Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992), pp. 1593-1599
Effects of Antiulcer Drugs on Phosphatidylcholine Synthesis in Isolated Guinea Pig Gastric Glands HOGARA NISHISAKI, CHOITSU SAKAMOTO, YOSHITAKA KONDA, OSAMU NAKANO, TAKASHI MATOZAKI, MUNEHIKO NAGAO, KOHEI MATSUDA, KEN WADA, and MASATO KASUGA
To better understand phosphatidylcholine synthesis in the stomach, we isolated guinea pig gastric glands and examined their [3H]choline incorporation into phosphatidylcholine in response to either antiulcer drugs such as geranylgeranylacetone (GGA) and H 2receptor antagonists or agents that cause phosphatidylcholine synthesis in other tissues. [3H]Choline incorporation was stimulated by GGA, palmitate, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Dibutyryl cyclic-AMP had no effect. By contrast with GGA, famotidine, ranitidine, and cimetidine equipotently inhibited [3H]choline incorporation into phosphatidylcholine. GGA, palmitate, and TPA increased phosphatidyl-[3H]choline and decreased phosphoryi-[3H]choline as compared with control in tissues that had been pulsed with [3H]choline. On the other hand, no more decrease in [3H]choline incorporation at chase periods was observed in pulse-labeled glands in response to each H2-receptor antagonist. The particulate fraction o f glands that had been incubated with GGA or palmitate had more CTP-phosphocholine cytidylyltransferase activity than that of glands incubated without agents. A decrease in choline kinase activity was not observed in the cytosolic fraction of glands that had been incubated with cimetidine. These results suggest that GGA and palmitate stimulate phosphatidylcholine synthesis by activating cytidylyltransferase, and H2-receptor antagonists may affect phosphatidytcholine synthesis by inhibiting choline uptake in the gastric glands. KEY WORDS: ulcer; antiulcer drugs; phosphatidyicholine synthesis; gastric glands.
It has been suggested recently that not only the mucus layer adjacent to the wall of the gastrointestinal tract but also surface active phospholipids chemically similar to pulmonary surfactants may play an important role in protecting the mucosal surface of the gastrointestinal tract against the meManuscript received September 17, 1990; revised manuscript received October 23, 1991; accepted December 16, 1991. From the Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Address for reprint requests: Dr. Choitsu Sakamoto, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
chanical forces of digestion or acidic luminal fluids (1-3). Exogenously administered liposomal surfactant suspension has been shown to reduce acidinduced gastric ulcerogenesis and bleeding in rats (4). Furthermore, the protection of gastric mucosa by prostaglandin has been suggested to be, at least in part, due to the maintenance of a nonwettable hydrophobic lining on the gastric epithelium (5). Surfactant phospholipids in the lung have been well characterized, and their ability to reduce surface tension at the interface between air and liquid has been shown to be essential for pulmonary mechanics and homeostasis.
Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992) 0163-2116/92/1000-1593506.50/0 9 1992PlenumPublishingCorporation
1593
NISHISAKI ET AL Dipalmitoylphosphatidylcholine, a the major component of these surfactant lipids that is synthesized in alveolar type II cells, has been shown to be secreted by at least two different mechanisms, one of which is dependent on cellular cyclic AMP and the other involving protein kinase C (6-8). By contrast with pulmonary surfactants, however, not only the mechanism by which surfactant like phospholipids are synthesized but also whether they are secreted in the stomach are not yet known. Furthermore, if gastric surfactant like phospholipids are secreted, it is also not yet known what cells in the stomach secrete them. Accordingly, as an initial step, we investigated the mechanism by which phosphatidylcholine, a major component of gastric mucosal phospholipids (9) is synthesized in the isolated guinea pig gastric glands in vitro. Furthermore, we examined whether phosphatidylcholine biosynthesis is influenced by both antiulcer drugs and an agent that stimulates the activation of protein kinase C or cyclic AMP-dependent protein kinase. We tested as antiulcer drugs Hzreceptor antagonists and a new acyclic isoprenoid that has been commonly used for treating the patients with gastric ulcer in Japan (10, 11). We show in the present study that antiulcer drugs may affect the biosynthetic pathway to decrease or increase phosphatidylcholine synthesis in gastric glands in vitro.
MATERIALS AND METHODS Materials. [methyl-3H]Choline chloride (80 Ci/mmol) and phosphoryl[methyl-lnC]choline (50 mCi/mmol) were obtained from Amersham Japan; authentic phospholipid standards from Sigma; silica gel 60 plates from Merck; 12-O-tetradecanoyl-phorbol-13-acetate (TPA), CTP, and type I collagenase were from Sigma. Geranylgeranylacetone (6,10,14,18-tetramethyl5,9,13,17-nonadecatetraene-2-one; purity 99.2%) (GGA) was synthesized and donated by Eisai Co., Ltd., Tokyo, Japan (12, 13). Preparation of Guinea Pig Gastric Glands. The isolated guinea pig gastric glands were prepared as described previously (14). Briefly, the minced fundic and corpus mucosa was digested for 50 min at 37~ C in a solution consisting of 132 mM NaC1, 4.7 mM KC1, 11.1 mM MgCI2, 5 mM Na2HPO 4, 1 mM NaH2PO4, 11.1 mM glucose, essential amino acids, and 0.5% bovine serum albumin and containing 0.1% collagenase. At the end of incubation, the glands were filtered through nylon mesh to remove coarse fragments and rinsed. Finally, the glands were suspended in the aforementioned solution containing 1.28 mM CaC12 at pH 7.4 (KRP buffer). Incorporation of [all]Choline into Gastric Glands and Extraction of Phospholipids. Gastric glands suspended in
the aforementioned KRP buffer were incubated with 0.5 ~Ci/ml [3H]choline in the presence of test agents. We
1594
used as test agents palmitate, TPA, dibutyryl cyclic-AMP (dbcAMP), and antiulcer drugs GGA and H2-receptor antagonists such as famotidine, ranitidine, and cimetidine. TPA was dissolved in dimethyl sulfoxide and the final concentration of dimethyl sulfoxide at 0.01% was added for control experiments. GGA added into the buffer was sonicated to prepare micelles. After incubation for up to 180 min at 37~ C, the glands were washed twice with ice-cold KRP buffer and then labeled phospholipids were extracted by adding 1.88 ml chloroform-methanol (1 : 2, v/v) into 0.5 ml of the gland suspension according to the method of Bligh and Dyer (15). Each phospholipid was resolved from the organic phase by thin-layer chromatography (TLC) using chloroform-methanol-acetic acid-H20 (50: 30: 8: 4, v/v) as solvent. Pulse-Chase Study. Isolated gastric glands suspended in KRP buffer were incubated with 1.0 IxCi/ml [3H]choline for 15 min. Thereafter, the medium was removed, the glands were washed, and then the aforementioned test agents added into the gland suspension followed by further incubation. At the indicated time, the reaction was stopped by adding 1.88 ml chloroform-methanol (1:2, v/v) to the suspension. Radioactivity in the aqueous upper phase and organic lower phase of the extractions was determined by liquid scintillation counting. The distribution of radioactivity among water-soluble choline metabolites in the upper phase was examined by TLC using methanol-0.5% NaC1-NH3 (50:50:1, v/v) as solvent and choline-containing phospholipids in the lower phase examined as described above. Determination of Cytidylyltransferase Activity in Particulate Fraction of Gastric Glands. Isolated gastric glands
were incubated for 180 min at 37~ C in KRP buffer in the presence of test agents. At the end of incubation, the glands were washed, resuspended in 50 mM Tris buffer (pH 7.4), and homogenized with a Teflon-glass homogenizer. The homogenate was initially centrifuged until the speed attained 2000 rpm in a Tomy Seiko RL-7000 centrifuge and then switched off. After the supernatant was centrifuged at 100,000g for 60 min, the resulting pellets were used for the enzyme assay. CTP-phosphocholine cytidylyltransferase activity was determined by measuring the incorporation of radioactivity from [methyl-14C]phosphocholine into CDP choline (16). The incubation mixture contained 50 mM Tris HC1, 5 mM CTP, 10 mM MgC12, 0.6 mM [methyl-14C]phospho choline, and up to 100 t*g protein in a final volume of 50 pA. Incubation was carried out at 37~ C over 15 min. The phosphocholine and CDP choline generated were separated by TLC on silica gel 60 plates with the solvent system described above. Spots were visualized, scraped off, and assayed for radioactivity. Determination of Choline Kinase Activity in Cytosolic Fraction of Gastric Glands. Choline kinase activity was
assayed according to the method previously described (17). The formation of [3H]phosphocholine from [methyl3H]choline was determined. The assay buffer contained 50 mM NaC1, 50 mM ATP, 67 mM Tris HC1 (pH 8.5), 100 mM MgC12, 5 mM [3H]choline, and 30 ixg of cytosolic protein, which was prepared by centrifugation at 100,000g for 60 min of homogenate of gastric glands that had been incubated with or without cimetidine for 180 min. After 10 rain Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
PHOSPHATIDYLCHOLINE SYNTHESIS IN GASTRIC GLANDS TABLE 1. INCORPORATION OF [3H]CHOLINE INTO VARIOUS PHOSPHOLIPIDS IN GASTRIC GLANDS*
Incubation time (min) Phospholipids
30
60
120
180
Phosphatidylcholine Lysophosphatidylcholine Sphingomyelin
1585 -+ 21 27 -+ 10 14 _+ 3
3073 -+ 10 62 -+ 3 38 -+ 3
4147 -+ 14 91 -+ 5 50 --- 5
5027 +- 74 261 -+ 23 170 -+ 51
*Gastric glands were incubated with [3H]choline for the indicated time. Thereafter phospholipids were extracted and separated by T L C . Values are m e a n -+ SE o f four separate e x p e r i m e n t s and e x p r e s s e d as c p m per sample.
