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Effects of acid-inhibitory antiulcer drugs on mucin biosynthesis in the rat stomach
European Journal of Pharmacology, 251 (1994) 107-111 © 1994 Elsevier Science B.V. All rights reserved 0014-2999/94/$07.00
107
EJP 21407
Short communication
Effects of acid-inhibitory antiulcer drugs on mucin biosynthesis in the rat stomach T a k a f u m i I c h i k a w a a, K a z u h i k o I s h i h a r a b, K a t s u n o r i Saigenji c a n d K y o k o H o t t a *'a Departments of "Biochemistry, b Chemistry and c Internal Medicine, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa 228, Japan Received 8 September 1993, accepted 9 November 1993
The effects of the anti-acid secretory agents, cimetidine (N-cyano-N'-methyl-N"-(2-([(5-methyl-lH-imidazol-4yl)methyl]thio)ethyl)guanidine), ranitidine (N-(2-(((-5-[(dimethy•amin•)methy•]-2-furany•)methy•)thi•)ethy•)-N'-methy•-2-nitr•1,1-ethene-diamine), roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide hydrochloride), FRG-8813 (2-(furfury•su••ny•)-N-(4-[4-(piperidin•methy•)-2-pyridy•]•xy-(z)-2-buteny•)acetamide)• omeprazole (5-methoxy-2-([(4-methoxy3,5-dimethylpyridinyl)methyl]sulfinyl)-lH-benzimidazole) and NC-1300-O-3 (2-([2-(isobutylmethylamino)benzyl]sulfinyl)-lHbenzimidazole), on mucin biosynthesis were studied in rat gastric mucosa by using an organ culture technique. [3H]Glucosamine incorporation was stimulated in the corpus region by the histamine H 2 receptor antagonists which have a six-membered aromatic ring, roxatidine and FRG-8813, and the new H+,K+-ATPase inhibitor, NC-1300-O-3. Thus, these drugs not only inhibit acid secretion but may also promote gastric mucosal protective actions. The present observations also demonstrate that the determination of mucin biosynthesis may be a useful tool for evaluation of mucosal protective activity. Histamine H 2 receptor antagonist; H+,K+-ATPase inhibitor; Gastric mucin biosynthesis; Organ culture; (Rat)
1. Introduction
Medical therapy for peptic ulcer disease has been targeted at inhibiting acid secretion, based on the belief that ulcers occur as a result of an imbalance between luminal aggressive and mucosal defensive factors. The introduction of histamine H e receptor antagonists has provided effective non-surgical therapy for duodenal and gastric ulcers by inhibiting acid-peptic activity. More recently, omeprazole (5-methoxy-2-([(4methoxy-3,5-dimethylpyridinyl)methyl]sulfinyl)-1 H-benzimidazole), an H+,K+-ATPase inhibitor, showed the ability to inhibit acid secretion totally and to heal a duodenal ulcer within 2 weeks (Borg et al., 1986). Compared with the aggressive factors, little attention has been paid to the mucosal defensive factors in ulcer therapy and the role of these drugs in gastric mucosal protection has not been well characterized. Mucin, a major component of gastric mucus, is a high-molecular-weight compound of unique structure and is considered to be an important mucosal defensive factor (Allen et al., 1986). The purpose of this
* Corresponding author. Tel. 81-427-78-9267, fax 81-427-78-8441.
SSDI 0 0 1 4 - 2 9 9 9 ( 9 3 ) E 0 8 2 1 - 9
study was to compare the effects of the following histamine H 2 receptor antagonists and H+,K+-ATPase inhibitors on mucin biosynthesis in an organ culture system of the rat stomach. Four established drugs, cimetidine ( N-cyano-N '-methyl-N "-(2-([(5-methyl- l Himidazol-4-yl)methyl]thio)ethyl)guanidine), ranitidine (N-(2-(((-5-[(dimethylamino)methyl]-2-furanyl)methyl)thio)ethyl)-N'-methyl-2-nitro-l,l-ethene-diamine), roxatidine (2-acetoxy-N-(3-[ m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide hydrochloride) and omeprazole, and two new drugs, FRG-8813 (2-(furfurylsulfinyl)-N-(4-[4-(piperidinomethyl)-2-pyridyl]oxy-(z)-2butenyl)acetamide) and NC-1300-O-3 (2-([2-(isobutylmethylamino)benzyl]sulfinyl)-1H-benz-imidazole) (Ishihara et al., 1992), were studied (fig. 1).
2. Materials and methods
2.1. Experimental animals Seven-week-old male Wistar rats (SLC, Shizuoka, Japan) each weighing approximately 160 g were used. All were fasted for 24 h before the experiments and had free access to water during this time.
