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Synthesis of thromboxane B2 and prostaglandins by bovine gastric mucosal microsomes
PROSTAGLANDINS
SYNTHESIS OF THROMBOXANE B2 AND PROSTAGLANDINS BY BOVINE GASTRIC MUCOSAL MICROSOMES Ali, M., Zamecnik, J., Cerskus, A.L., Stoessl, A.J., Barnett, W.H., and McDonald, J.W.D. Departments of Biochemistry, Obstetrics and Gynecology, and Medicine University of Western Ontario London, Ontario, Canada. N6A 5A5
ABSTRACT Bovine gastric mucosal microsomes synthesize prostaglandins from arachidonic acid but thromboxane B2 is the principal product. Thromboxane B2 synthesis occurs at an appreciable rate from endogenous precursor but more rapidly with added arachidonate. Nonsteroidal antiinflammatory drugs inhibited synthesis of prostaglandins and thromboxanes with the following decreasing order of potency: indomethacin, fenoprofen, acetylsalicylic acid, phenylbutazone, sulfinpyrazone, and acetaminophen. INTRODUCTION Nonsteroidal antiinflammatory drugs (NSAID), inhibitors of prostaglandin synthesis in a variety of tissues, produce gastrointestinal symptoms and gastric ulceration in animals and man. This serious side effect may interfere with treatment. These agents also inhibit prostaglandin synthesis in a variety of tissues. PGE2 and synthetic analogues inhibit gastric acid secretion in man (1,2). The extensive gastrointestinal ulceration induced in rats by single injections of indomethacin can be completely prevented by PGE2 and other prostaglandins (3). This is also true of the damage induced by aspirin and indomethacin in human gastric mucosa (4). A synthetic analogue of PGE2 greatly reduces the incidence of gastric mucosal hemorrhage induced in rats by a combination of bile acid and hydrochloric acid (5). These observations suggest that the ulcerogenic side effect of NSAID may be due to removal of the protective action of prostaglandins on the gastrointestinal tract. Although the nature of this protective effect is not known it may relate to stimulation of gastric mucus production (6). Peskar (7) has recently reported synthesis of PGE2 and PGF2u by microsomes from human gastric mucosa. We have studied synthesis of prostaglandins and thromboxane BP (TXB2) by bovine gastric mucosal microsomes and the effects of NSAID on synthesis of these products.
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1977 VOL. 14 NO. 5
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PROSTAGLANDINS
METHODS AND MATERIALS Bovine Gastric Mucosal Microsomes The mucosa was stripped from the whole fresh bovine stomach and chilled on ice. All subsequent preparative studies were carried out at O'C. Histologic confirmation confirmed that the tissue was gastric epithelium with some muscularis mucosa but no submucosal elements. The tissue was cut into small strips, homogenized in 3 volumes of phosphate buffer (0.1 M, pH 7.3) containing 0.1 M dithiothreitol in a Sorval Omnihomogenizor at top speed for 3 minutes and filtered through cheese cloth. Mitochondria were sedimented at 10,000 x g for 15 minutes, and the supernatant was than centrifuged at 100,000 x g for 60 minutes. The microsomal pellet was resuspended and washed twice in phosphate buffer. The final pellet was resuspended in phosphate buffer and stored in small aliquots at -8OOC. Approximately 4 mg of microsomal protein were obtained per gram gastric mucosa. Enzyme Incubation Prostaglandin and thromboxane synthesizing activity was stable for several weeks. Frozen samples were thawed immediately prior to incubation and diluted in 0.05M tris-HCl buffer (pH 8.0) containing 0.66 mM phenol to a protein concentration of 8 mgfml. Samples of the microsomal preparation were equilibrated for 3 minutes at 37'C in 12 x 75 mm disposable polypropylene tubes. After addition of solutions of inhibitors or saline , preincubation was continued and prostaglandin synthesis was initiated by the addition of Cl-14Clarachidonic acid. Each 0.75 ml reaction mixture contained 4 mg of microsomal protein, inhibitors or saline, and arachidonic acid in 0.05M tris-HCl buffer (pH 8.0). Extraction and Chromatography Incubation was terminated after 10 minutes by addition of 2.5 ml of saline and 4.5 ml of absolute ethanol. The pH was adjusted to 3 with 9.2% formic acid and the lipids were extracted into chloroform and chromatographed on silicic acid as described previously (8), except that elution of prostaglandins and TXB2 was accomplished with 10 ml of 10% methanol in chloroform. Aliquot samples were counted in a liquid scintillation counter. Recovery of radioactivity was greater than 95%. Zero time controls in which arachidonic acid was added after the absolute ethanol were carried through the extraction and chromatography procedures in parallel with incubated samples. Values for radioactivity eluted with 10% methanol in chloroform from these samples (approximately 0.5% of the total radioactivity) were subtracted from values obtained for incubated samples. All
NOVEMBER 1977 VOL. 14 NO. 5
PROSTAGLANDINS