of incubation at 37~ C, the reaction was stopped by boiling. Choline and phosphocholine were separated by TLC as described above. Protein was measured with the Bio-Rad reagent as described by Bradford (18). Trypan Blue Exclusion Test. Gastric glands before or after incubation with drugs for 3 hr were washed with and resuspended in phosphate-buffered saline, and thereafter 0.1 ml trypan blue solution (0.4 g/dl) was added to the suspension. The number of stained or nonstained cells was counted within 10 rain. Statistical Analysis. All values are given mean -+ sE. An analysis of variance was used for statistical evaluation. With these analyses, an associated probability (P value) of less than 5% was considered statistically significant. All experiments were performed at least three times in duplicate unless otherwise indicated. RESULTS [3H]Choline uptake by isolated guinea pig gastric glands into phospholipids increased with time of incubation. H o w e v e r , at any time point, nearly 90% o f the lipid-associated radiolabel had been incorporated into phosphatidylcholine, with the remainder in sphingomyelin and lysophosphatidylcholine (Table 1). Therefore, the incorporation of [3H]choline into phosphatidylcholine was expressed as a percentage o f the radioactivity in the lipid fraction to total glandular radioactivity. The incorporation o f [3H]choline into phosphatidylcholine attained at 180 min was 21 --- 2% (mean - SE, N = 12) in controls. Palmitate (10 -3 M), TPA (1.6 x 10 -6 M), and G G A (10 -3 M) significantly increased the incorporation up to 37 -+ 4% (mean ----SE, N = 6; P < 0.01) 28 • 3% (mean • SE, N = 4; P < 0.05), and 31 • 2% (mean --- SE, N = 6; P < 0.05) at 180 min, respectively. On the other hand, the H2-receptor antagonist famotidine (10 -3 M), ranitidine (10 -3 M), and cimetidine (10 -3 M) significantly decreased the incorporation to 16 --- 2% (mean --- SE, N = 6; P < 0.05) 15 • 2% (mean • sE, N = 6; P < 0.05), and 14 • 2% (mean --- SE, N = 6; P < 0.05), respectively, d b c A M P had no effect on Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
the incorporation of [3H]choline into phosphatidylcholine (Figure 1). Palmitate, TPA, and G G A dose-dependently stimulated, but famotidine, ranitidine and cimetidine dose-dependently inhibited the incorporation of [3H]choline into phosphatidylcholine. Minimum effective doses of palmitate, TPA, and G G A were 3 x 10 -4 M, 3 • 10 -7 M, and 10 -4 M, respectively. Palmitate produced a 1.7-fold increase in the incorporation, with the maximal stimulation at 10 -3 M, whereas TPA and G G A produced a 1.4-fold increase at 3 x 10 -7 M and 10 -3 M, respectively. On the other hand, dbcAMP did not stimulate [3H]choline incorporation into phosphatidylcholine at all. Minimum effective doses of all tested H2-receptor antagonists
eo
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~
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~
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m 20 IM Z ..A 0
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Fig 1. Time-dependent increases in [3H]choline incorporation into phosphatidylcholine in gastric glands. Gastric glands were incubated in the absence ((3 O) or presence of either 10-3 M palmitate (tk------~), 1.6 • 10-6 M TPA (_A -'), 10 -3 M GGA (D------I1), 10-3 M dbcAMP (I-1 V1), 10-3 M cimetidine (~--(>), 10-3 M famotidine (A A), or 10-3 M ranitidine (V V) for 180 min. Thereafter gastric phospholipids were extracted, and the radioactivity in the phospholipid fraction was measured. [3H]Choline incorporation into phosphatidylcholine was expressed as a percentage of the radioactivity in the phospholipid fraction to total cellular radioactivity. Values indicated (*P < 0.05, **P < 0.01) are significantlydifferent from control.
1595
NISHISAKI ET AL ~O 50
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Fig 2. Dose-dependent stimulation or inhibition of [3H]choline incorporation into phosphatidylcholine by various drugs. Gastric glands were incubated in the presence or absence of varying doses of test agents for 180 rain at 37~ C. Thereafter gastric phospholipids were extracted, and the radioactivity in the phospholipid fraction was measured. [3H]Choline incorporation was expressed as a percentage of control. Symbols used are the same as those in Figure 1. Each value is the mean - SE of at least four separate experiments. Values indicated (*P < 0.05, **P < 0.01)
are significantlydifferentfrom control. to inhibit the incorporation were the same, ie, 10-3 M. Furthermore, all H2-receptor antagonists equipotently decreased the incorporation with the maximal reduction at 10-2 M to 50% of control (Figure 2). To eliminate the possibility that either an increase or a decrease in [3H]choline uptake into gastric glands in response to drugs may be due to nonspecific injury of gastric cells exposed to high concentrations of the drugs, we examined gastric cell viability by using the trypan blue dye exclusion test. Although viability of gastric cells exposed to 10-2 M of an Hz-receptor antagonist for 180 min decreased from 95 -+ 3% to 75 - 4%, there was no significant difference in viability between the gastric cells exposed to an H2-receptor antagonist and gastric cells incubated for 180 min without drugs. Palmitate partially restored H2-receptor antagonist-induced reduction of the [3H]choline incorporation into phosphatidylcholine when the glands were incubated simultaneously with palmitate and an H2-receptor antagonist. The incorporation was 27 -+ 2% (mean -+ SE, N = 6) with palmitate plus cimetidine, the value of which was significantly greater than 21 --+ 2% in control (P < 0.01) and smaller than 37 +- 4% with palmitate alone (P < 0.01) (Figure 3). To further explore which enzymatic step regulated [3H]choline incorporation into phosphatidylcholine, gastric glands were pulsed with [3H]choline, then medium aspirated, and the glands chased in the ab-
1596
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20
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~--, plus 10 -3 M cimetidine; A A, plus 10-3 M famotidine; ~7 V, plus 10 -3 M ranitidine for 180 min. [3H]Choline incorporation was expressed as a percentage of the radioactivity in the phospholipid fraction to total cellular radioactivity. Each value is the mean -+ SE of at least six separate experiments. Values indicated (*P < 0.05, **P < 0.01) are significantly different from control and values (*P < 0.05, t t P < 0.01) are significantly different from palmitate alone.