108
2.2. Organ culture The stomachs of the rats were excised immediately after they were killed by light anesthetization followed by exsanguination from the carotid artery. The stomachs were then cut along the greater curvature, and the luminal surface was gently washed with Ca2+/Mg 2+free phosphate-buffered saline (PBS(-)). The glandular part was selected, separated into the corpus and antrum, and cut into small 2 x 2 mm sections. The tissue culture method of Eastwood and Trier (1973) was used with a modification (Ishihara et al., 1988). Eight tissue fragments, the mucosal surface facing up, were placed on a stainless steel grid in the central well of a plastic culture dish (60 × 15 mm, Falcon, USA) containing 0.75 ml of culture medium. The medium consisted of 90% Eagle's minimum essential medium and 10% dialyzed fetal calf serum, with 370 kBq/ml of D-[1,6-3H(N)]glucosamine hydrochloride (1950 G B q / mmol, New England Nuclear). For the addition of anti-acid secretory agents to the culture medium, the drug was dissolved in dimethyl sulfoxide (DMSO) and added at concentrations of 10 -8 , 10 -7 , 10 -6 and 10 -5 M to the dishes, making the final concentration of
N y CI-13 Cimetldine
N ~ ~. CH2.S.CH2-CH2-N=C.NH-CH3 /
NH-CN
CH3.~.
Ranitidine
CH31N-CH2-~ O":~CH2-S-CH2-CH2-NH-CIINH-CH 3 CH-NO2 HC1
- c.2--~,~,,o_c~2_c.2.c.~.~..c.2.o~c~, •
FRG-8813
2.3. Isolation of labeled mucin and radioactivity measurements Upon completion of the culture period, the tissue fragments on a grid were harvested from the medium, gently rinsed with P B S ( - ) and boiled at 100°C for 3 rain in 0.4 ml of 0.05 M Tris-HCl buffer, pH 7.2. The extraction and isolation of mucin were performed as previously described (Azuumi et al., 1980). The tissue fragments were homogenized with a Physcotron microhomogenizer (Niti-On, Chiba, Japan). Triton X-100 was added to a 2% (v/v) concentration and the homogenate was shaken for 1 h at 37°C. The homogenate thus obtained was centrifuged at 8000 × g for 30 min to obtain the supernatant. A 0.4-ml portion of the supernatant was applied onto a Bio-Gel A-1.5m column (1 X 30 cm) previously equilibrated with the Tris buffer containing 2% Triton X-100, and the column was eluted with this buffer. Finally, fractions of 0.8 ml each were collected and the radioactivity was measured by using a scintillation counter (Beckman, Model LS2800, USA) with Aquasol-2 (New England Nuclear, USA) as the scintillant. The radioactivity incorporated into the void volume of the column, which had been demonstrated to be the synthesized mucin (Ichikawa et al., in press), was determined. To compare the synthesis of mucin, the total radioactivity of this fraction was divided by the tissue protein content of each homogenate to give the value relative to that of the control. 2.4. Drugs
•
Roxatidine
DMSO 0.01%. A DMSO solution without drug was added to the medium in the control well. All the dishes were maintained at 37°C for 5 h in 5% CO 2 and 95% air.
HC1
ell2 ~.CH2.CH=CH.CH2.NH.~I.CH2.~I.CH 2 " ~
The following drugs were obtained for use in this study: cimetidine (Smith-Kline Beecham, Japan); ranitidine" hydrochloride (Sankyo, Japan); roxatidine acetate hydrochloride (Takeda, Japan); FRG-8813 (Fujirebio, Japan); omeprazole (Fujisawa-Astra, Japan); NC-1300-O-3 (Nippon Chemiphar, Japan).
2.5. Protein determination N Omeprazole
,,,~.,~
CH3-O
N/
H
N II
O
dn~ \ O-CH3
Protein content in the tissue homogenate was determined by using the Pierce BCA protein assay kit with bovine serum albumin as the standard.
2.6. Statistical analysis Ne-1300-O-3
'/~,,~'~'N" ~ H
CH3-N-CH2-CH-CH3 I CH3 Fig. I. Structures of the drugs used in this study. Upper panel: histamine t t 2 receptor antagonists; lower panel: H+,K+-ATPase inhibitors.
The results were expressed as means + S.D. A oneway analysis of variance (ANOVA) with Dunnett's test was used for statistical analysis with P < 0.05 taken as significant.