incubations were performed in duplicate, and mean values were calculated. Thin Layer Chromatography Eluates from the silicic acid columns were taken to dryness under nitrogen and dissolved in methanol. Chromatography was carried out on thin layer plates of silica gel GF254 (type 60) developed with benzene:dioxane:acetic acid (2O:lO:l) (v/v/v). Authentic standards of PGEr and PGFaa were detected by spraying with 10% phosphomolybdic acid in ethanol. Thin layer chromatograms were scanned on a Packard Model 7201 Radiochromatogram Scanner. Radioactively-labelled products were eluted with methanol and counted. Recovery from thin layer plates was approximately 80%. Gas Chromatography-Mass Spectrometry of TXBz The LKB 9000-S gas chromatograph-mass spectrometer was used in these studies. TXBl eluted from thin layer chromatograms and converted to the tritrimethylsilyl ether methyl ester was identified by comparison with the same derivative of authentic TXBp and quantified using 2H8-TXB2 as described previously (9). Materials Authentic prostaglandins and TXB, were kindly supplied by Dr. John Pike, The Upjohn Company. Arachidonic acid-l-14C was obtained from Amersham/Searle Corporation. Inhibitors were kindly supplied by the following companies: fenoprofen - Eli Lilly and Company (Canada) Ltd.; acetaminophen McNeil Laboratories (Canada) Ltd.; indomethacin - Merck Frosst Laboratories; sulfinpyrazone and phenylbutazone - Ciba Geigy Canada Ltd. RESULTS Prostaglandin and Thromboxane Synthesis Figure 1 shows the time course of prostaglandin and thromboxane synthesis and the effect of concentration of added arachidonic acid. Experiments with inhibitors were carried out using 33 uM arachiddnate for 10 minutes. Figure 2 is a radiochromatogram scan of a thin layer chromatogram of reaction products eluted from a silicic acid column. The major radioactive peak chromatographing with authentic TXB2 was not always completely separated from PGE2. Radioactivity cochromatographing with authentic markers was measured in 2 independent experiments with results shown in Table 1. Because thromboxane synthesis by gastric
NOVEMBER 1977 VOL. 14 NO. 5
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PROSTAGLANDINS
Figure 1:
Prostaglandin and thromboxane synthesis by bovine gastric mucosal microsomes
Figure 2:
Radiochromatogram scan of thin layer chromatogram of radioactively-labelled products following incubation of gastric mucosal microsomes with arachidonic acid-14C.
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Table 1:
Reaction products presumptively identified on thin layer chromatograms from extracts of gastric mucosal microsomes incubated with arachidonate-r4C. Radioactivity in Products (cpm)
PGF2a PGEz Tm2
PGDz
Experiment 1
Experiment 2
2020 2800 13800 2070
3520 2040 15900 2230
mucosa has not been previously reported, the identify of TXB:!was confirmed by gas chromatography-mass spectrometry (GC-MS). The material exhibited a retention time and mass spectrum identical to that reported by Wolfe (10). Pace-Asciak (11) showed that 6-ketoPGFlo was a major reaction product of arachidonic acid in homogenates of rat stomach. We examined eluates from the silicic acid columns and from the labelled peaks of thin layer chromatograms for the presence of 6-keto-PGFlo. Under conditions in which we prepared and isolated the methyl oxhe ditrimethylsilyl ether methyl ester of 6-keto-PGFla formed from PGH2 by pig aortic microsomes we found no evidence by GC-MS for this material in the gastric mucosal preparation. The minor products, presumptively identified on thin layer chromatograms as PGE2, PGFza, and PGD2, were not studied further. Estimations of amounts of products (nMoles/mg protein) formed based on the specific radioactivity of added arachidonate were: PGF2u, 0.03; PGE2, 0.03; TXB2, 0.17; PGD2, 0.03. Calculations based on the specific radioactivity of added arachidonic acid assuming no dilution with endogenous arachidonate under estimate the rates of synthesis of prostaglandins and thromboxanes. TXB2 synthesis was estimated by GC-MS in 2 independent experiments in the presence and absence of added 33 uM arachidonate (Table 2). Table 2:
TXB2 synthesis by bovine gastric mucosal microsomes TXB2
(nMoles/mg protein)
Microsomes + 33 pM arachidonate + 33 uM arachidonate or 6 uM indomethacin
NOVEMBER 1977 VOL. 14 NO. 5
Exp. 1
Exp. 2
0.22 0.51
0.22 0.54
0.05
-----
823
PROSTAGLANDINS
TXBz synthesis proceeded at an appreciable rate in the absence of added arachidonate. The level of TXB, in samples incubated with indomethacin was only 20% of that observed in microsomal preparations incubated without added substrate or indomethacin. Therefore, TXB, estimated in samples incubated without added substrate largely represented synthesis from endogenous precursor rather than preformed TXB,. Whether free arachidonate is present in the microsomes or generated by phospholipase activity during incubation has not been determined. Effects of Inhibitors The inhibition of prostaglandin synthesis by acetylsalicylic acid increased for 30 minutes. Preincubation had no effect with the other inhibitors. Dose response lines for the inhibitors tested are shown in Figure 3. The order of potency of the drugs was indomethacin > fenoprofen > aspirin > phenylbutazone > sulfinpyrazone > acetaminophen.