sence or presence of a test agent. Palmitate (i0 -3 M), TPA (1.6 x 10 -6 M), and GGA (10 -3 M) significantly stimulated [3H]choline incorporation into phosphatidylcholine in the glands as compared with control (39 -+ 7% in palmitate-treated glands at 180 min, mean +-SE, N = 6, P < 0.01; 29 +- 1% of TPA-treated glands, mean -+ SE, N = 6, P < 0.05; 24 +- 1% of GGA-treated glands, mean -+ sv., N = 6, P < 0.05). On the other hand, decreased [3H]choline incorporation at any time point of the chase periods was observed in response to each H2-receptor antagonist (Figure 4). Furthermore, each H2-receptor antagonist did not reduce the palmitate-induced increase in [3H]choline incorporation of a chase period (Figure 5). The radioactivity associated with phosphocholine at the end of a pulse period was 58 -+ 2% (mean -+ SE, N = 7) of the total glandular radioactivity. The radioactivity in the phosphocholine significantly decreased at 60 min of the chase period and later in response to either palmitate, TPA, or GGA. On the other hand, [3H]CDP choline significantly increased at 180 min of the chase period in response to either palmitate, TPA, or GGA. These results suggest that the radioactivity lost from phosphocholine appears in CDP choline and phosphatidylcholine (Figure 6). We next measured the activity of CTP-phosphoDigestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
PHOSPHATIDYLCHOLINE SYNTHESIS IN GASTRIC GLANDS
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Fig 4. Pulse-chase study on the metabolism of choline in gastric glands. Gastric glands were pulsed with [3H]choline for 15 min and subsequently chased for the indicated periods in the absence or presence of 10-3 M palmitate, 1.6 • 10 -6 M TPA, 10 -3 M GGA, 10 -3 M cimetidine, 10-3 M famotidine, or 10 -3 M ranitidine. All symbols used are the same as those in Figure 1. Each value is the mean -+ SE of six separate experiments. Values indicated (*P < 0.05, **P < 0.01) are significantly different from control.
choline cytidylyltransferase, a well-known ratelimiting enzyme of phosphatidylcholine biosynthesis, in the particulate fraction of palmitate, TPA-, or GGA-treated gastric glands. The cytidylyltransferase activity in the fraction of glands that had been incubated with GGA or palmitate was significantly higher than that of glands incubated without 50
i
0
a'o 8'o '1 0 '1 0 '
180
' 120
(rain)
TIME
I
I
180
I
(min)
Fig 6. Effect of various agents on water-solublecholine metabolites after pulse labeling of gastric glands with [3H]choline. Gastric glands were chased for the indicated periods in the absence or presence of 10 -3 M palmitate, 1.6 • 10-6 M TPA, 10 -3 M GGA, 10 -3 M cimetidine, 10 -3 M famotidine, or 10 -3 M ranitidine. Symbols used are the same as those in Figure 1. Thereafter, water-soluble fractions were extracted, and choline metabolites in the fractions were separated on TLC. Each value, the mean -+ SE of at least six separate experiments, was expressed as a percentage of total cellular radioactivity. Values (*P < 0.05, **P < 0.01) are significantly different from control.
agents (Table 2). On the other hand, choline kinase activity was not inhibited in the cytosolic fraction of gastric glands that had been incubated with cimetidine (10 -3 M) for 180 min (Table 3). Cimetidine at 10 -3 M, even when added into the assay medium, did not inhibit choline kinase activity of cytosolic fraction of glands (data not shown).
"6
DISCUSSION 4O
W tO
Phospholipid, chemically similar to pulmonary surfactants, is a constituent of the mucus gel, and it has been suggested to play an important role in
~x
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30
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TABLE 2.
CYTIDYLYLTRANSFERASE FRACTIONS
W Z
ACTIVITY
OF
GASTRIC
IN
PARTICULATE
GLANDS*
Activity (nrnol/min/mg protein)
d"i- 10 0
I
1
0
30
I
I
I
60 120 T I M E (rain)
I
I
180
Fig 5. Effect of simultaneous treatment with palmitate and one of H2-receptor antagonists on [3H]choline incorporation in gastric glands pulse-labeled with [3Hlcholine. Gastric glands were pulsed with [3H]choline for 15 min and subsequently chased for the indicated periods in the absence or presence of palmitate or palmitate plus one of the H2-receptor antagonists. Doses of agents used and symbols are the same as those in Figure 3. Each value is the mean +- SE of at least six separate experiments. Values indicated (**P < 0.01) are significantly different from control. Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
Control Palmitate TPA GGA
0 . 9 2 -+ 0.10 1.15 - 0 . 0 9 I
1.08 _+ 0.18 1.69 +-- 0 . 3 9 9
*Gastric glands were incubated for 180 min in the absence or presence of either 10 -3 M palmitate, 10 -6 M TPA, or 10 -3 M GGA. Then, particulate fractions were prepared and cytidylyltransferase activities in the fractions were measured as described in Materials and Methods. Each value is the mean +-- SE of four separate experiments. tValues indicated (P < 0.05) are significantly different from control.
1597
NISHISAKI ET AL TABLE 3. CHOLINE KINASE ACTIVITY IN CYTOSOLIC FRACTIONS OF GASTRIC GLANDS* Activity (nmol/min/mg protein) Control Cimetidine
75.4 -+ 15.8 68.2 - 17.7
*Gastric glands were incubated for 180 min in the absence or presence of 10-3 M cimetidine. Then, cytosolic fractions were prepared and choline kinase activities in the fractions were measured as described in Materials and Methods. Each value is the mean -+ SE of six separate experiments.
protecting the mucosa against either hydrochloric acid-, ethanol-, or drug-induced ulcerogenesis in the stomach (4, 19). In the present study, we showed that isolated guinea pig gastric glands were capable of increasing phosphatidylcholine synthesis in response to either palmitate, TPA, or GGA treatment. Of interest is the observation that phosphatidylcholine biosynthesis was stimulated by GGA, a new acyclic isoprenoid that is a novel antiulcer drug. A variety of antiulcer actions of GGA have been reported previously. GGA has been shown to protect gastric mucosa against experimental ulcers induced by hydrochloric acid, ethanol, or drugs (10-13). Furthermore, it has been considered recently that GGA may exert antiulcer action by enhancing the defense factors such as glycoprotein and phospholipids in the stomach (20, 21). However, since GGA has been administered perorally in those studies, it has not been as yet clarified whether GGA directly stimulates phospholipid synthesis in the gastric mucosa. We showed in the present study that GGA stimulated the particulate CTP-phosphocholine cytidylyltransferase activity, a rate-limiting enzyme for phosphatidylcholine biosynthesis (22). Current studies imply that the membrane-bound cytidylyltransferase activity rather than the soluble activity governs the overall rate of phosphatidylcholine biosynthesis (17, 22). Protein phosphorylation of cytidylyltransferase and free fatty acids have been suggested to be involved in the regulation of translocation of the enzyme to the endoplasmic reticulum (22). By contrast with GGA, all HE-receptor antagonists equipotently decreased [3H]choline incorporation into phosphatidylcholine in the gastric glands. Furthermore, HE-receptor antagonists reduced palmitate-stimulated [3H]choline incorporation into phosphatidylcholine in the glands. When gastric glands were prelabeled with [3H]choline and thereafter chased in the presence of each H2-receptor antago-
1598
nist, no decrease in phosphatidylcholine biosynthesis could be detected, suggesting that H2-receptor antagonists specifically regulate choline metabolism rather than exert their toxic effects on gastric glands. Analysis of phosphocholine and CDP choline in the glands prelabeled with [3H]choline revealed that H2-receptor antagonists may inhibit the initial step of phosphatidylcholine biosynthesis rather than the pathway distal to cytidylyltransferase. However, choline kinase activity in the glands that had been incubated with cimetidine was not decreased compared with that of control. Further, cimetidine, even when added into the assay medium, did not inhibit choline kinase activity, suggesting that cimetidine may affect choline transport from extracellular medium into the cytosol in the glands. It should be noted that a minimum effective dose of each H Ereceptor antagonist to inhibit [3H]choline incorporation into phosphatidylcholine is 10 - 3 M , with a maximal inhibitory concentration at 10 -2 M. When compared with the dose of each H2-receptor antagonist to inhibit l0 -3 M histamine-stimulated cAMP accumulation in guinea pig gastric glands, the ability of each HE-receptor antagonist required for an inhibition of [3H]choline incorporation is about l03- to 10g-fold less potent (23). It has been shown already that there are several biological effects of H Ereceptor antagonists other than the action as the receptor antagonists. Cimetidine has been reported to inhibit gastric alcohol dehydrogenase activity noncompetitively in the rat (24). It has been also shown to bind to liver microsomes, thereby inhibiting cytochrome P-450 competitively in the rat and humans (25-27). Therefore, the results in the present study suggest that HE-receptor antagonists may inhibit [3H]choline incorporation through the mechanism other than as the receptor antagonists. In hepatocytes and alveolar type II pneumocytes, fatty acids such as palmitate and oleate already have been shown to stimulate the biosynthesis of phosphatidylcholine (28, 29). This effect has been shown to be mediated by an activation of the synthesis of CDP choline catalyzed by CTPphosphocholine cytidylyltransferase. Other earlier studies have documented an increase in cytidylyltransferase activity in response to TPA (30, 31). TPA-activated protein kinase C has been suggested to stimulate both phosphatidylcholine biosynthesis and turnover through the activation of cytidylyltransferase and phospholipase C, respectively. However, in the present study, no clear correlation was observed between palmitate- or TPA-stimuDigestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