109
3. Results
3.1. Influence of anti-acid secretory agents on mucin biosynthesis in the corpus region Fig. 2 shows the mucin biosynthetic activity of the corpus as measured by [3H]glucosamine incorporation. The biosynthesis of mucin in the control was 18.3 + 1.8 dpm//zg tissue protein. The changes in the biosynthesis of mucin by the addition of each histamine H 2 receptor antagonist are shown in fig. 2a. Roxatidine and FRG-8813 significantly and dose dependently increased [3H]glucosamine incorporation into mucin. On the other hand, the addition of cimetidine and ranitidine yielded no significant change in mucin biosynthesis (fig. 2a). Fig. 2b shows the biosynthesis of mucin in the corpus after the addition of omeprazole or NC-1300-O-3. The addition of NC-1300-O-3 enhanced [3H]glucosamine incorporation into mucin by about 20 ~ 25% (P < 0.05), but omeprazole had no effect on mucin biosynthesis.
3.2. Influence of anti-acid secretory agents on mucin biosynthesis in the antral region No significant change could be detected in the mucin biosynthesis of the antrum after addition of 10 - 8 ~ % " (a) 180. 160.
ROX
----h--.
FRG Cim
•
Ran
---4P--
140. 120. >
--I...........
100. .o
80. 6O %
w
140
(b) •
.... @...
NC Opz
**
*
120 100. 80 60 C o n c e n t r a t i o n (M)
Fig. 2. Effects of anti-acid secretory agents on mucin biosynthesis. (a) Influence of the histamine H 2 receptor antagonists on [3H]glucosamine incorporation into mucins in the corpus region. (b) Influence of the H+,K+-ATPase inhibitors on [3H]glucosamine incorporation into mucins in the corpus region. Cim: cimetidine, Ran: ranitidine, Rox: roxatidine, FRG: FRG-8813, Opz: omeprazole, NC: NC-13000-3. Values are expressed as percentages of the control and represent means + S.D. from 8 different samples. * P < 0.05 and ** P < 0.01 as compared with the control value.
10 -5 M of each histamine H 2 receptor antagonist (specific activity: 28.9 ~ 33.5 dpm//zg tissue protein). The same results were obtained after the addition of omeprazole and NC-1300-O-3 (data not shown).
4. Discussion
Since the concept of 'cytoprotection' was introduced by Robert et al. (1979) as an ability of prostaglandins which is independent of the anti-acid secretory effect, many drugs have been investigated for their activity to protect the gastric mucosa from a variety of necrotizing agents. Although the exact mechanism of mucosal cytoprotection is unknown, it presumably involves one or more of the naturally occurring defensive factors of gastric mucosa such as mucin metabolism. Previously, we reported that exogenous prostaglandins, which are capable of inducing direct cytoprotection, significantly increase the biosynthesis of mucins in the corpus and antrum of the rat stomach (Ishihara et al., 1988). Moreover, our recent studies indicated that tetragastrin protects the gastric mucosa from necrotizing agents (Komuro et al., 1992) and significantly increases the biosynthesis of mucin (Ichikawa et al., in press). From these results, it is suggested that the activation of gastric mucin biosynthesis by a drug is closely related to the presence of mucosal protective activity or the so-called cytoprotection. Current peptic ulcer therapy relies largely on potent anti-acid secretory agents such as the histamine H z receptor antagonists and the H+,K+-ATPase inhibitor. These agents are thought to promote ulcer healing by removing the aggressive actions of gastric acid on the gastroduodenal mucosa. However, the gastric cytoprotective properties of these agents are still controversial. In this study, for clarification of the effects of anti-acid secretory agents on gastric mucin metabolism, rat gastric tissue was cultured in the presence of these drugs and an assessment was made of mucin biosynthesis. Of the four histamine H a receptor antagonists used in the present work, roxatidine has been reported to prevent the formation of gastric mucosal lesions induced by necrotizing agents in rats (Shiratsuchi et al., 1988), and this prevention may have been due not only to the inhibition of aggressive factors such as acid but also to the maintenance of defensive factors such as the gastric mucosal hexosamine content and gastric mucosal potential difference. Our recent data indicate that the administration of FRG-8813 inhibits ethanolinduced gastric injury accompanied by the conservation of mucin content (Ishihara et al., 1992). In this study, 3H-labeled mucin in the corpus tissue increased by about 30 ~ 50% above that of the control after the addition of roxatidine and FRG-8813. These results confirm that roxatidine and FRG-8813 not only inhibit
110