Figure 3.
Effects of nonsteroidal antiinflammatory drugs on synthesis of prostaglandins and thromboxanes by gastric mucosal microsomes. Microsomes (4 mg) were preincubated for 30 minutes with acetylsalicylic acid (o--O) or for 10 minutes with indomethacin (@-@>, fenoprofen (h), phenylbutazone (wo), sulfinpyrazone (&A), or acetaminophen (W). Control tubes were preincubated with saline. Incubation with 33 PM arachidonate containing arachidonate-"C was continued for 10 minutes.
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Table 3 compares the relative potency of the 6 drugs tested in terms of concentrations required for 50% inhibition of prostaglandins and thromboxanes. The differences between effects of all drugs were statistically significant (p < 0.05) except for the comparison of aspirin with phenylbutazone. Table 3:
Relative potency of inhibitors and synthesis of prostaglandins and thromboxanes by gastric mucosal microsomes.
Mean
S.E.M.
Indomethacin
0.16
+
0.042
Fenoprofen
3.21
f
0.32
Aspirin
13
+
3.0
Phenylbutazone
31
f
14
Sulfinpyrazone
120
?r
27
410
f
90
Acetaminophen
DISCUSSION Synthesis of thromboxanes by gastric mucosa has not been previously reported. Previous reports by Pace-Asciak and Wolfe (11) have demonstrated the synthesis of PGE2, PGFzu, and 6-keto-PGFla by rat stomach homogenate. PGE2 and PGF@ were shown by Peskar'by radioimmunoassay to be synthesized by human gastric mucosa (7). The amount of PGE2 formed in our experiments was similar tc that reported by Peskar while PGF2u appeared to accumulate to a greater extent in our experiments. It appears that TXB2 is quantitatively a more important product of arachidonate metabolism in gastric mucosa. The possible physiological importance of thromboxane synthesis in gastric mucosa is uncertain. It remains to be determined whether TXB;1or its immediate precursor TXA2, like PGEp and its analogues, exert a protective effect on the gastrointestinal mucosa, and whether TXB2 is secreted into the gastric juice. The relative potency of indomethacin, aspirin and phenylbutazone as inhibitors of prostaglandin and thromboxane synthesis compares rather closely with the potency of these agents as ulcerogenic drugs reported by others (12). Similar data for ulcerogenic potency are not available for fenoprofen. The very low potency of acetaminophen as an inhibitor is interesting in view of its relative freedom from gastrointestinal side effects in human subjects (13). Peskar found
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PROSTAGLANDINS
it to be without effect on human gastric mucosal prostaglandin synthesis (7). Sulfinpyrazone has an appreciable, but acceptably low, incidence of gastrointestinal side effects in gouty patients (14) but is said to be as ulcerogenic as phenylbutazone in rats (15). In our experiments it was a weaker inhibitor of prostaglandin synthesis than aspirin and phenylbutazone, although at higher concentrations its potency approached that of the other drugs. It may not be entirely valid to compare the inhibition of enzyme activity in one species with the ulcerogenic effect in rats or humans. However, Flower has pointed out that there is little interspecies variation in the effects of NSAID on prostaglandin synthesis (16). The tissue of origin appears to be a more important factor in determining relative susceptibility of cyclooxygenase to inhibition by drugs. The similarity of the relative potency of NSAID as inhibitors of gastric mucosal prostaglandin synthesis, on one hand, and as ulcerogenic agents on the other, supports the hypothesis that the gastrointestinal side effects of these drugs may be due to inhibition of synthesis of prostaglandins. NSAID have been shown recently to inhibit synovial microsomal prostaglandin synthesis (17). This action may account for the efficacy of these agents in inflammatory joint disease. The inhibition by sulfinpyrazone of platelet cyclooxygenase activity (8) may account for the apparent antithrombotic activity of this weakly antiinflammatory drug. In vitro systems may be useful to predict the therapeutic ratio of NSAID. i.e., agents which are potent with respect to effects on synovial microsomal or platelet prostaglandin synthesis relative to their effects on gastric mucosal microsomes may be superior clinically. ACKNOWLEDGEMENT This work was supported by the Medical Research Council of Canada, the Ontario Heart Foundation, and Ciba-Geigy Canada Ltd. The technical assistance of Ms. Carolyn Armstrong is greatly appreciated. REFERENCES 1.