PHOSPHATIDYLCHOLINE SYNTHESIS IN GASTRIC GLANDS lated cytidylyltransferase activity and phosphatidylcholine biosynthesis in the gastric glands. Although, at present, we can not account for the reason, a possible explanation is that the activity of other enzymes that contribute to the biosynthesis of phosphatidylcholine may be influenced by the treatment of the drugs. In any case, further work is clearly required to elucidate the precise mechanism by which these drugs stimulate phosphatidylcholine biosynthesis in the gastric glands. REFERENCES I. Hills BA, Butler BD, Lichtenberger LM: Gastric mucosal Barrier: hydrophobic lining to the lumen of the stomach. Am J Physiol 244:G561-G568, 1983 2. Slomiany A, Galicki NI, Kojima K, Banas-Gruszka Z, Slomiany BL: Glyceroglucolipids of the mucous barrier of dog Stomach. Biochim Biophys Acta 665:88-91, 1981 3. Duane WC, Wiegand DM, Sievert CE: Bile acid and bile salt disrupt gastric mucosal barrier in the dog by different mechanisms. Am J Physiol 242:G95-G99, 1982 4. Lichtenberger LM, Graziani LA, Dial EJ, Butler BD, Hills BA: Role of surface-active phospholipids in gastric cytoprotection. Science 219:1327-1329, 1983 5. Lichtenberger LM, Richards JE, Hills BA: Effect of 16,16dimethyl prostaglandin E E o n the surface hydrophohicity of aspirin-treated canine gastric mucosa. Gastroenterology 88:308-314, 1985 6. Dobbs LG, Mason RJ: Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to 13-adrenergicstimulation. J Clin Invest 63:378-387, 1979 7. Brown LAS, Longmore WJ: Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture. J Biol Chem 256:66-72, 1981 8. Sano K, Voelker DR, Mason RJ: Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells. J Biol Chem 260:12725-12729, 1985 9. Wassef MK, Lin YN, Horowitz MI: Molecular species of phosphatidylcholine from rat gastric mucosa. Biochim Biophys Acta 573:222-226, 1979 10. Terano A, Shiga J, Hiraishi H, Ota S, Sugimoto T: Protective action of tetraprenylacetone against ethanol-induced damage in rat gastric mucosa. Digestion 35:182-188, 1986 1I. Arakawa T, Yamada H, Nakamura A, Nebiki M, Fukuda T, Nakamura H, Kobayashi K: Gastric cytoprotection by tetraprenylacetone in human subjects. Digestion 39: I 11-117, 1988 12. Fujimoto M, Yamanaka T, Bessho M, Igarashi T: Effects of geranylgeranylacetone on gastrointestinal secretion in rats. Eur J Phamacol 77:113-118, 1982 13. Murakami M, Oketani K, Fujisaki H, Wakabayash T, Ohgo T: Antiulcer effect of geranylgeranylacetone, a new acyclic polyisoprenoid on experimentally induced gastric and duodenal ulcers in rats. Arzneim Forsch 31:799-804, 198i 14. Sakamoto C, Matozaki T, Nagao M, Baba S" Combined effect of phorbol ester and A23187 or dibutyryl cyclic AMP on pepsinogen secretion from isolated gastric glands. Biochem Biophys Res Commun 131:314-319, 1985
Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
15. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911-917, 1959 16. Aeberhard EE, Barrett CT, Kaplan SA, Scott ML: Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II penumocytes. Biochim Biophys Acta 833:473-483, 1985 17. Burkhardt R, Von Wichert P, Batenburg JJ, Van Golde LMG: Fatty acids stimulate phosphatidylcholine synthesis and CTP: choline-phosphate cytidylyltransfemse in iype II pneumocytes isolated from adult rat lung. Biochem J 254:495-500, i988 18. Bradford MA: A rapid and sensitive method for the quantiration of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248-254, 1976 19. Martin GP, Marriott C: Membrane damage by bile salts: The protective function of phospholipids. J Pharm Pharmacol 31:754-759, 1981 20. Oketani K, Murakami M, Fujisaki H, Wakabayashi T, Hotta K: Effect of geranylgeranylacetone on aspirin-induced changes in gastric glycoprotein. Jpn J Pharmacoi 33:593-601, 1983 21. Bilski J, Sarosiek J, Murty ULN, Aono M, Moriga M, Slomiany A, Slomiany BL: Enhancement of the lipid Content and physical properties of gastric mucus by geranylgeranylacetone. Biochem Pharmacol 36:4059-4065, 1987 22. Pelech SL, Vance DE: Regulation of phosphatidylcholine biosynthesis. Biochim Biophys Acta 779:217=251, 1984 23. Tanaka A, Nishihara S, Misawa T, Ibayashi H: Effects of HE-receptor antagonists of 3H-cimetidine binding and histamine-stimulation of cellular cAMP in isolated guinea pig gastric glands. Jpn J Pharmacol 45:97-105, 1987 24. Caballeria J, Baraona E, Rodamilans M, Lieber CS: Effects of cimetidine on gastric alcohol deliydrogenase activity and blood ethanol levels. Gastroenterology 96:388-392, 1989 25. Knodell RG, Holtzman JL, Crankshaw DL, Steele NH, Stanley LN: Drug metabolism by rat and human hepatic microsomes in response to interaction with HE-receptor antagonists. Gastroenterology 82:84-88, 1982 26. Speeg KU, Patwardhan RV, Avant GR, Mitchell MC, Schenker S: Inhibition of microsomal drug metabolism by histamine HE-receptor antagonists studied in vivo and in vitro in rodents. Gastroenterology 82:89-96, 1982 27. Seitz H, Veith S, Czygan P: In vivo interactions between HE-receptor antagonists and ethanol metabolism in man and in rats. Hepatology 6:1231-1234, 1984 28. Aeberhard EE, Barrett CT, Kaplan SA, Scott ML: Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes. Biochim Biophys Acta 875:6-11, 1986 29. Pelech SL, Haydn Pritchard P, Brindley DN, Vance DE: Fatty acids promote translocati0n Of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and stimulate rat hepatic phosphatidylcholine Synthesis. J Biol Chem 258:6782-6788, 1983 30. Liscovitch M, Slack B, Blusztajn JK, Wurtman RJ: Differential regulation of phosphatidylcholine biosynthesis by 12O-tetradecanoylphorbol-13-acetate and diacylglycerol in NG108-15 neuroblastoma • glioma hybrid cells. J Biol Chem 262:17487-17491, 1987 31. Rosenberg IL, Smart DA, Gilfillan AM, Rooney SA: Effect of l-oleoyl-2-acetylglycerol and other lipids On phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type Ii pneumocytes. Biochim Biophys Acta 921:473-480, 1987
1599
Effects of Antiulcer Drugs on Phosphatidylcholine Synthesis in Isolated Guinea Pig Gastric Glands HOGARA NISHISAKI, CHOITSU SAKAMOTO, YOSHITAKA KONDA, OSAMU NAKANO, TAKASHI MATOZAKI, MUNEHIKO NAGAO, KOHEI MATSUDA, KEN WADA, and MASATO KASUGA
To better understand phosphatidylcholine synthesis in the stomach, we isolated guinea pig gastric glands and examined their [3H]choline incorporation into phosphatidylcholine in response to either antiulcer drugs such as geranylgeranylacetone (GGA) and H 2receptor antagonists or agents that cause phosphatidylcholine synthesis in other tissues. [3H]Choline incorporation was stimulated by GGA, palmitate, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Dibutyryl cyclic-AMP had no effect. By contrast with GGA, famotidine, ranitidine, and cimetidine equipotently inhibited [3H]choline incorporation into phosphatidylcholine. GGA, palmitate, and TPA increased phosphatidyl-[3H]choline and decreased phosphoryi-[3H]choline as compared with control in tissues that had been pulsed with [3H]choline. On the other hand, no more decrease in [3H]choline incorporation at chase periods was observed in pulse-labeled glands in response to each H2-receptor antagonist. The particulate fraction o f glands that had been incubated with GGA or palmitate had more CTP-phosphocholine cytidylyltransferase activity than that of glands incubated without agents. A decrease in choline kinase activity was not observed in the cytosolic fraction of glands that had been incubated with cimetidine. These results suggest that GGA and palmitate stimulate phosphatidylcholine synthesis by activating cytidylyltransferase, and H2-receptor antagonists may affect phosphatidytcholine synthesis by inhibiting choline uptake in the gastric glands. KEY WORDS: ulcer; antiulcer drugs; phosphatidyicholine synthesis; gastric glands.
