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Eicosanoid formation by mammalian intestine. Effects of some intestinal secretagogues
European Journal of Pharmacology, 138 (1987) 107-113 Elsevier
107
EJP 00770
Eicosanoid formation by mammalian intestine. Effects of some intestinal secretagogues F r a n c e s c o C a p a s s o 1, I g n a t i u s A. T a v a r e s , R a c h a e l T s a n g , J o h n A. R e n n i e a n d A l a n B e n n e t t * • Department of Experimental Pharmacology, Universityof Naples, Via L. Rodino 22, Naples, Italy, andDepartment of Surgery, King's CollegeSchool of Medicine and Dentistry, The Rayne Institute, 123 ColdharbourLane, London SE5 9NU, England Received 20 November 1986, revised MS received 24 February 1987, accepted 17 March 1987
Intestinal tissues of man, rat, mouse, guinea-pig and rabbit were preincubated with laxatives, homogenised, and incubated with [14C]arachidonic acid. After extraction into chloroform, the eicosanoids were separated by thin layer chromatography. Metabolism of [14C]arachidonic acid into prostaglandins (PGs), and the lipoxygenase products LTB4 and 5-HETE, was stimulated by ricinoleic acid (100/~g/ml) or phenolphthalein (100/~g/ml), and to a lesser extent by picosulphate (125 /~g/ml) and sulfosuccinate (200 #g/ml). Mannitol (500 ~tg/ml) had no effect. Indomethacin (1 ttg/ml) inhibited the stimulation of PG formation. The dual pathway inhibitor BW755C (1 #g/ml) reduced the formation of prostaglandins, LTB4 and 5-HETE. In some experiments on rat colon, prostanoids were separated from lipoxygenase products, characterised by their chromatographic mobility and quantitated (relative amounts PGE 2 > PGF2a > TXB 2 > PGD2). Their formation was enhanced by ricinoleic acid (100 /~g/ml) and inhibited by either indomethacin or BW 755C (1 /~g/ml). The present results indicate that mammalian isolated gut tissue can convert [14C]arachidonic acid into both cyclo-oxygenase and lipoxygenase products, and support the suggestion that eicosanoids may participate in the laxative effect of some secretagogues. Intestinal secretagogues; Gut tissue; Indomethacin; BW 755C
1. I n t r o d u c t i o n
The digestive tract can form substantial amounts of prostaglandins (PGs) and can release them in response to various stimuli (Bennett et al., 1967; Coceani et al., 1967; Collier et al., 1976; H e r m a n n and Vane, 1976). Some types of PGs cause accumulation of water and electrolytes in the lumen of the small intestine, increased intestinal propulsion, and diarrhoea (Misiewicz et al., 1969; Pierce et al., 1971; A1-Awqati and
* To whom all correspondence should be addressed: Department of Surgery, King's College School of Medicine and Dentistry, The Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, England.
Greenough, 1972; Matuchansky and Bernier, 1973; Karim, 1976; Rask-Madsen and Bukhave., 1983). These findings led to research on the possibility that some intestinal secretagogues exert effects on the digestive tract through stimulation of PG biosynthesis. Some secretagogues act in this way (Collier et al., 1976; Beubler and Juan, 1979; Cohen, 1982; Capasso et al., 1983), and their effects are reduced by indomethacin or aspirin which inhibit P G biosynthesis (Capasso et al., 1984a; Capasso et al., 1986). However, numerous other substances can be formed from PG precursors, including the lipoxygenase products hydroperoxy- and hydroxy-eicosatetraenoates (HPETEs, HETEs) and leukotrienes (Samuelsson, 1983). Many arachidonate cyclo-oxygenase and lipo-
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
108
xygenase products can potently stimulate gut smooth muscle and secretion (Lewis and Austin, 1981; Goetzl and Pickett, 1980; Goldberg and Subers, 1982; Musch et al., 1982). Lipoxygenase products as well as PGs seem to be important in the diarrhoea of patients with colonic inflammatory lesions (Musch et al., 1982), but there is little information about the generation and functions of these compounds in the gastrointestinal tract. Preliminary experiments, some of which have been reported briefly (Capasso et al., 1984b; 1985a,b; Bennett et al., 1985) showed that various intestinal secretagogues stimulate the formation of arachidonic acid metabolites, probably by an effect on phospholipase A 2 (Capasso et al., 1986). We now describe further studies on the generation of lipoxygenase and cyclo-oxygenase products by intestinal tissues in vitro by drugs that affect gut function.