Chen, F_.W.K.,H.S. Teck and S.S.M. Karim. The Effect of 15(R) 15 Methyl Prostaglandin E on Gastric Acid Secretion in Duodenal Ulcer Patients, Prostaglandins -13:115, 1977.
2.
Nylander, B. and S. Andersson. Gastric Secretory Inhibition Induced by Three Methyl Analogs of Prostaglandin E Administered Intragastrically to Man, Stand. J. Gastroenterology 2:759, 1974.
826
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3.
Whittle, B.J.R. Relationship Between the Prevention of Rat Gastric Erosions and the Inhibition of Acid Secretion by European J. Pharmac. %:233, 1976. Prostaglandins,
4.
Cohen, M.M. Aspirin and Surg. Form
5.
Carmichael, H.A., L. Nelson, R.I. Russell, A. Lyon, and V. Chandra. The Effect of the Synthetic Prostaglandin Analog 15 (R) 15 Methyl-PGE, Methyl Ester on Gastric Mucosal Hemorrhage Induced in Rats by Taurocholic Acid and Hydrochloric Acid, Am. J. Dig. Dis. -22:411, 1977.
6.
Bolton, J.R., D. Palmer and M.M. Cohen. Effect of the Ez Prostaglandins on Gastric Mucus Production in Rats, Surg. Forum -27:402, 1976.
7.
Peskar, B.M. On the Synthesis of Prostaglandins by Human Gastric Mucosa and Its Modification by Drugs, Biochim. Biophys. Acta 487:307, 1977.
8.
Ali, M. and J.W.D. McDonald. Effects of Sulfinpyrazone on Platelet Prostaglandin Synthesis and Platelet Release of Serotonin. J. Lab. Clin. Med. -89:868, 1977.
9.
Ali, M., A.L. Cerskus, J. Zamecnik and J.W.D. McDonald. Synthesis of Prostaglandin D2 and Thromboxane B2 by Human Platelets, Thrombosis Research. October 1977. IN PRESS.
and J.M. Pollett. Prostaglandin E2 Prevents Indomethacin Damage to Human Gastric Mucosa, -27:400, 1976.
10.
Wolfe, L.S., K. Rostworowski, and J. Marion. Endogenous Formation of the Prostaglandin Endoperoxide Metabolite, Thromboxane BP, by Brain Tissue, Biochem. Biophys. Res. Comm. -70:907, 1976.
11.
Pace-Asciak, C. and L.S. Wolfe. A Novel Prostaglandin Derivative Formed from Arachidonic Acid by Rat Stomach Homogenates, Biochemistry -10:3657, 1971.
12.
Rainsford, K.D. The Biochemical Pathology of Aspirin-Induced Gastric Damage, Agents and Actions 1:326, 1975.
13.
Koch-Weser, J. Acetaminophen.
14.
Burns, J.J., T.F. Yu, P.G. Dayton, A.B. Gutman and B.B. Brodie. Biochemical Pharmacological Considerations of Phenylbutasone and Its Analogues. Ann. NY Acad. Sci. 86:253, 1960.
15.
Domenjoz, R. The Pharmacology of Phenylbutasone Analogues, Ann. NY Acad. Sci. -86:263, 1960.
16.
Flower, R.J. Drugs Which Inhibit Prostaglandin Biosynthesis. Pharmacol. Rev. -26:33, 1974.
17.
Crook, D., A.J. Collins, P.A. Bacon and R. Chan. Prostaglandin Synthetase Activity from Human Rheumatoid Synovial Microsomes. Effect of "Aspirin-Like" Drug Therapy, Ann. Rheum. Die.. -35:327, 1976.
Received
g/2/77
Medical Intelligence. Drug Therapy N. Engl. J. Med. -295:1297, 1976.