It has been suggested recently that not only the mucus layer adjacent to the wall of the gastrointestinal tract but also surface active phospholipids chemically similar to pulmonary surfactants may play an important role in protecting the mucosal surface of the gastrointestinal tract against the meManuscript received September 17, 1990; revised manuscript received October 23, 1991; accepted December 16, 1991. From the Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Address for reprint requests: Dr. Choitsu Sakamoto, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
chanical forces of digestion or acidic luminal fluids (1-3). Exogenously administered liposomal surfactant suspension has been shown to reduce acidinduced gastric ulcerogenesis and bleeding in rats (4). Furthermore, the protection of gastric mucosa by prostaglandin has been suggested to be, at least in part, due to the maintenance of a nonwettable hydrophobic lining on the gastric epithelium (5). Surfactant phospholipids in the lung have been well characterized, and their ability to reduce surface tension at the interface between air and liquid has been shown to be essential for pulmonary mechanics and homeostasis.
Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992) 0163-2116/92/1000-1593506.50/0 9 1992PlenumPublishingCorporation
1593
NISHISAKI ET AL Dipalmitoylphosphatidylcholine, a the major component of these surfactant lipids that is synthesized in alveolar type II cells, has been shown to be secreted by at least two different mechanisms, one of which is dependent on cellular cyclic AMP and the other involving protein kinase C (6-8). By contrast with pulmonary surfactants, however, not only the mechanism by which surfactant like phospholipids are synthesized but also whether they are secreted in the stomach are not yet known. Furthermore, if gastric surfactant like phospholipids are secreted, it is also not yet known what cells in the stomach secrete them. Accordingly, as an initial step, we investigated the mechanism by which phosphatidylcholine, a major component of gastric mucosal phospholipids (9) is synthesized in the isolated guinea pig gastric glands in vitro. Furthermore, we examined whether phosphatidylcholine biosynthesis is influenced by both antiulcer drugs and an agent that stimulates the activation of protein kinase C or cyclic AMP-dependent protein kinase. We tested as antiulcer drugs Hzreceptor antagonists and a new acyclic isoprenoid that has been commonly used for treating the patients with gastric ulcer in Japan (10, 11). We show in the present study that antiulcer drugs may affect the biosynthetic pathway to decrease or increase phosphatidylcholine synthesis in gastric glands in vitro.
MATERIALS AND METHODS Materials. [methyl-3H]Choline chloride (80 Ci/mmol) and phosphoryl[methyl-lnC]choline (50 mCi/mmol) were obtained from Amersham Japan; authentic phospholipid standards from Sigma; silica gel 60 plates from Merck; 12-O-tetradecanoyl-phorbol-13-acetate (TPA), CTP, and type I collagenase were from Sigma. Geranylgeranylacetone (6,10,14,18-tetramethyl5,9,13,17-nonadecatetraene-2-one; purity 99.2%) (GGA) was synthesized and donated by Eisai Co., Ltd., Tokyo, Japan (12, 13). Preparation of Guinea Pig Gastric Glands. The isolated guinea pig gastric glands were prepared as described previously (14). Briefly, the minced fundic and corpus mucosa was digested for 50 min at 37~ C in a solution consisting of 132 mM NaC1, 4.7 mM KC1, 11.1 mM MgCI2, 5 mM Na2HPO 4, 1 mM NaH2PO4, 11.1 mM glucose, essential amino acids, and 0.5% bovine serum albumin and containing 0.1% collagenase. At the end of incubation, the glands were filtered through nylon mesh to remove coarse fragments and rinsed. Finally, the glands were suspended in the aforementioned solution containing 1.28 mM CaC12 at pH 7.4 (KRP buffer). Incorporation of [all]Choline into Gastric Glands and Extraction of Phospholipids. Gastric glands suspended in
the aforementioned KRP buffer were incubated with 0.5 ~Ci/ml [3H]choline in the presence of test agents. We
1594
used as test agents palmitate, TPA, dibutyryl cyclic-AMP (dbcAMP), and antiulcer drugs GGA and H2-receptor antagonists such as famotidine, ranitidine, and cimetidine. TPA was dissolved in dimethyl sulfoxide and the final concentration of dimethyl sulfoxide at 0.01% was added for control experiments. GGA added into the buffer was sonicated to prepare micelles. After incubation for up to 180 min at 37~ C, the glands were washed twice with ice-cold KRP buffer and then labeled phospholipids were extracted by adding 1.88 ml chloroform-methanol (1 : 2, v/v) into 0.5 ml of the gland suspension according to the method of Bligh and Dyer (15). Each phospholipid was resolved from the organic phase by thin-layer chromatography (TLC) using chloroform-methanol-acetic acid-H20 (50: 30: 8: 4, v/v) as solvent. Pulse-Chase Study. Isolated gastric glands suspended in KRP buffer were incubated with 1.0 IxCi/ml [3H]choline for 15 min. Thereafter, the medium was removed, the glands were washed, and then the aforementioned test agents added into the gland suspension followed by further incubation. At the indicated time, the reaction was stopped by adding 1.88 ml chloroform-methanol (1:2, v/v) to the suspension. Radioactivity in the aqueous upper phase and organic lower phase of the extractions was determined by liquid scintillation counting. The distribution of radioactivity among water-soluble choline metabolites in the upper phase was examined by TLC using methanol-0.5% NaC1-NH3 (50:50:1, v/v) as solvent and choline-containing phospholipids in the lower phase examined as described above. Determination of Cytidylyltransferase Activity in Particulate Fraction of Gastric Glands. Isolated gastric glands
were incubated for 180 min at 37~ C in KRP buffer in the presence of test agents. At the end of incubation, the glands were washed, resuspended in 50 mM Tris buffer (pH 7.4), and homogenized with a Teflon-glass homogenizer. The homogenate was initially centrifuged until the speed attained 2000 rpm in a Tomy Seiko RL-7000 centrifuge and then switched off. After the supernatant was centrifuged at 100,000g for 60 min, the resulting pellets were used for the enzyme assay. CTP-phosphocholine cytidylyltransferase activity was determined by measuring the incorporation of radioactivity from [methyl-14C]phosphocholine into CDP choline (16). The incubation mixture contained 50 mM Tris HC1, 5 mM CTP, 10 mM MgC12, 0.6 mM [methyl-14C]phospho choline, and up to 100 t*g protein in a final volume of 50 pA. Incubation was carried out at 37~ C over 15 min. The phosphocholine and CDP choline generated were separated by TLC on silica gel 60 plates with the solvent system described above. Spots were visualized, scraped off, and assayed for radioactivity. Determination of Choline Kinase Activity in Cytosolic Fraction of Gastric Glands. Choline kinase activity was
assayed according to the method previously described (17). The formation of [3H]phosphocholine from [methyl3H]choline was determined. The assay buffer contained 50 mM NaC1, 50 mM ATP, 67 mM Tris HC1 (pH 8.5), 100 mM MgC12, 5 mM [3H]choline, and 30 ixg of cytosolic protein, which was prepared by centrifugation at 100,000g for 60 min of homogenate of gastric glands that had been incubated with or without cimetidine for 180 min. After 10 rain Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
PHOSPHATIDYLCHOLINE SYNTHESIS IN GASTRIC GLANDS TABLE 1. INCORPORATION OF [3H]CHOLINE INTO VARIOUS PHOSPHOLIPIDS IN GASTRIC GLANDS*
Incubation time (min) Phospholipids
30
60
120
180
Phosphatidylcholine Lysophosphatidylcholine Sphingomyelin
1585 -+ 21 27 -+ 10 14 _+ 3
3073 -+ 10 62 -+ 3 38 -+ 3
4147 -+ 14 91 -+ 5 50 --- 5
5027 +- 74 261 -+ 23 170 -+ 51
*Gastric glands were incubated with [3H]choline for the indicated time. Thereafter phospholipids were extracted and separated by T L C . Values are m e a n -+ SE o f four separate e x p e r i m e n t s and e x p r e s s e d as c p m per sample.