2. Materials and methods
2.1. Tissue source and experimental procedures
Segments of intestinal tissue were removed immediately from male Wistar rats, Swiss mice, guinea-pigs and rabbits after cervical dislocation or ether anaesthesia, and were used without separation of the different layers. Human colon was obtained from at operation for diverticular disease or carcinoma of the colon or rectum, and separated into the muscle and mucosal layers. After collection, the specimens were immediately washed with ice-cold Krebs solution and cut into small pieces. Weighed tissue samples (100 mg wet weight) were pre-incubated in 5 ml Krebs solution (37 ° C, 20 rain) without (control) or with a test drug (/~g/ml; mannitol 500, phenolphthalein 100, picosulphate 125, ricinoleic acid 100, sulfosuccinate 200). After adding [14C] arachidonic acid (0.1/LCi, 1.7 nM) the tissue was homogenised at room temperature for 30 sec using a Silverson homogeniser, and the samples were further incubated with shaking at 37°C for 60 min, as described by Capasso et al. (1984b). Human tissue was rinsed and incubated (100 mg) in a similar manner. In some experiments indometha-
cin (1 #g/ml) or the cyclo-oxygenase/lipoxygenase inhibitor BW755C (1 /zg/ml) were incubated with the tissue for 20 min at 37 °C before adding the arachidonic acid. Enzymic activity was terminated with methanol/formic acid (7.5 m1:145 /tl) and the fatty acids were extracted with chloroform (15 ml × 2). Following evaporation to dryness each extract was chromatographed on a 1 g column of silicic acid and eluted with hexane/diethyl ether (80: 20, 30 ml) followed by ethyl acetate (30 ml). The ethyl acetate fraction, which contained the arachidonic acid metabolites, was evaporated to dryness, redissolved in 75/tl of chloroform/methanol (1 : 1) and further chromatographed on silica gel thin layer plates (Eastman Kodak) developed in diethyl ether/hexane/acetic acid (40 : 60 : 3). In this system PGs and other prostanoids chromatograph together close to the origin (PGE z was therefore used as the standard for the group), but the lipoxygenase products are separated). In some experiments, the organic phase of ethyl acetate/ hexane/acetic acid/water (56 : 24 : 12 : 60) was also used to facilitate the separation of the individual prostanoids (PGE 2, PGF2~, PGD 2 and TXB2) from the lipoxygenase metabolites (LTB4, 5-HETE). Radioactive products were located using autoradiography (21 days contact with Kodak NS-2T photographic film). The silica gel zone corresponding to each radioactive band was removed, and the radioactivity determined by liquid scintillation counting (Packard scintillation counter). Arachidonic acid metabolites were characterised by co-chromatography with standard PGE 2, PGF2~ , PGD2, TXB2, LTB4 and 5-HETE, which were visualised by exposure of the thin layer chromatography plate to iodine. 2.2. Data analysis
The results were calculated as percentage of [14C]arachidonic acid conversion/100 mg tissue, and as the percentage increase compared to controls. Student's t-test for paired data was used to determine significance of the difference from controls (2-tailed tests).
109 [14C]arachidonic acid into p r o d u c t s w h i ch chrom a t o g r a p h e d with PGs, L T B 4 a n d 5 - H E T E (table 1). M e t a b o l i s m was greater in the large intestine than in the small intestine, and was greater via the cy cl o - o x y g en ase p a t h w a y than via lipoxygenase. T h e rank o r d e r of % c o n v e r s i o n was: colon (2.8 ± 0.6 S.E.), c a e c u m (2.6 ± 0.4), r e c t u m (2.6 _ 0.5), d u o d e n u m (1.5 ± 0.4), j e j u n u m (1.4 ± 0.4), i l e u m (1.2 + 0.4).