- Approved
10/25/77
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827
SYNTHESIS OF THROMBOXANE B2 AND PROSTAGLANDINS BY BOVINE GASTRIC MUCOSAL MICROSOMES Ali, M., Zamecnik, J., Cerskus, A.L., Stoessl, A.J., Barnett, W.H., and McDonald, J.W.D. Departments of Biochemistry, Obstetrics and Gynecology, and Medicine University of Western Ontario London, Ontario, Canada. N6A 5A5
ABSTRACT Bovine gastric mucosal microsomes synthesize prostaglandins from arachidonic acid but thromboxane B2 is the principal product. Thromboxane B2 synthesis occurs at an appreciable rate from endogenous precursor but more rapidly with added arachidonate. Nonsteroidal antiinflammatory drugs inhibited synthesis of prostaglandins and thromboxanes with the following decreasing order of potency: indomethacin, fenoprofen, acetylsalicylic acid, phenylbutazone, sulfinpyrazone, and acetaminophen. INTRODUCTION Nonsteroidal antiinflammatory drugs (NSAID), inhibitors of prostaglandin synthesis in a variety of tissues, produce gastrointestinal symptoms and gastric ulceration in animals and man. This serious side effect may interfere with treatment. These agents also inhibit prostaglandin synthesis in a variety of tissues. PGE2 and synthetic analogues inhibit gastric acid secretion in man (1,2). The extensive gastrointestinal ulceration induced in rats by single injections of indomethacin can be completely prevented by PGE2 and other prostaglandins (3). This is also true of the damage induced by aspirin and indomethacin in human gastric mucosa (4). A synthetic analogue of PGE2 greatly reduces the incidence of gastric mucosal hemorrhage induced in rats by a combination of bile acid and hydrochloric acid (5). These observations suggest that the ulcerogenic side effect of NSAID may be due to removal of the protective action of prostaglandins on the gastrointestinal tract. Although the nature of this protective effect is not known it may relate to stimulation of gastric mucus production (6). Peskar (7) has recently reported synthesis of PGE2 and PGF2u by microsomes from human gastric mucosa. We have studied synthesis of prostaglandins and thromboxane BP (TXB2) by bovine gastric mucosal microsomes and the effects of NSAID on synthesis of these products.
NOVEMBER
1977 VOL. 14 NO. 5
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PROSTAGLANDINS
METHODS AND MATERIALS Bovine Gastric Mucosal Microsomes The mucosa was stripped from the whole fresh bovine stomach and chilled on ice. All subsequent preparative studies were carried out at O'C. Histologic confirmation confirmed that the tissue was gastric epithelium with some muscularis mucosa but no submucosal elements. The tissue was cut into small strips, homogenized in 3 volumes of phosphate buffer (0.1 M, pH 7.3) containing 0.1 M dithiothreitol in a Sorval Omnihomogenizor at top speed for 3 minutes and filtered through cheese cloth. Mitochondria were sedimented at 10,000 x g for 15 minutes, and the supernatant was than centrifuged at 100,000 x g for 60 minutes. The microsomal pellet was resuspended and washed twice in phosphate buffer. The final pellet was resuspended in phosphate buffer and stored in small aliquots at -8OOC. Approximately 4 mg of microsomal protein were obtained per gram gastric mucosa. Enzyme Incubation Prostaglandin and thromboxane synthesizing activity was stable for several weeks. Frozen samples were thawed immediately prior to incubation and diluted in 0.05M tris-HCl buffer (pH 8.0) containing 0.66 mM phenol to a protein concentration of 8 mgfml. Samples of the microsomal preparation were equilibrated for 3 minutes at 37'C in 12 x 75 mm disposable polypropylene tubes. After addition of solutions of inhibitors or saline , preincubation was continued and prostaglandin synthesis was initiated by the addition of Cl-14Clarachidonic acid. Each 0.75 ml reaction mixture contained 4 mg of microsomal protein, inhibitors or saline, and arachidonic acid in 0.05M tris-HCl buffer (pH 8.0). Extraction and Chromatography Incubation was terminated after 10 minutes by addition of 2.5 ml of saline and 4.5 ml of absolute ethanol. The pH was adjusted to 3 with 9.2% formic acid and the lipids were extracted into chloroform and chromatographed on silicic acid as described previously (8), except that elution of prostaglandins and TXB2 was accomplished with 10 ml of 10% methanol in chloroform. Aliquot samples were counted in a liquid scintillation counter. Recovery of radioactivity was greater than 95%. Zero time controls in which arachidonic acid was added after the absolute ethanol were carried through the extraction and chromatography procedures in parallel with incubated samples. Values for radioactivity eluted with 10% methanol in chloroform from these samples (approximately 0.5% of the total radioactivity) were subtracted from values obtained for incubated samples. All
NOVEMBER 1977 VOL. 14 NO. 5
PROSTAGLANDINS