gastric acid secretion as histamine H 2 receptor antagonists but may also promote gastric mucosal protective action as well. On the other hand, many reports have indicated that cimetidine and ranitidine lack a protective effect against necrotizing agent-induced gastric mucosal damage in the rat (Tarnawski et al., 1985; Shiratsuchi et al., 1988). The present results show that cimetidine and ranitidine failed to stimulate mucin biosynthesis. A similar result for ranitidine was obtained by Heim et al. (1990), using isolated pig gastric mucosal cells. This result, together with our results, confirms that cimetidine and ranitidine may lack effects on the defensive factors of the gastric mucosa although they reduce the aggressive forces such as acid secretion. Heim et al. (1990) found histamine to function via histamine H 2 receptors as a stimulant of protein and glycoprotein production in isolated pig gastric nonparietal cells. In this study, the effect of roxatidine or FRG-8813 in the corpus was not due to H 2 antagonism since the other H 2 antagonists did not affect mucin production. Our present results also show that none of the drugs examined in this study caused any significant change in the mucin biosynthesis of the antrum. As previously mentioned, prostaglandins significantly increase mucin biosynthesis in both the corpus and antral regions, and consequently, the stimulation of mucin biosynthesis by anti-acid secretory agents might not be considered to be mediated by prostaglandins. The mechanisms responsible for regulating mucin biosynthesis are currently not well understood. If roxatidine or FRG-8813 has a direct effect on the mucus producing cells in the oxyntic region, they might be very useful tools for the further clarification of the regulatory mechanism of mucin synthesis. It has been suggested that the critical structural feature of histamine H 2 receptor antagonists is the imidazole ring or ethylthiomethyl ring side chain of the various guanidine derivatives (Durant et al., 1978). Roxatidine and FRG-8813 differ considerably in chemical structure from the other two conventional histamine H 2 receptor antagonists and have neither of these structural components. They have a unique sixmembered aromatic ring, such as the benzene (roxatidine) and pyridine (FRG-8813) derivatives, instead of a five-membered heterocyclic ring (fig. 1). It may be assumed that the difference in the effects on the cytoprotective action is due to these structural differences. Therefore, we postulate that if a new histamine H 2 receptor antagonist has a six-membered aromatic ring, the drug might have gastric mucosal protective actions as well as accelerate mucus production. The H +,K+-ATPase inhibitors, omeprazole and NC1300-O-3, have been reported to inhibit strongly gastric acid secretion. Ohara et al. (1989) indicated that NC1300, the prototype drug of NC-1300-O-3, significantly
increased the mucin content of the gastric gel layer in rats, whereas omeprazole did not cause any significant change in the mucin content after low-dose administration and caused a slight reduction in corpus mucin content following high-dose administration. Our preliminary study (Ichikawa et al., 1991) and the present results clearly demonstrate the difference in the effect on mucin biosynthesis of omeprazole and NC-1300-O-3. In conclusion, the present findings demonstrated that the histamine H 2 receptor antagonists which have a six-membered aromatic ring, roxatidine and FRG8813, and the new H+,K+-ATPase inhibitor, NC-13000-3, not only inhibit acid secretion but might also promote gastric mucosal protective actions.
Acknowledgements This work was supported in part by Grants-in-Aid from the Research Funds of Kitasato Gakuen and the Japanese Ministry of Education.
References Allen, A., D. Hutton, A. Leonard, J. Pearson and L. Sellers, 1986, The role of mucus in the protection of the gastroduodenal mucosa, Scand. J. Gastroenterol. 21 (Suppl. 125), 71. Azuumi, Y., S. Ohara, K. Ishihara, H. Okabe and K. Hotta, 1980, Correlation of quantitative changes of gastric mucosal glycoproteins with aspirin-induced gastric damage in rats, Gut 21,533. Borg, K., L. Olbe, S. Rune and A. Walan, eds., 1986, Proceedings of the First International Symposium on Omeprazole, Scand. J. Gastroenterol. 20 (Suppl. 