TABLE 1 [14C]Arachidonie acid metabolism by rat intestinal tissue in vitro. Results are expressed as % conversion, means ± S.E. Tissue
n
PGs
LTB4
5-HETE
Duodenum Jejunum Ileum Caecum Colon Rectum
7 7 7 7 7 7
0.87 ± 0.09 0.78 _+0.07 0.69 4-0.06 1.78 + 0.15 1.83 + 0.34 1.75 5:0.18
0.27 4-0.03 0.274- 0.03 0.21 5:0.03 0.39 ± 0.04 0.45 + 0.06 0.39_+0.07
0.34 _+0.03 0.30 :t:0.04 0.275:0.04 0.405:0.04 0.494-0.04 0.42 ± 0.06
3.2. Effects of intestinal secretagogues on [14C] arachidonic acid metabolism by rat and human colon
2.3. Drugs Mannitol, phenolphthalein, sulfosuccinate, r i c i n o l e i c acid, u n l a b e l l e d a r a c h i d o n i c acid (Sigma); [14C]arachidonic acid ( R a d i o c h e m i c a l Centre, A m e r s h a m ) . T h e o th e r substances were generously d o n a t e d : p i c o s u l p h a t e ( D e Angel±); P G D 2 , P G E 2 , PGF2~ a n d T X B 2 (Glaxo, U p j o h n ) ; L T B 4 an d 5 - H E T E ( M e r c k - F r o s s t ) ; B W 7 5 5 C (W e ll co m e) ; i n d o m e t h a c i n ( M e r c k Sharp a n d D o h m e ) . O t h e r chemicals were reagent grade.
As sh o w n in table 2, a r a c h i d o n i c acid m e t a b o lism via the cyclo-oxygenase an d lipoxygenase p a t h w a y s in rat w h o l e co l o n an d h u m a n co l o n muscle an d m u c o s a (studied separately) was stimulated b y several secretagogues. In b o t h species ricinoleic acid a n d p h e n o l p h t h a l e i n were the m o s t potent, p i c o s u l p h a t e an d sulfosuccinate usually caused s o m e stimulation, an d m a n n i t o l h ad little or no effect. C o n v e r s i o n of [14C]arachidonic acid to r a d i o a c tive p r o d u c t s by c o n t r o l h o m o g e n a t e s of rat c o l o n was 3.9 + 0.7% ( m e a n + S.E., n = 11) of the a d d e d substrate; the co n v er si o n s by h o m o g e n a t e s of hum a n colonic muscle an d m u c o s a were respectively 5.3 ___0.9% ( m ean + S.E., n = 14) and 2.9 + 0.7% (n = 11).
3. Results
3.1. Metabolism of exogenous [14Cjarachidonic acid by rat gastrointestinal tissues Segments of rat intestine (whole wall) inc u b a t e d in K r e b s solution c o n v e r t e d exogenous TABLE 2
Effect of laxatives on [14C]arachidonic acid metabolism by rat and human colon in vitro. Ricinoleic acid and phenolphthalein strongly stimulated the formation of prostaglandins and lipoxygenase products in rat and human colon. Picosulphate and sulfosuccinate were weaker and mannitol had little or no effect. Results are expressed as % increase and analysed by Student's t-test for paired data. n ~-8-11 for rat colon, 8-14 for human colon muscle, and 8-11 for human colon mucosa. P values: a < 0.1; b < 0.05; c < 0.02; d < 0.01.