incubations were performed in duplicate, and mean values were calculated. Thin Layer Chromatography Eluates from the silicic acid columns were taken to dryness under nitrogen and dissolved in methanol. Chromatography was carried out on thin layer plates of silica gel GF254 (type 60) developed with benzene:dioxane:acetic acid (2O:lO:l) (v/v/v). Authentic standards of PGEr and PGFaa were detected by spraying with 10% phosphomolybdic acid in ethanol. Thin layer chromatograms were scanned on a Packard Model 7201 Radiochromatogram Scanner. Radioactively-labelled products were eluted with methanol and counted. Recovery from thin layer plates was approximately 80%. Gas Chromatography-Mass Spectrometry of TXBz The LKB 9000-S gas chromatograph-mass spectrometer was used in these studies. TXBl eluted from thin layer chromatograms and converted to the tritrimethylsilyl ether methyl ester was identified by comparison with the same derivative of authentic TXBp and quantified using 2H8-TXB2 as described previously (9). Materials Authentic prostaglandins and TXB, were kindly supplied by Dr. John Pike, The Upjohn Company. Arachidonic acid-l-14C was obtained from Amersham/Searle Corporation. Inhibitors were kindly supplied by the following companies: fenoprofen - Eli Lilly and Company (Canada) Ltd.; acetaminophen McNeil Laboratories (Canada) Ltd.; indomethacin - Merck Frosst Laboratories; sulfinpyrazone and phenylbutazone - Ciba Geigy Canada Ltd. RESULTS Prostaglandin and Thromboxane Synthesis Figure 1 shows the time course of prostaglandin and thromboxane synthesis and the effect of concentration of added arachidonic acid. Experiments with inhibitors were carried out using 33 uM arachiddnate for 10 minutes. Figure 2 is a radiochromatogram scan of a thin layer chromatogram of reaction products eluted from a silicic acid column. The major radioactive peak chromatographing with authentic TXB2 was not always completely separated from PGE2. Radioactivity cochromatographing with authentic markers was measured in 2 independent experiments with results shown in Table 1. Because thromboxane synthesis by gastric
NOVEMBER 1977 VOL. 14 NO. 5
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PROSTAGLANDINS
Figure 1:
Prostaglandin and thromboxane synthesis by bovine gastric mucosal microsomes
Figure 2:
Radiochromatogram scan of thin layer chromatogram of radioactively-labelled products following incubation of gastric mucosal microsomes with arachidonic acid-14C.
822
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Table 1:
Reaction products presumptively identified on thin layer chromatograms from extracts of gastric mucosal microsomes incubated with arachidonate-r4C. Radioactivity in Products (cpm)
PGF2a PGEz Tm2
PGDz
Experiment 1
Experiment 2
2020 2800 13800 2070
3520 2040 15900 2230
mucosa has not been previously reported, the identify of TXB:!was confirmed by gas chromatography-mass spectrometry (GC-MS). The material exhibited a retention time and mass spectrum identical to that reported by Wolfe (10). Pace-Asciak (11) showed that 6-ketoPGFlo was a major reaction product of arachidonic acid in homogenates of rat stomach. We examined eluates from the silicic acid columns and from the labelled peaks of thin layer chromatograms for the presence of 6-keto-PGFlo. Under conditions in which we prepared and isolated the methyl oxhe ditrimethylsilyl ether methyl ester of 6-keto-PGFla formed from PGH2 by pig aortic microsomes we found no evidence by GC-MS for this material in the gastric mucosal preparation. The minor products, presumptively identified on thin layer chromatograms as PGE2, PGFza, and PGD2, were not studied further. Estimations of amounts of products (nMoles/mg protein) formed based on the specific radioactivity of added arachidonate were: PGF2u, 0.03; PGE2, 0.03; TXB2, 0.17; PGD2, 0.03. Calculations based on the specific radioactivity of added arachidonic acid assuming no dilution with endogenous arachidonate under estimate the rates of synthesis of prostaglandins and thromboxanes. TXB2 synthesis was estimated by GC-MS in 2 independent experiments in the presence and absence of added 33 uM arachidonate (Table 2). Table 2:
TXB2 synthesis by bovine gastric mucosal microsomes TXB2
(nMoles/mg protein)
Microsomes + 33 pM arachidonate + 33 uM arachidonate or 6 uM indomethacin
NOVEMBER 1977 VOL. 14 NO. 5
Exp. 1
Exp. 2
0.22 0.51
0.22 0.54
0.05
-----
823
PROSTAGLANDINS
TXBz synthesis proceeded at an appreciable rate in the absence of added arachidonate. The level of TXB, in samples incubated with indomethacin was only 20% of that observed in microsomal preparations incubated without added substrate or indomethacin. Therefore, TXB, estimated in samples incubated without added substrate largely represented synthesis from endogenous precursor rather than preformed TXB,. Whether free arachidonate is present in the microsomes or generated by phospholipase activity during incubation has not been determined. Effects of Inhibitors The inhibition of prostaglandin synthesis by acetylsalicylic acid increased for 30 minutes. Preincubation had no effect with the other inhibitors. Dose response lines for the inhibitors tested are shown in Figure 3. The order of potency of the drugs was indomethacin > fenoprofen > aspirin > phenylbutazone > sulfinpyrazone > acetaminophen.