108), 1. Durant, G.J., W.A.M. Duncan, C.R. Ganellin, M.E. Parsons, R.C. Blakemore and A.C. Rasmussen, 1978, Impromidine (SK&F 92676) is a very potent and specific agonist for histamine H 2 receptors, Nature 276, 403. Eastwood, G.L. and J.S. Trier, 1973, Organ culture of human rectal mucosa, Gastroenterology 64, 375. Heim, H., A. Oestmann and K. Sewing, 1990, Effects of histamine on protein and glycoprotein production of isolated pig gastric mucosal cells, Pharmacology 40, 265. Ichikawa, T., K. Ishihara, K. Saigenji and K. Hotta, 1991, Effects of omeprazole on gastric mucus glycoprotein biosynthesis, Gastroenterol. Jpn. 26, 390. Ichikawa, T., K. Ishihara, K. Saigenji and K. Hotta, 1993, Stimulation of mucus glycoprotein biosynthesis in rat gastric mucosa by gastrin, Biochem. Pharmacol. (in press). Ishihara, K., H. Kuwata, S. Ohara, H. Okabe and K. Hotta, 1988, Changes of rat gastric mucus glycoproteins in cytoprotection: influences of prostaglandin derivatives, Digestion 39, 162. Ishihara, K., Y. Komuro, T. Ichikawa and K. Hotta, 1992, Effects of antiulcer drugs, NC-1300-O-3, FRG-8813 and Z-300 on ethanolinduced damage and gastric mucin content in rat stomach, Hell. J. Gastroenterol. 5 (Suppl.), 131 (Abstract No. 523). Komuro, Y., K. Ishihara, S. Ohara, K. Saigenji and K. Hotta, 1992, Effects of tetragastrin on mucus glycoprotein in rat gastric mucosal protection, Gastroenterol. Jpn. 27, 597. Ohara, K., H. Ohkawa, K. Ishihara, K. Hotta, Y. Komuro and H. Okabe, 1989, Effect of the proton pump inhibitor, NC-1300, on
111 gastric mucus secretion in rats, Scand. J. Gastroenterol. 24 (Suppl. 162), 186. Robert, A., J. Nezamis, C. Lancaster and A. Hanchar, 1979, Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCI, NaOH, hypertonic NaCI, and thermal injury, Gastroenterology 77, 433. Shiratsuchi, K., H. Fuse, M. Hagiwara, T. Mikami, K. Miyasaka and
H. Sakuma, 1988, Cytoprotective action of roxatidine acetate HCI, Arch. Int. Pharmacodyn. 294, 295. Tarnawski, A., D. Hollander, H. Gergely and J. Stachura, 1985, Comparison of antacid, sucralfate, cimetidine, and ranitidine in protection of the gastric mucosa against ethanol injury, Am. J. Med. 79 (Suppl. 2C), 19.
107
EJP 21407
Short communication
Effects of acid-inhibitory antiulcer drugs on mucin biosynthesis in the rat stomach T a k a f u m i I c h i k a w a a, K a z u h i k o I s h i h a r a b, K a t s u n o r i Saigenji c a n d K y o k o H o t t a *'a Departments of "Biochemistry, b Chemistry and c Internal Medicine, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa 228, Japan Received 8 September 1993, accepted 9 November 1993
The effects of the anti-acid secretory agents, cimetidine (N-cyano-N'-methyl-N"-(2-([(5-methyl-lH-imidazol-4yl)methyl]thio)ethyl)guanidine), ranitidine (N-(2-(((-5-[(dimethy•amin•)methy•]-2-furany•)methy•)thi•)ethy•)-N'-methy•-2-nitr•1,1-ethene-diamine), roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide hydrochloride), FRG-8813 (2-(furfury•su••ny•)-N-(4-[4-(piperidin•methy•)-2-pyridy•]•xy-(z)-2-buteny•)acetamide)• omeprazole (5-methoxy-2-([(4-methoxy3,5-dimethylpyridinyl)methyl]sulfinyl)-lH-benzimidazole) and NC-1300-O-3 (2-([2-(isobutylmethylamino)benzyl]sulfinyl)-lHbenzimidazole), on mucin biosynthesis were studied in rat gastric mucosa by using an organ culture technique. [3H]Glucosamine incorporation was stimulated in the corpus region by the histamine H 2 receptor antagonists which have a six-membered aromatic ring, roxatidine and FRG-8813, and the new H+,K+-ATPase inhibitor, NC-1300-O-3. Thus, these drugs not only inhibit acid secretion but may also promote gastric mucosal protective actions. The present observations also demonstrate that the determination of mucin biosynthesis may be a useful tool for evaluation of mucosal protective activity. Histamine H 2 receptor antagonist; H+,K+-ATPase inhibitor; Gastric mucin biosynthesis; Organ culture; (Rat)
1. Introduction
Medical therapy for peptic ulcer disease has been targeted at inhibiting acid secretion, based on the belief that ulcers occur as a result of an imbalance between luminal aggressive and mucosal defensive factors. The introduction of histamine H e receptor antagonists has provided effective non-surgical therapy for duodenal and gastric ulcers by inhibiting acid-peptic activity. More recently, omeprazole (5-methoxy-2-([(4methoxy-3,5-dimethylpyridinyl)methyl]sulfinyl)-1 H-benzimidazole), an H+,K+-ATPase inhibitor, showed the ability to inhibit acid secretion totally and to heal a duodenal ulcer within 2 weeks (Borg et al., 1986). Compared with the aggressive factors, little attention has been paid to the mucosal defensive factors in ulcer therapy and the role of these drugs in gastric mucosal protection has not been well characterized. Mucin, a major component of gastric mucus, is a high-molecular-weight compound of unique structure and is considered to be an important mucosal defensive factor (Allen et al., 1986). The purpose of this
* Corresponding author. Tel. 81-427-78-9267, fax 81-427-78-8441.