Drug
#g/ml
Rat whole colon PGs
LTB4
5-HETE
PGs
Mannitol
500
Picosulphate
125
Sulfosuccinate
200
Phenolphthalein
100
Ricinoleic acid
100
2.2 +11.8 76.5 b _+15.1 44.7 a 5:22.2 104.6 ¢ +22.3 118.0 ¢ + 18.9
9.3 -1-11.2 71.6 a +16.4 37.3 +12.6 75.8 b 5:20.0 85.5 b 5:15.2
2.6 5:5.1 65.2 5:26.1 50.6 a 5-17.4 68.6 a 5:11.0 88.1 b ± 12.4
8.9 4.0 +8.8 +7.3 77.7 a 70.4 +19.1 _+30.0 59.8 a 18.7 +10.3 +10.3 155.2 e 87.5 b ±36.8 ±18.3 197.1 a 131.2e ± 44.7 ~ + 33.8
Human colon muscle LTB4
Human colon mucosa 5-HETE
PGs
LTB4
5-HETE
2.0 +0.7 57.3 _+15.5 51.5 ±13.4 96.0 b +20.8 116.0b + 36.1
- 13.2 5:13.1 20.0 5:8.1 85.7 a ±36.8 139.2 d +26.8 150.9 d -t-38.6
7.6 5:6.8 25.3 5:6.8 39.6 ±7.0 52.1 a 5:10.8 69.5 b 5:13.1
14.2 5:10.0 71.4 a ±33.1 8().4 b ±30.0 66.6 b +11.0 94.4 c 5:24.6
110 TABLE 3 Stimulation by ricinoleic acid (100/~g/ml) of [14C]arachidonic acid metabolism by rat colon and human colon muscle in vitro. Effect of indomethacin (1/tg/ml) or BW755C (1 /~g/ml). Results are expressed as % increase and analysed by Student's t-test for paired data. n = 6 in both cases. P values: a < 0.05; b < 0.02; c < 0.01 compared to controls; d < 0.01 compared to ricinoleic acid alone. Drug
Whole rat colon
Indomethacin BW 755C Ricinoleic acid Ricinoleic acid + indomethacin Ricinoleic acid + BW 755C
Human colon muscle
PGs
LTB4
5-HETE
PGs
LTB4
5-HETE
- 55.3 c -+4.0 - 47.2 c -+4.7 93.9 c -+11.8 26.1 d -+ 11.2 15.6 d -+ 18.3
- 2.4 -+3.1 - 41.1 c _+5.0 62.2 b -+8.2 54.0 _+6.5 -- 31.2 d -+7.0
18.3 -+7.6 - 46.3 ~ _+2.7 118.6 a -+17.0 75.0 _+24.7 15.9 d -+ 13.8
- 64.9 b -+4.5 - 59.7 b _+3.3 115.0 b -+2.1 69.3 ~ -+ 8.7 71.8 ~ -+ 7.1
-- 12.1 -+6.6 -- 53.2 b _+4.0 103.7 a -+10.4 52.6 -+ 10.6 10.7 ~ _+6.4
14.4 _+6.6 - - 62.0 b _+2.7 112.9 a +14.0 57.2 ___5.3 12.0 d -+7.8
3.3. Effects of indomethacin and BW755C on [:4C]arachidonic acid metabolism stimulated by ricinoleic acid in human and rat colon
cyclo-oxygenase/lipoxygenase inhibitor BW755C 1 t t g / m l ( t a b l e 3). T h e l a t t e r d r u g , b u t n o t i n d o m e t h a c i n , also i n h i b i t e d the f o r m a t i o n of the lipoxygenase products.
Both the normal and the stimulated formation of cyclo-oxygenase products by rat and human colon were i n h i b i t e d by the c y c l o - o x y g e n a s e inhibitor indomethacin
3.4. Metabolism of [:4C]arachidonic acid by colon and ileum tissue from different species
1 /~g/ml and by the dual Arachidonic
acid
metabolism
was
markedly
g r e a t e r in t h e c o l o n t h a n i n t h e i l e u m o f g u i n e a - p i g s TABLE 4
a n d rabbits, but m a i n l y similar in these 2 regions
Effect of ricinoleic acid (100/~g/ml) on [14C]arachidonic acid metabolism by intestinal tissue from different species. Results are expressed as % increase and analysed by Student's t-test for paired data. n = 5-8; P values: a < 0.1; b < 0 . 0 5 ; c < 0.02; d < 0.01. Control values: guinea-pig ileum (PGs 1.80-+0.17; LTB4 0.30_+0.06, 5-HETE 0.39+0.05) and colon (PGs 2.50-+ 0.29; LTB4 0.42 _+0.08; 5-HETE 0.36 + 0.04; mouse ileum (PGs 1.26-+0.16; LTB4 0.32-+0.11; 5-HETE 0.30+0.07; and colon (PG 1.52 + 0.14; LTB4 0.28 + 0.06; 5-HETE 0.38 -+0.06); rabbit ileum (PGs 2.60+0.40; LTB4 0.40+0.2; 5-HETE (0.40-+0.11) and colon (PGs 3.60+0.61; LTB4 0.58-+0.20; 5-HETE 0.68-+ 0.18).