Figure 3.
Effects of nonsteroidal antiinflammatory drugs on synthesis of prostaglandins and thromboxanes by gastric mucosal microsomes. Microsomes (4 mg) were preincubated for 30 minutes with acetylsalicylic acid (o--O) or for 10 minutes with indomethacin (@-@>, fenoprofen (h), phenylbutazone (wo), sulfinpyrazone (&A), or acetaminophen (W). Control tubes were preincubated with saline. Incubation with 33 PM arachidonate containing arachidonate-"C was continued for 10 minutes.
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Table 3 compares the relative potency of the 6 drugs tested in terms of concentrations required for 50% inhibition of prostaglandins and thromboxanes. The differences between effects of all drugs were statistically significant (p < 0.05) except for the comparison of aspirin with phenylbutazone. Table 3:
Relative potency of inhibitors and synthesis of prostaglandins and thromboxanes by gastric mucosal microsomes.
Mean
S.E.M.
Indomethacin
0.16
+
0.042
Fenoprofen
3.21
f
0.32
Aspirin
13
+
3.0
Phenylbutazone
31
f
14
Sulfinpyrazone
120
?r
27
410
f
90
Acetaminophen
DISCUSSION Synthesis of thromboxanes by gastric mucosa has not been previously reported. Previous reports by Pace-Asciak and Wolfe (11) have demonstrated the synthesis of PGE2, PGFzu, and 6-keto-PGFla by rat stomach homogenate. PGE2 and PGF@ were shown by Peskar'by radioimmunoassay to be synthesized by human gastric mucosa (7). The amount of PGE2 formed in our experiments was similar tc that reported by Peskar while PGF2u appeared to accumulate to a greater extent in our experiments. It appears that TXB2 is quantitatively a more important product of arachidonate metabolism in gastric mucosa. The possible physiological importance of thromboxane synthesis in gastric mucosa is uncertain. It remains to be determined whether TXB;1or its immediate precursor TXA2, like PGEp and its analogues, exert a protective effect on the gastrointestinal mucosa, and whether TXB2 is secreted into the gastric juice. The relative potency of indomethacin, aspirin and phenylbutazone as inhibitors of prostaglandin and thromboxane synthesis compares rather closely with the potency of these agents as ulcerogenic drugs reported by others (12). Similar data for ulcerogenic potency are not available for fenoprofen. The very low potency of acetaminophen as an inhibitor is interesting in view of its relative freedom from gastrointestinal side effects in human subjects (13). Peskar found
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PROSTAGLANDINS
it to be without effect on human gastric mucosal prostaglandin synthesis (7). Sulfinpyrazone has an appreciable, but acceptably low, incidence of gastrointestinal side effects in gouty patients (14) but is said to be as ulcerogenic as phenylbutazone in rats (15). In our experiments it was a weaker inhibitor of prostaglandin synthesis than aspirin and phenylbutazone, although at higher concentrations its potency approached that of the other drugs. It may not be entirely valid to compare the inhibition of enzyme activity in one species with the ulcerogenic effect in rats or humans. However, Flower has pointed out that there is little interspecies variation in the effects of NSAID on prostaglandin synthesis (16). The tissue of origin appears to be a more important factor in determining relative susceptibility of cyclooxygenase to inhibition by drugs. The similarity of the relative potency of NSAID as inhibitors of gastric mucosal prostaglandin synthesis, on one hand, and as ulcerogenic agents on the other, supports the hypothesis that the gastrointestinal side effects of these drugs may be due to inhibition of synthesis of prostaglandins. NSAID have been shown recently to inhibit synovial microsomal prostaglandin synthesis (17). This action may account for the efficacy of these agents in inflammatory joint disease. The inhibition by sulfinpyrazone of platelet cyclooxygenase activity (8) may account for the apparent antithrombotic activity of this weakly antiinflammatory drug. In vitro systems may be useful to predict the therapeutic ratio of NSAID. i.e., agents which are potent with respect to effects on synovial microsomal or platelet prostaglandin synthesis relative to their effects on gastric mucosal microsomes may be superior clinically. ACKNOWLEDGEMENT This work was supported by the Medical Research Council of Canada, the Ontario Heart Foundation, and Ciba-Geigy Canada Ltd. The technical assistance of Ms. Carolyn Armstrong is greatly appreciated. REFERENCES 1.