SSDI 0 0 1 4 - 2 9 9 9 ( 9 3 ) E 0 8 2 1 - 9
study was to compare the effects of the following histamine H 2 receptor antagonists and H+,K+-ATPase inhibitors on mucin biosynthesis in an organ culture system of the rat stomach. Four established drugs, cimetidine ( N-cyano-N '-methyl-N "-(2-([(5-methyl- l Himidazol-4-yl)methyl]thio)ethyl)guanidine), ranitidine (N-(2-(((-5-[(dimethylamino)methyl]-2-furanyl)methyl)thio)ethyl)-N'-methyl-2-nitro-l,l-ethene-diamine), roxatidine (2-acetoxy-N-(3-[ m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide hydrochloride) and omeprazole, and two new drugs, FRG-8813 (2-(furfurylsulfinyl)-N-(4-[4-(piperidinomethyl)-2-pyridyl]oxy-(z)-2butenyl)acetamide) and NC-1300-O-3 (2-([2-(isobutylmethylamino)benzyl]sulfinyl)-1H-benz-imidazole) (Ishihara et al., 1992), were studied (fig. 1).
2. Materials and methods
2.1. Experimental animals Seven-week-old male Wistar rats (SLC, Shizuoka, Japan) each weighing approximately 160 g were used. All were fasted for 24 h before the experiments and had free access to water during this time.
108
2.2. Organ culture The stomachs of the rats were excised immediately after they were killed by light anesthetization followed by exsanguination from the carotid artery. The stomachs were then cut along the greater curvature, and the luminal surface was gently washed with Ca2+/Mg 2+free phosphate-buffered saline (PBS(-)). The glandular part was selected, separated into the corpus and antrum, and cut into small 2 x 2 mm sections. The tissue culture method of Eastwood and Trier (1973) was used with a modification (Ishihara et al., 1988). Eight tissue fragments, the mucosal surface facing up, were placed on a stainless steel grid in the central well of a plastic culture dish (60 × 15 mm, Falcon, USA) containing 0.75 ml of culture medium. The medium consisted of 90% Eagle's minimum essential medium and 10% dialyzed fetal calf serum, with 370 kBq/ml of D-[1,6-3H(N)]glucosamine hydrochloride (1950 G B q / mmol, New England Nuclear). For the addition of anti-acid secretory agents to the culture medium, the drug was dissolved in dimethyl sulfoxide (DMSO) and added at concentrations of 10 -8 , 10 -7 , 10 -6 and 10 -5 M to the dishes, making the final concentration of
N y CI-13 Cimetldine
N ~ ~. CH2.S.CH2-CH2-N=C.NH-CH3 /
NH-CN
CH3.~.
Ranitidine
CH31N-CH2-~ O":~CH2-S-CH2-CH2-NH-CIINH-CH 3 CH-NO2 HC1
- c.2--~,~,,o_c~2_c.2.c.~.~..c.2.o~c~, •
FRG-8813
2.3. Isolation of labeled mucin and radioactivity measurements Upon completion of the culture period, the tissue fragments on a grid were harvested from the medium, gently rinsed with P B S ( - ) and boiled at 100°C for 3 rain in 0.4 ml of 0.05 M Tris-HCl buffer, pH 7.2. The extraction and isolation of mucin were performed as previously described (Azuumi et al., 1980). The tissue fragments were homogenized with a Physcotron microhomogenizer (Niti-On, Chiba, Japan). Triton X-100 was added to a 2% (v/v) concentration and the homogenate was shaken for 1 h at 37°C. The homogenate thus obtained was centrifuged at 8000 × g for 30 min to obtain the supernatant. A 0.4-ml portion of the supernatant was applied onto a Bio-Gel A-1.5m column (1 X 30 cm) previously equilibrated with the Tris buffer containing 2% Triton X-100, and the column was eluted with this buffer. Finally, fractions of 0.8 ml each were collected and the radioactivity was measured by using a scintillation counter (Beckman, Model LS2800, USA) with Aquasol-2 (New England Nuclear, USA) as the scintillant. The radioactivity incorporated into the void volume of the column, which had been demonstrated to be the synthesized mucin (Ichikawa et al., in press), was determined. To compare the synthesis of mucin, the total radioactivity of this fraction was divided by the tissue protein content of each homogenate to give the value relative to that of the control. 2.4. Drugs
•
Roxatidine
DMSO 0.01%. A DMSO solution without drug was added to the medium in the control well. All the dishes were maintained at 37°C for 5 h in 5% CO 2 and 95% air.