of incubation at 37~ C, the reaction was stopped by boiling. Choline and phosphocholine were separated by TLC as described above. Protein was measured with the Bio-Rad reagent as described by Bradford (18). Trypan Blue Exclusion Test. Gastric glands before or after incubation with drugs for 3 hr were washed with and resuspended in phosphate-buffered saline, and thereafter 0.1 ml trypan blue solution (0.4 g/dl) was added to the suspension. The number of stained or nonstained cells was counted within 10 rain. Statistical Analysis. All values are given mean -+ sE. An analysis of variance was used for statistical evaluation. With these analyses, an associated probability (P value) of less than 5% was considered statistically significant. All experiments were performed at least three times in duplicate unless otherwise indicated. RESULTS [3H]Choline uptake by isolated guinea pig gastric glands into phospholipids increased with time of incubation. H o w e v e r , at any time point, nearly 90% o f the lipid-associated radiolabel had been incorporated into phosphatidylcholine, with the remainder in sphingomyelin and lysophosphatidylcholine (Table 1). Therefore, the incorporation of [3H]choline into phosphatidylcholine was expressed as a percentage o f the radioactivity in the lipid fraction to total glandular radioactivity. The incorporation o f [3H]choline into phosphatidylcholine attained at 180 min was 21 --- 2% (mean - SE, N = 12) in controls. Palmitate (10 -3 M), TPA (1.6 x 10 -6 M), and G G A (10 -3 M) significantly increased the incorporation up to 37 -+ 4% (mean ----SE, N = 6; P < 0.01) 28 • 3% (mean • SE, N = 4; P < 0.05), and 31 • 2% (mean --- SE, N = 6; P < 0.05) at 180 min, respectively. On the other hand, the H2-receptor antagonist famotidine (10 -3 M), ranitidine (10 -3 M), and cimetidine (10 -3 M) significantly decreased the incorporation to 16 --- 2% (mean --- SE, N = 6; P < 0.05) 15 • 2% (mean • sE, N = 6; P < 0.05), and 14 • 2% (mean --- SE, N = 6; P < 0.05), respectively, d b c A M P had no effect on Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
the incorporation of [3H]choline into phosphatidylcholine (Figure 1). Palmitate, TPA, and G G A dose-dependently stimulated, but famotidine, ranitidine and cimetidine dose-dependently inhibited the incorporation of [3H]choline into phosphatidylcholine. Minimum effective doses of palmitate, TPA, and G G A were 3 x 10 -4 M, 3 • 10 -7 M, and 10 -4 M, respectively. Palmitate produced a 1.7-fold increase in the incorporation, with the maximal stimulation at 10 -3 M, whereas TPA and G G A produced a 1.4-fold increase at 3 x 10 -7 M and 10 -3 M, respectively. On the other hand, dbcAMP did not stimulate [3H]choline incorporation into phosphatidylcholine at all. Minimum effective doses of all tested H2-receptor antagonists
eo
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o
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~
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Fig 1. Time-dependent increases in [3H]choline incorporation into phosphatidylcholine in gastric glands. Gastric glands were incubated in the absence ((3 O) or presence of either 10-3 M palmitate (tk------~), 1.6 • 10-6 M TPA (_A -'), 10 -3 M GGA (D------I1), 10-3 M dbcAMP (I-1 V1), 10-3 M cimetidine (~--(>), 10-3 M famotidine (A A), or 10-3 M ranitidine (V V) for 180 min. Thereafter gastric phospholipids were extracted, and the radioactivity in the phospholipid fraction was measured. [3H]Choline incorporation into phosphatidylcholine was expressed as a percentage of the radioactivity in the phospholipid fraction to total cellular radioactivity. Values indicated (*P < 0.05, **P < 0.01) are significantlydifferent from control.
1595
NISHISAKI ET AL ~O 50
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Fig 2. Dose-dependent stimulation or inhibition of [3H]choline incorporation into phosphatidylcholine by various drugs. Gastric glands were incubated in the presence or absence of varying doses of test agents for 180 rain at 37~ C. Thereafter gastric phospholipids were extracted, and the radioactivity in the phospholipid fraction was measured. [3H]Choline incorporation was expressed as a percentage of control. Symbols used are the same as those in Figure 1. Each value is the mean - SE of at least four separate experiments. Values indicated (*P < 0.05, **P < 0.01)
are significantlydifferentfrom control. to inhibit the incorporation were the same, ie, 10-3 M. Furthermore, all H2-receptor antagonists equipotently decreased the incorporation with the maximal reduction at 10-2 M to 50% of control (Figure 2). To eliminate the possibility that either an increase or a decrease in [3H]choline uptake into gastric glands in response to drugs may be due to nonspecific injury of gastric cells exposed to high concentrations of the drugs, we examined gastric cell viability by using the trypan blue dye exclusion test. Although viability of gastric cells exposed to 10-2 M of an Hz-receptor antagonist for 180 min decreased from 95 -+ 3% to 75 - 4%, there was no significant difference in viability between the gastric cells exposed to an H2-receptor antagonist and gastric cells incubated for 180 min without drugs. Palmitate partially restored H2-receptor antagonist-induced reduction of the [3H]choline incorporation into phosphatidylcholine when the glands were incubated simultaneously with palmitate and an H2-receptor antagonist. The incorporation was 27 -+ 2% (mean -+ SE, N = 6) with palmitate plus cimetidine, the value of which was significantly greater than 21 --+ 2% in control (P < 0.01) and smaller than 37 +- 4% with palmitate alone (P < 0.01) (Figure 3). To further explore which enzymatic step regulated [3H]choline incorporation into phosphatidylcholine, gastric glands were pulsed with [3H]choline, then medium aspirated, and the glands chased in the ab-
1596
x~tt
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~--, plus 10 -3 M cimetidine; A A, plus 10-3 M famotidine; ~7 V, plus 10 -3 M ranitidine for 180 min. [3H]Choline incorporation was expressed as a percentage of the radioactivity in the phospholipid fraction to total cellular radioactivity. Each value is the mean -+ SE of at least six separate experiments. Values indicated (*P < 0.05, **P < 0.01) are significantly different from control and values (*P < 0.05, t t P < 0.01) are significantly different from palmitate alone.