o f t h e m o u s e ( t a b l e 4). I n all c a s e s r i c i n o l e i c a c i d or phenolphthalein stimulated the conversion of
Tissue
PGs
LTB4
100.3_+16.2b 159.0+16.3 c
67.5_+ 7.7 a 75.5_+15.4a
55.5_+12.9b 109.0+24.7 b
u e s b e i n g T X B 2 0.18, P G E 2 0.23, P G F 2 , 0.30, P G D 2 0.41. T h e a m o u n t s o f c y c l o - o x y g e n a s e p r o d u c t s c h r o m a t o g r a p h i n g at t h e a p p r o p r i a t e R e
162.7+69.8 c 235.2+48.5 d
84.8+41.0 b 154.1_+32.9 c
72.2+11.1 b 91.8+28.1 b
v a l u e s w e r e : P G E 2 > P G F 2 , > T X B 2 > P G D 2. Ricinoleic acid enhanced their formation, whereas
76.9_+19.7a 118.3-+18.8 b
105.2_+28.7 b 103.4-+22.3 b
90.0--+23.1 a 73.5-+10.8 a
i n d o m e t h a c i n or B W 7 5 5 C 1 / ~ g / m l i n h i b i t e d their b a s a l a n d r i c i n o l e i c a c i d - s t i m u l a t e d s y n t h e s i s (tab l e 5).
Rabbit Ileum Colon
Table 5 shows the formation of cyclo-oxygenase p r o d u c t s f r o m e x o g e n o u s a r a c h i d o n i c acid. P G E 2, PGFz,, PGD 2 and TXB z were characterised by c o m p a r i s o n w i t h a u t h e n t i c s t a n d a r d s , t h e R e val-
Mouse Ileum Colon
3.5. Effects of ricinoleic acid on the formation of different prostanoids by rat colon
5-HETE
Guinea-pig Ileum Colon
exogenous arachidonic acid into cyclo-oxygenase and lipoxygenase products.
111
TABLE 5 S t i m u l a t i o n b y ricinoleic a c i d (100 ~ t g / m l ) o f [ 1 4 C ] a r a c h i d o n i c a c i d m e t a b o l i s m b y r a t c o l o n in vitro. E f f e c t o f i n d o m e t h a c i n (1 / ~ g / m l ) o r B W 7 5 5 C (1 ~ g / m l ) . R e s u l t s a r e e x p r e s s e d as % i n c r e a s e a n d a n a l y s e d b y S t u d e n t ' s t-test f o r p a i r e d d a t a , n = 5; P values: a < 0.1; b < 0.05; c < 0.02; d < 0.01. Drug
PGE 2
PGF2a
PGD 2
TXB 2
Indomethacin BW755C Ricinoleic acid Ricinoleic acid + indomethacin Ricinoleic acid + BW755C
-58.6+ 6.8 0 -41.8+ 7.0 d 147.0 + 30.0 c
-38.4+ 6.5 a - - 3 5 . 2 + 6.6 a 105.4 + 18.8 ¢
-36.2+ 7.1 a - - 4 1 . 6 + 5.7 111.1 + 36.6
-39.2+ 9.1 a - - 4 0 . 0 + 4.7 a 90.7 -t- 15.7 b
56.4 _ 21.7 d
36.4 + 20.7 e
36.1 + 17.4 b
33.8 + 15.1 a
54.1 + 20.7 d
32.4 + 18.8 ¢
27.7 _+ 18.7 a
29.2 + 10.0 a
4. Discussion
Our results confirm that arachidonic acid cyclo-oxygenase and lipoxygenase products can be formed by rat, guinea-pig, mouse, rabbit and human intestine. The products have been characterised by their chromatographic mobility and by the inhibitory effect of indomethacin and the dual cyclo-oxygenase/lipoxygenase inhibitor BW755C. In the rat we found that PGE 2 is the major product. Ahlquist et al. (1982) and BoughtonSmith et al. (1983) found that human colonic mucosa also produced mostly PGE2, whereas Bennett et al. (1981, 1987) found that PGI 2 was the most abundant metabolite formed when the human colonic mucosa or muscles were homogenised in Krebs solution. Arachidonic acid metabolism via the cyclooxygenase pathway was quantitatively greater than via the lipoxygenase pathway, and the large intestine was more active than the small intestine except in the mouse where the tissues were mainly equi-active. Other investigators have also observed regional metabolic variation in PG generation, possibly due to regional differences in muscle cyclo-oxygenase activity (Ahlquist et al., 1982; Ali and McDonald, 1980). We have found a similar profile for the lipoxygenase products LTB4 and 5-HETE, presumably because of regional differences in lipoxygenase activity. Our results also demonstrate that the conversion of exogenous [14C]arachidonic acid was stimulated by all the laxatives except mannitol,