HC1
ell2 ~.CH2.CH=CH.CH2.NH.~I.CH2.~I.CH 2 " ~
The following drugs were obtained for use in this study: cimetidine (Smith-Kline Beecham, Japan); ranitidine" hydrochloride (Sankyo, Japan); roxatidine acetate hydrochloride (Takeda, Japan); FRG-8813 (Fujirebio, Japan); omeprazole (Fujisawa-Astra, Japan); NC-1300-O-3 (Nippon Chemiphar, Japan).
2.5. Protein determination N Omeprazole
,,,~.,~
CH3-O
N/
H
N II
O
dn~ \ O-CH3
Protein content in the tissue homogenate was determined by using the Pierce BCA protein assay kit with bovine serum albumin as the standard.
2.6. Statistical analysis Ne-1300-O-3
'/~,,~'~'N" ~ H
CH3-N-CH2-CH-CH3 I CH3 Fig. I. Structures of the drugs used in this study. Upper panel: histamine t t 2 receptor antagonists; lower panel: H+,K+-ATPase inhibitors.
The results were expressed as means + S.D. A oneway analysis of variance (ANOVA) with Dunnett's test was used for statistical analysis with P < 0.05 taken as significant.
109
3. Results
3.1. Influence of anti-acid secretory agents on mucin biosynthesis in the corpus region Fig. 2 shows the mucin biosynthetic activity of the corpus as measured by [3H]glucosamine incorporation. The biosynthesis of mucin in the control was 18.3 + 1.8 dpm//zg tissue protein. The changes in the biosynthesis of mucin by the addition of each histamine H 2 receptor antagonist are shown in fig. 2a. Roxatidine and FRG-8813 significantly and dose dependently increased [3H]glucosamine incorporation into mucin. On the other hand, the addition of cimetidine and ranitidine yielded no significant change in mucin biosynthesis (fig. 2a). Fig. 2b shows the biosynthesis of mucin in the corpus after the addition of omeprazole or NC-1300-O-3. The addition of NC-1300-O-3 enhanced [3H]glucosamine incorporation into mucin by about 20 ~ 25% (P < 0.05), but omeprazole had no effect on mucin biosynthesis.
3.2. Influence of anti-acid secretory agents on mucin biosynthesis in the antral region No significant change could be detected in the mucin biosynthesis of the antrum after addition of 10 - 8 ~ % " (a) 180. 160.
ROX
----h--.
FRG Cim
•
Ran
---4P--
140. 120. >
--I...........
100. .o
80. 6O %
w
140
(b) •
.... @...
NC Opz
**
*
120 100. 80 60 C o n c e n t r a t i o n (M)
Fig. 2. Effects of anti-acid secretory agents on mucin biosynthesis. (a) Influence of the histamine H 2 receptor antagonists on [3H]glucosamine incorporation into mucins in the corpus region. (b) Influence of the H+,K+-ATPase inhibitors on [3H]glucosamine incorporation into mucins in the corpus region. Cim: cimetidine, Ran: ranitidine, Rox: roxatidine, FRG: FRG-8813, Opz: omeprazole, NC: NC-13000-3. Values are expressed as percentages of the control and represent means + S.D. from 8 different samples. * P < 0.05 and ** P < 0.01 as compared with the control value.
10 -5 M of each histamine H 2 receptor antagonist (specific activity: 28.9 ~ 33.5 dpm//zg tissue protein). The same results were obtained after the addition of omeprazole and NC-1300-O-3 (data not shown).