sence or presence of a test agent. Palmitate (i0 -3 M), TPA (1.6 x 10 -6 M), and GGA (10 -3 M) significantly stimulated [3H]choline incorporation into phosphatidylcholine in the glands as compared with control (39 -+ 7% in palmitate-treated glands at 180 min, mean +-SE, N = 6, P < 0.01; 29 +- 1% of TPA-treated glands, mean -+ SE, N = 6, P < 0.05; 24 +- 1% of GGA-treated glands, mean -+ sv., N = 6, P < 0.05). On the other hand, decreased [3H]choline incorporation at any time point of the chase periods was observed in response to each H2-receptor antagonist (Figure 4). Furthermore, each H2-receptor antagonist did not reduce the palmitate-induced increase in [3H]choline incorporation of a chase period (Figure 5). The radioactivity associated with phosphocholine at the end of a pulse period was 58 -+ 2% (mean -+ SE, N = 7) of the total glandular radioactivity. The radioactivity in the phosphocholine significantly decreased at 60 min of the chase period and later in response to either palmitate, TPA, or GGA. On the other hand, [3H]CDP choline significantly increased at 180 min of the chase period in response to either palmitate, TPA, or GGA. These results suggest that the radioactivity lost from phosphocholine appears in CDP choline and phosphatidylcholine (Figure 6). We next measured the activity of CTP-phosphoDigestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
PHOSPHATIDYLCHOLINE SYNTHESIS IN GASTRIC GLANDS
,0) i
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Fig 4. Pulse-chase study on the metabolism of choline in gastric glands. Gastric glands were pulsed with [3H]choline for 15 min and subsequently chased for the indicated periods in the absence or presence of 10-3 M palmitate, 1.6 • 10 -6 M TPA, 10 -3 M GGA, 10 -3 M cimetidine, 10-3 M famotidine, or 10 -3 M ranitidine. All symbols used are the same as those in Figure 1. Each value is the mean -+ SE of six separate experiments. Values indicated (*P < 0.05, **P < 0.01) are significantly different from control.
choline cytidylyltransferase, a well-known ratelimiting enzyme of phosphatidylcholine biosynthesis, in the particulate fraction of palmitate, TPA-, or GGA-treated gastric glands. The cytidylyltransferase activity in the fraction of glands that had been incubated with GGA or palmitate was significantly higher than that of glands incubated without 50
i
0
a'o 8'o '1 0 '1 0 '
180
' 120
(rain)
TIME
I
I
180
I
(min)
Fig 6. Effect of various agents on water-solublecholine metabolites after pulse labeling of gastric glands with [3H]choline. Gastric glands were chased for the indicated periods in the absence or presence of 10 -3 M palmitate, 1.6 • 10-6 M TPA, 10 -3 M GGA, 10 -3 M cimetidine, 10 -3 M famotidine, or 10 -3 M ranitidine. Symbols used are the same as those in Figure 1. Thereafter, water-soluble fractions were extracted, and choline metabolites in the fractions were separated on TLC. Each value, the mean -+ SE of at least six separate experiments, was expressed as a percentage of total cellular radioactivity. Values (*P < 0.05, **P < 0.01) are significantly different from control.
agents (Table 2). On the other hand, choline kinase activity was not inhibited in the cytosolic fraction of gastric glands that had been incubated with cimetidine (10 -3 M) for 180 min (Table 3). Cimetidine at 10 -3 M, even when added into the assay medium, did not inhibit choline kinase activity of cytosolic fraction of glands (data not shown).
"6
DISCUSSION 4O
W tO
Phospholipid, chemically similar to pulmonary surfactants, is a constituent of the mucus gel, and it has been suggested to play an important role in
~x
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TABLE 2.
CYTIDYLYLTRANSFERASE FRACTIONS
W Z
ACTIVITY
OF
GASTRIC
IN
PARTICULATE
GLANDS*
Activity (nrnol/min/mg protein)
d"i- 10 0
I
1
0
30
I
I
I
60 120 T I M E (rain)
I
I
180
Fig 5. Effect of simultaneous treatment with palmitate and one of H2-receptor antagonists on [3H]choline incorporation in gastric glands pulse-labeled with [3Hlcholine. Gastric glands were pulsed with [3H]choline for 15 min and subsequently chased for the indicated periods in the absence or presence of palmitate or palmitate plus one of the H2-receptor antagonists. Doses of agents used and symbols are the same as those in Figure 3. Each value is the mean +- SE of at least six separate experiments. Values indicated (**P < 0.01) are significantly different from control. Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
Control Palmitate TPA GGA
0 . 9 2 -+ 0.10 1.15 - 0 . 0 9 I
1.08 _+ 0.18 1.69 +-- 0 . 3 9 9
*Gastric glands were incubated for 180 min in the absence or presence of either 10 -3 M palmitate, 10 -6 M TPA, or 10 -3 M GGA. Then, particulate fractions were prepared and cytidylyltransferase activities in the fractions were measured as described in Materials and Methods. Each value is the mean +-- SE of four separate experiments. tValues indicated (P < 0.05) are significantly different from control.
1597
NISHISAKI ET AL TABLE 3. CHOLINE KINASE ACTIVITY IN CYTOSOLIC FRACTIONS OF GASTRIC GLANDS* Activity (nmol/min/mg protein) Control Cimetidine
75.4 -+ 15.8 68.2 - 17.7
*Gastric glands were incubated for 180 min in the absence or presence of 10-3 M cimetidine. Then, cytosolic fractions were prepared and choline kinase activities in the fractions were measured as described in Materials and Methods. Each value is the mean -+ SE of six separate experiments.
protecting the mucosa against either hydrochloric acid-, ethanol-, or drug-induced ulcerogenesis in the stomach (4, 19). In the present study, we showed that isolated guinea pig gastric glands were capable of increasing phosphatidylcholine synthesis in response to either palmitate, TPA, or GGA treatment. Of interest is the observation that phosphatidylcholine biosynthesis was stimulated by GGA, a new acyclic isoprenoid that is a novel antiulcer drug. A variety of antiulcer actions of GGA have been reported previously. GGA has been shown to protect gastric mucosa against experimental ulcers induced by hydrochloric acid, ethanol, or drugs (10-13). Furthermore, it has been considered recently that GGA may exert antiulcer action by enhancing the defense factors such as glycoprotein and phospholipids in the stomach (20, 21). However, since GGA has been administered perorally in those studies, it has not been as yet clarified whether GGA directly stimulates phospholipid synthesis in the gastric mucosa. We showed in the present study that GGA stimulated the particulate CTP-phosphocholine cytidylyltransferase activity, a rate-limiting enzyme for phosphatidylcholine biosynthesis (22). Current studies imply that the membrane-bound cytidylyltransferase activity rather than the soluble activity governs the overall rate of phosphatidylcholine biosynthesis (17, 22). Protein phosphorylation of cytidylyltransferase and free fatty acids have been suggested to be involved in the regulation of translocation of the enzyme to the endoplasmic reticulum (22). By contrast with GGA, all HE-receptor antagonists equipotently decreased [3H]choline incorporation into phosphatidylcholine in the gastric glands. Furthermore, HE-receptor antagonists reduced palmitate-stimulated [3H]choline incorporation into phosphatidylcholine in the glands. When gastric glands were prelabeled with [3H]choline and thereafter chased in the presence of each H2-receptor antago-