with rinicoleic acid being the most potent. The effect of sulfosuccinate and picosulphate were at most weak, particularly on the formation of lipoxygenase products. It is not clear from the experiments of Beubler and Juan (1979) whether the laxative effect of sulfosuccinate is mediated by PGs, but our results with this drug indicate that PGs are unlikely to be important. This is in contrast to phenolphthalein whose laxative effect is suppressed by PG synthesis inhibitors (Capasso et al., 1984a). The activity of the laxatives varied in different tissues. Cohen (1982) showed regional differences in the effect of laxatives on PG synthesis by rat gut. We found that ricinoleic acid stimulated arachidonic acid metabolism more in the large intestine than in the small intestine. With phenolphthalein a similar difference occurred in the rat (Capasso et al., 1984b) and rabbit (unpublished), but the reverse occurred in the mouse and guineapig (unpublished). Hypotheses on the mechanisms by which secretagogues cause laxation include stimulation of cAMP (see Beubler and Juan, 1979) or inhibition of Na/K-ATPase (see Gaginella and Bass, 1978). In rats, laxatives increase the luminal amount of PGs in the colon (Autore et al., 1984; Beubler and Juan, 1979; Cohen, 1982; Capasso et al., 1983, 1986) and the net secretion of water and electrolytes into the small intestine (Beubler and Juan, 1979). Increased intestinal formation of histamine and 5-hydroxytryptamine by laxatives (Autore et al., 1984; Capasso et al., 1986), possibly due to mucosal injury (Beubler and Juan, 1979),
112
might also contribute to the cathartic effect. Histamine and 5-HT increase the availability of calcium to phospholipase A 2 and consequently increase PG production (see Capasso et al., 1986). Some laxatives require calcium for their effects on electrolyte transport (Donowitz et al., 1984) and arachidonic acid metabolism (Capasso et al., 1985a,b). Since lipoxygenase and cyclo-oxygenase metabolites can stimulate intestinal secretion, these products are likely to play an important part in the effect of some laxatives. The stimulation of eicosanoid formation by the muscle may also make a substantial primary contribution to the cathartic effect.
Acknowledgements This research was supported by Wellcome Trust, N.A.T.O. and Consiglio Nazionale delle Ricerche.
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113 Karim, S.M.M., 1976, The effect of polyphloretin phosphate and other compounds on prostaglandin-induced diarrhoea in man, Ann. Acad. Med. 3, 201. Lewis, R.A. and K.F. Austin, 1981, Mediation of local homeostasis and inflammation by leukotrienes and other mast cell-dependent compounds, Nature 293, 103. Matuchansky, C. and J.J. Bernier, 1973, Effect of prostaglandin E 1 on glucose, water, and electrolyte absorption in human jejunum, Gastroenterology 64, 1111. Misiewicz, J.J., S.L. Waller, N. Kiley and E.W. Horton, 1969, Effect of oral prostaglandin E 1 on intestinal transit in man, Lancet i, 648. Musch, M.W., R.J. Miller, H. Field and H.I. Siegel, 1982, Stimulation of colonic secretion by lipoxygenase metabolites of arachidonic acid, Science 217, 1253.