4. Discussion
Since the concept of 'cytoprotection' was introduced by Robert et al. (1979) as an ability of prostaglandins which is independent of the anti-acid secretory effect, many drugs have been investigated for their activity to protect the gastric mucosa from a variety of necrotizing agents. Although the exact mechanism of mucosal cytoprotection is unknown, it presumably involves one or more of the naturally occurring defensive factors of gastric mucosa such as mucin metabolism. Previously, we reported that exogenous prostaglandins, which are capable of inducing direct cytoprotection, significantly increase the biosynthesis of mucins in the corpus and antrum of the rat stomach (Ishihara et al., 1988). Moreover, our recent studies indicated that tetragastrin protects the gastric mucosa from necrotizing agents (Komuro et al., 1992) and significantly increases the biosynthesis of mucin (Ichikawa et al., in press). From these results, it is suggested that the activation of gastric mucin biosynthesis by a drug is closely related to the presence of mucosal protective activity or the so-called cytoprotection. Current peptic ulcer therapy relies largely on potent anti-acid secretory agents such as the histamine H z receptor antagonists and the H+,K+-ATPase inhibitor. These agents are thought to promote ulcer healing by removing the aggressive actions of gastric acid on the gastroduodenal mucosa. However, the gastric cytoprotective properties of these agents are still controversial. In this study, for clarification of the effects of anti-acid secretory agents on gastric mucin metabolism, rat gastric tissue was cultured in the presence of these drugs and an assessment was made of mucin biosynthesis. Of the four histamine H a receptor antagonists used in the present work, roxatidine has been reported to prevent the formation of gastric mucosal lesions induced by necrotizing agents in rats (Shiratsuchi et al., 1988), and this prevention may have been due not only to the inhibition of aggressive factors such as acid but also to the maintenance of defensive factors such as the gastric mucosal hexosamine content and gastric mucosal potential difference. Our recent data indicate that the administration of FRG-8813 inhibits ethanolinduced gastric injury accompanied by the conservation of mucin content (Ishihara et al., 1992). In this study, 3H-labeled mucin in the corpus tissue increased by about 30 ~ 50% above that of the control after the addition of roxatidine and FRG-8813. These results confirm that roxatidine and FRG-8813 not only inhibit
110
gastric acid secretion as histamine H 2 receptor antagonists but may also promote gastric mucosal protective action as well. On the other hand, many reports have indicated that cimetidine and ranitidine lack a protective effect against necrotizing agent-induced gastric mucosal damage in the rat (Tarnawski et al., 1985; Shiratsuchi et al., 1988). The present results show that cimetidine and ranitidine failed to stimulate mucin biosynthesis. A similar result for ranitidine was obtained by Heim et al. (1990), using isolated pig gastric mucosal cells. This result, together with our results, confirms that cimetidine and ranitidine may lack effects on the defensive factors of the gastric mucosa although they reduce the aggressive forces such as acid secretion. Heim et al. (1990) found histamine to function via histamine H 2 receptors as a stimulant of protein and glycoprotein production in isolated pig gastric nonparietal cells. In this study, the effect of roxatidine or FRG-8813 in the corpus was not due to H 2 antagonism since the other H 2 antagonists did not affect mucin production. Our present results also show that none of the drugs examined in this study caused any significant change in the mucin biosynthesis of the antrum. As previously mentioned, prostaglandins significantly increase mucin biosynthesis in both the corpus and antral regions, and consequently, the stimulation of mucin biosynthesis by anti-acid secretory agents might not be considered to be mediated by prostaglandins. The mechanisms responsible for regulating mucin biosynthesis are currently not well understood. If roxatidine or FRG-8813 has a direct effect on the mucus producing cells in the oxyntic region, they might be very useful tools for the further clarification of the regulatory mechanism of mucin synthesis. It has been suggested that the critical structural feature of histamine H 2 receptor antagonists is the imidazole ring or ethylthiomethyl ring side chain of the various guanidine derivatives (Durant et al., 1978). Roxatidine and FRG-8813 differ considerably in chemical structure from the other two conventional histamine H 2 receptor antagonists and have neither of these structural components. They have a unique sixmembered aromatic ring, such as the benzene (roxatidine) and pyridine (FRG-8813) derivatives, instead of a five-membered heterocyclic ring (fig. 1). It may be assumed that the difference in the effects on the cytoprotective action is due to these structural differences. Therefore, we postulate that if a new histamine H 2 receptor antagonist has a six-membered aromatic ring, the drug might have gastric mucosal protective actions as well as accelerate mucus production. The H +,K+-ATPase inhibitors, omeprazole and NC1300-O-3, have been reported to inhibit strongly gastric acid secretion. Ohara et al. (1989) indicated that NC1300, the prototype drug of NC-1300-O-3, significantly
increased the mucin content of the gastric gel layer in rats, whereas omeprazole did not cause any significant change in the mucin content after low-dose administration and caused a slight reduction in corpus mucin content following high-dose administration. Our preliminary study (Ichikawa et al., 1991) and the present results clearly demonstrate the difference in the effect on mucin biosynthesis of omeprazole and NC-1300-O-3. In conclusion, the present findings demonstrated that the histamine H 2 receptor antagonists which have a six-membered aromatic ring, roxatidine and FRG8813, and the new H+,K+-ATPase inhibitor, NC-13000-3, not only inhibit acid secretion but might also promote gastric mucosal protective actions.
Acknowledgements This work was supported in part by Grants-in-Aid from the Research Funds of Kitasato Gakuen and the Japanese Ministry of Education.
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