Chen, F_.W.K.,H.S. Teck and S.S.M. Karim. The Effect of 15(R) 15 Methyl Prostaglandin E on Gastric Acid Secretion in Duodenal Ulcer Patients, Prostaglandins -13:115, 1977.
2.
Nylander, B. and S. Andersson. Gastric Secretory Inhibition Induced by Three Methyl Analogs of Prostaglandin E Administered Intragastrically to Man, Stand. J. Gastroenterology 2:759, 1974.
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3.
Whittle, B.J.R. Relationship Between the Prevention of Rat Gastric Erosions and the Inhibition of Acid Secretion by European J. Pharmac. %:233, 1976. Prostaglandins,
4.
Cohen, M.M. Aspirin and Surg. Form
5.
Carmichael, H.A., L. Nelson, R.I. Russell, A. Lyon, and V. Chandra. The Effect of the Synthetic Prostaglandin Analog 15 (R) 15 Methyl-PGE, Methyl Ester on Gastric Mucosal Hemorrhage Induced in Rats by Taurocholic Acid and Hydrochloric Acid, Am. J. Dig. Dis. -22:411, 1977.
6.
Bolton, J.R., D. Palmer and M.M. Cohen. Effect of the Ez Prostaglandins on Gastric Mucus Production in Rats, Surg. Forum -27:402, 1976.
7.
Peskar, B.M. On the Synthesis of Prostaglandins by Human Gastric Mucosa and Its Modification by Drugs, Biochim. Biophys. Acta 487:307, 1977.
8.
Ali, M. and J.W.D. McDonald. Effects of Sulfinpyrazone on Platelet Prostaglandin Synthesis and Platelet Release of Serotonin. J. Lab. Clin. Med. -89:868, 1977.
9.
Ali, M., A.L. Cerskus, J. Zamecnik and J.W.D. McDonald. Synthesis of Prostaglandin D2 and Thromboxane B2 by Human Platelets, Thrombosis Research. October 1977. IN PRESS.
and J.M. Pollett. Prostaglandin E2 Prevents Indomethacin Damage to Human Gastric Mucosa, -27:400, 1976.
10.
Wolfe, L.S., K. Rostworowski, and J. Marion. Endogenous Formation of the Prostaglandin Endoperoxide Metabolite, Thromboxane BP, by Brain Tissue, Biochem. Biophys. Res. Comm. -70:907, 1976.
11.
Pace-Asciak, C. and L.S. Wolfe. A Novel Prostaglandin Derivative Formed from Arachidonic Acid by Rat Stomach Homogenates, Biochemistry -10:3657, 1971.
12.
Rainsford, K.D. The Biochemical Pathology of Aspirin-Induced Gastric Damage, Agents and Actions 1:326, 1975.
13.
Koch-Weser, J. Acetaminophen.
14.
Burns, J.J., T.F. Yu, P.G. Dayton, A.B. Gutman and B.B. Brodie. Biochemical Pharmacological Considerations of Phenylbutasone and Its Analogues. Ann. NY Acad. Sci. 86:253, 1960.
15.
Domenjoz, R. The Pharmacology of Phenylbutasone Analogues, Ann. NY Acad. Sci. -86:263, 1960.
16.
Flower, R.J. Drugs Which Inhibit Prostaglandin Biosynthesis. Pharmacol. Rev. -26:33, 1974.
17.
Crook, D., A.J. Collins, P.A. Bacon and R. Chan. Prostaglandin Synthetase Activity from Human Rheumatoid Synovial Microsomes. Effect of "Aspirin-Like" Drug Therapy, Ann. Rheum. Die.. -35:327, 1976.
Received
g/2/77
Medical Intelligence. Drug Therapy N. Engl. J. Med. -295:1297, 1976.
- Approved
10/25/77
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