l l y treated animals. Several studies have demonstrated that s h o r t - t e r m t r e a t m e n t (30 d a y s ) w i t h H2 a n t a g o n i s t s i n c l u d i n g c i m e t i d i n e a n d r a n t i d i n e fails to elicit a n i n c r e a s e i n e i t h e r I R G or G C N , b u t b o t h of t h e s e i n c r e a s e signifi c a n t l y a t 8 w e e k s [21-23]. A n o t h e r p o s s i b i l i t y is t h a t h i g h e r or m o r e f r e q u e n t d o s i n g m a y be n e e d e d i n r a t s to achieve complete c o n t i n u o u s acid inhibition with famotidine. That the increase in GCN and AG in the omeprazolet r e a t e d g r o u p was n o t s i g n i f i c a n t m a y h a v e b e e n d u e to t h e s h o r t - t e r m t r e a t m e n t (21 days) of t h e a n i m a l s . C r e u t z f e l d t e t al. [24] h a v e d e m o n s t r a t e d t h a t s h o r t - t e r m t r e a t m e n t (30 days) of r a t s w i t h o m e p r a z o l e u s i n g t h e

We thank Marion Baxter for assistance in preparation of this manuscript. This research was supported in part by a Veterans Administration RAG grant, Project No. 0001. REFERENCES 1. Edkins, J. S. On the chemical mechanism of gastric secretion. Proc. R. Soc. Lond. 76: 376, 1905. 2. Gregory, R. A., and Tracy, H. J. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 5: 103, 1964. 3. McGuigan, J. E., and Trudeau, W. Immunochemicalmeasurement of elevated levels of gastrin in the serum of patients with pancreatic tumors of the Zollinger-Ellison variety. N. Engl. J. Med. 2 8 7 : 1308, 1968. 4. Walsh, J. H. Peptides as regulators of gastric acid secretion. Annu. Rev. Physiol. 50: 41, 1988. 5. Larsson, L-I., Goltermann, N., DeMagistris, L., Rehfeld, J. F., and Schwartz, T. O. Somatostatin cell processes as pathways for paracrine secretion. Science 205: 1393, 1979. 6. Dockray, G. J., Vaillant, C., and Walsh, J. H. The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract. Neuroscience 4: 1561, 1979. 7. Azuma, T., Kawai, T., Inokuchi, H., and Kawai, K. Neural and paracrine regulation of gastrin release using rat antral mucosa in tissue culture--The effect of carbachol, bombesin, and anti-somatostatin antibody on gastrin release. Gastroenterol. Japan 21 (5): 459, 1986. 8. Saffouri, J. W., DuVal, J. W., and Makhlouf, G. M. Stimulation of gastrin secretion in vitro by intraluminal chemicals: Regulation by intramural cholinergic and noncholinergic neurons. Gastroenterology 87: 557, 1984. 9. Lichtenberger, L. M., Shaw, L. S., and Bailey, R. B. Influence of calcium on the release of gastrin from isolated rodent G-cells. Proc. Soc. Exp. Biol. Med. 166: 587-591, 1981. 10. Bertaccini, G., Erspamer, V., Melchiorri, P., and Sopranzi, N. Gastrin release by bombesin in the dog. Brit. J. Pharmacol. 52: 219, 1974. 11. McDonald, T. J., Nilsson, G., Vagne, M., Ghatei, M., Bloom, S. R., and Mutt, V. A gastrin releasing peptide from the porcine non-antral gastric tissue. Gut 19: 767, 1978. 12. DuVal, J. W., Saffouri, B., Weir, G. C., Walsh, J. H., Arimura, A., and Makhlouf, G. M. Stimulation of gastrin and somatostatin secretion from the isolated rat stomach by bombesin. Amer. J. Physiol. 241: G242, 1981.

FABRI E T AL.: FUNDIC FACTOR REGULATES G CELLS 13.

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