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nist, no decrease in phosphatidylcholine biosynthesis could be detected, suggesting that H2-receptor antagonists specifically regulate choline metabolism rather than exert their toxic effects on gastric glands. Analysis of phosphocholine and CDP choline in the glands prelabeled with [3H]choline revealed that H2-receptor antagonists may inhibit the initial step of phosphatidylcholine biosynthesis rather than the pathway distal to cytidylyltransferase. However, choline kinase activity in the glands that had been incubated with cimetidine was not decreased compared with that of control. Further, cimetidine, even when added into the assay medium, did not inhibit choline kinase activity, suggesting that cimetidine may affect choline transport from extracellular medium into the cytosol in the glands. It should be noted that a minimum effective dose of each H Ereceptor antagonist to inhibit [3H]choline incorporation into phosphatidylcholine is 10 - 3 M , with a maximal inhibitory concentration at 10 -2 M. When compared with the dose of each H2-receptor antagonist to inhibit l0 -3 M histamine-stimulated cAMP accumulation in guinea pig gastric glands, the ability of each HE-receptor antagonist required for an inhibition of [3H]choline incorporation is about l03- to 10g-fold less potent (23). It has been shown already that there are several biological effects of H Ereceptor antagonists other than the action as the receptor antagonists. Cimetidine has been reported to inhibit gastric alcohol dehydrogenase activity noncompetitively in the rat (24). It has been also shown to bind to liver microsomes, thereby inhibiting cytochrome P-450 competitively in the rat and humans (25-27). Therefore, the results in the present study suggest that HE-receptor antagonists may inhibit [3H]choline incorporation through the mechanism other than as the receptor antagonists. In hepatocytes and alveolar type II pneumocytes, fatty acids such as palmitate and oleate already have been shown to stimulate the biosynthesis of phosphatidylcholine (28, 29). This effect has been shown to be mediated by an activation of the synthesis of CDP choline catalyzed by CTPphosphocholine cytidylyltransferase. Other earlier studies have documented an increase in cytidylyltransferase activity in response to TPA (30, 31). TPA-activated protein kinase C has been suggested to stimulate both phosphatidylcholine biosynthesis and turnover through the activation of cytidylyltransferase and phospholipase C, respectively. However, in the present study, no clear correlation was observed between palmitate- or TPA-stimuDigestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)
PHOSPHATIDYLCHOLINE SYNTHESIS IN GASTRIC GLANDS lated cytidylyltransferase activity and phosphatidylcholine biosynthesis in the gastric glands. Although, at present, we can not account for the reason, a possible explanation is that the activity of other enzymes that contribute to the biosynthesis of phosphatidylcholine may be influenced by the treatment of the drugs. In any case, further work is clearly required to elucidate the precise mechanism by which these drugs stimulate phosphatidylcholine biosynthesis in the gastric glands. REFERENCES I. Hills BA, Butler BD, Lichtenberger LM: Gastric mucosal Barrier: hydrophobic lining to the lumen of the stomach. Am J Physiol 244:G561-G568, 1983 2. Slomiany A, Galicki NI, Kojima K, Banas-Gruszka Z, Slomiany BL: Glyceroglucolipids of the mucous barrier of dog Stomach. Biochim Biophys Acta 665:88-91, 1981 3. Duane WC, Wiegand DM, Sievert CE: Bile acid and bile salt disrupt gastric mucosal barrier in the dog by different mechanisms. Am J Physiol 242:G95-G99, 1982 4. Lichtenberger LM, Graziani LA, Dial EJ, Butler BD, Hills BA: Role of surface-active phospholipids in gastric cytoprotection. Science 219:1327-1329, 1983 5. Lichtenberger LM, Richards JE, Hills BA: Effect of 16,16dimethyl prostaglandin E E o n the surface hydrophohicity of aspirin-treated canine gastric mucosa. Gastroenterology 88:308-314, 1985 6. Dobbs LG, Mason RJ: Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to 13-adrenergicstimulation. J Clin Invest 63:378-387, 1979 7. Brown LAS, Longmore WJ: Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture. J Biol Chem 256:66-72, 1981 8. Sano K, Voelker DR, Mason RJ: Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells. J Biol Chem 260:12725-12729, 1985 9. Wassef MK, Lin YN, Horowitz MI: Molecular species of phosphatidylcholine from rat gastric mucosa. Biochim Biophys Acta 573:222-226, 1979 10. Terano A, Shiga J, Hiraishi H, Ota S, Sugimoto T: Protective action of tetraprenylacetone against ethanol-induced damage in rat gastric mucosa. Digestion 35:182-188, 1986 1I. Arakawa T, Yamada H, Nakamura A, Nebiki M, Fukuda T, Nakamura H, Kobayashi K: Gastric cytoprotection by tetraprenylacetone in human subjects. Digestion 39: I 11-117, 1988 12. Fujimoto M, Yamanaka T, Bessho M, Igarashi T: Effects of geranylgeranylacetone on gastrointestinal secretion in rats. Eur J Phamacol 77:113-118, 1982 13. Murakami M, Oketani K, Fujisaki H, Wakabayash T, Ohgo T: Antiulcer effect of geranylgeranylacetone, a new acyclic polyisoprenoid on experimentally induced gastric and duodenal ulcers in rats. Arzneim Forsch 31:799-804, 198i 14. Sakamoto C, Matozaki T, Nagao M, Baba S" Combined effect of phorbol ester and A23187 or dibutyryl cyclic AMP on pepsinogen secretion from isolated gastric glands. Biochem Biophys Res Commun 131:314-319, 1985
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15. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911-917, 1959 16. Aeberhard EE, Barrett CT, Kaplan SA, Scott ML: Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II penumocytes. Biochim Biophys Acta 833:473-483, 1985 17. Burkhardt R, Von Wichert P, Batenburg JJ, Van Golde LMG: Fatty acids stimulate phosphatidylcholine synthesis and CTP: choline-phosphate cytidylyltransfemse in iype II pneumocytes isolated from adult rat lung. Biochem J 254:495-500, i988 18. Bradford MA: A rapid and sensitive method for the quantiration of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248-254, 1976 19. Martin GP, Marriott C: Membrane damage by bile salts: The protective function of phospholipids. J Pharm Pharmacol 31:754-759, 1981 20. Oketani K, Murakami M, Fujisaki H, Wakabayashi T, Hotta K: Effect of geranylgeranylacetone on aspirin-induced changes in gastric glycoprotein. Jpn J Pharmacoi 33:593-601, 1983 21. Bilski J, Sarosiek J, Murty ULN, Aono M, Moriga M, Slomiany A, Slomiany BL: Enhancement of the lipid Content and physical properties of gastric mucus by geranylgeranylacetone. Biochem Pharmacol 36:4059-4065, 1987 22. Pelech SL, Vance DE: Regulation of phosphatidylcholine biosynthesis. Biochim Biophys Acta 779:217=251, 1984 23. Tanaka A, Nishihara S, Misawa T, Ibayashi H: Effects of HE-receptor antagonists of 3H-cimetidine binding and histamine-stimulation of cellular cAMP in isolated guinea pig gastric glands. Jpn J Pharmacol 45:97-105, 1987 24. Caballeria J, Baraona E, Rodamilans M, Lieber CS: Effects of cimetidine on gastric alcohol deliydrogenase activity and blood ethanol levels. Gastroenterology 96:388-392, 1989 25. Knodell RG, Holtzman JL, Crankshaw DL, Steele NH, Stanley LN: Drug metabolism by rat and human hepatic microsomes in response to interaction with HE-receptor antagonists. Gastroenterology 82:84-88, 1982 26. Speeg KU, Patwardhan RV, Avant GR, Mitchell MC, Schenker S: Inhibition of microsomal drug metabolism by histamine HE-receptor antagonists studied in vivo and in vitro in rodents. Gastroenterology 82:89-96, 1982 27. Seitz H, Veith S, Czygan P: In vivo interactions between HE-receptor antagonists and ethanol metabolism in man and in rats. Hepatology 6:1231-1234, 1984 28. Aeberhard EE, Barrett CT, Kaplan SA, Scott ML: Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes. Biochim Biophys Acta 875:6-11, 1986 29. Pelech SL, Haydn Pritchard P, Brindley DN, Vance DE: Fatty acids promote translocati0n Of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and stimulate rat hepatic phosphatidylcholine Synthesis. J Biol Chem 258:6782-6788, 1983 30. Liscovitch M, Slack B, Blusztajn JK, Wurtman RJ: Differential regulation of phosphatidylcholine biosynthesis by 12O-tetradecanoylphorbol-13-acetate and diacylglycerol in NG108-15 neuroblastoma • glioma hybrid cells. J Biol Chem 262:17487-17491, 1987 31. Rosenberg IL, Smart DA, Gilfillan AM, Rooney SA: Effect of l-oleoyl-2-acetylglycerol and other lipids On phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type Ii pneumocytes. Biochim Biophys Acta 921:473-480, 1987
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