Pierce, N.F., C.C.J. Carpenter and H.L. Elliott, 1971, Effects of prostaglandins, theophylline, and electrolyte exotoxin upon transmucosal water and electrolyte movement in the canine jejunum, Gastroenterology 60, 22. Rask-Madsen, J. and K. Buldaave, 1983, Prostaglandins and intestinal secretion, in: Intestinal Secretion, ed. L.A. Turnberg (Knapp Drewett and Sons Ltd, Great Britain) p. 76. Samuelsson, B., 1983, Leukotrienes: a new class of mediators of immediate hypersensitivity reactions and inflammation, in: Advances Prostaglandin and Thromboxane Research, eds. B. Samuelsson, R. Paoletti and P.W. Ramwell (Raven Press, New York) p. 1.
107
EJP 00770
Eicosanoid formation by mammalian intestine. Effects of some intestinal secretagogues F r a n c e s c o C a p a s s o 1, I g n a t i u s A. T a v a r e s , R a c h a e l T s a n g , J o h n A. R e n n i e a n d A l a n B e n n e t t * • Department of Experimental Pharmacology, Universityof Naples, Via L. Rodino 22, Naples, Italy, andDepartment of Surgery, King's CollegeSchool of Medicine and Dentistry, The Rayne Institute, 123 ColdharbourLane, London SE5 9NU, England Received 20 November 1986, revised MS received 24 February 1987, accepted 17 March 1987
Intestinal tissues of man, rat, mouse, guinea-pig and rabbit were preincubated with laxatives, homogenised, and incubated with [14C]arachidonic acid. After extraction into chloroform, the eicosanoids were separated by thin layer chromatography. Metabolism of [14C]arachidonic acid into prostaglandins (PGs), and the lipoxygenase products LTB4 and 5-HETE, was stimulated by ricinoleic acid (100/~g/ml) or phenolphthalein (100/~g/ml), and to a lesser extent by picosulphate (125 /~g/ml) and sulfosuccinate (200 #g/ml). Mannitol (500 ~tg/ml) had no effect. Indomethacin (1 ttg/ml) inhibited the stimulation of PG formation. The dual pathway inhibitor BW755C (1 #g/ml) reduced the formation of prostaglandins, LTB4 and 5-HETE. In some experiments on rat colon, prostanoids were separated from lipoxygenase products, characterised by their chromatographic mobility and quantitated (relative amounts PGE 2 > PGF2a > TXB 2 > PGD2). Their formation was enhanced by ricinoleic acid (100 /~g/ml) and inhibited by either indomethacin or BW 755C (1 /~g/ml). The present results indicate that mammalian isolated gut tissue can convert [14C]arachidonic acid into both cyclo-oxygenase and lipoxygenase products, and support the suggestion that eicosanoids may participate in the laxative effect of some secretagogues. Intestinal secretagogues; Gut tissue; Indomethacin; BW 755C
1. I n t r o d u c t i o n
The digestive tract can form substantial amounts of prostaglandins (PGs) and can release them in response to various stimuli (Bennett et al., 1967; Coceani et al., 1967; Collier et al., 1976; H e r m a n n and Vane, 1976). Some types of PGs cause accumulation of water and electrolytes in the lumen of the small intestine, increased intestinal propulsion, and diarrhoea (Misiewicz et al., 1969; Pierce et al., 1971; A1-Awqati and
* To whom all correspondence should be addressed: Department of Surgery, King's College School of Medicine and Dentistry, The Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, England.
Greenough, 1972; Matuchansky and Bernier, 1973; Karim, 1976; Rask-Madsen and Bukhave., 1983). These findings led to research on the possibility that some intestinal secretagogues exert effects on the digestive tract through stimulation of PG biosynthesis. Some secretagogues act in this way (Collier et al., 1976; Beubler and Juan, 1979; Cohen, 1982; Capasso et al., 1983), and their effects are reduced by indomethacin or aspirin which inhibit P G biosynthesis (Capasso et al., 1984a; Capasso et al., 1986). However, numerous other substances can be formed from PG precursors, including the lipoxygenase products hydroperoxy- and hydroxy-eicosatetraenoates (HPETEs, HETEs) and leukotrienes (Samuelsson, 1983). Many arachidonate cyclo-oxygenase and lipo-
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
108
xygenase products can potently stimulate gut smooth muscle and secretion (Lewis and Austin, 1981; Goetzl and Pickett, 1980; Goldberg and Subers, 1982; Musch et al., 1982). Lipoxygenase products as well as PGs seem to be important in the diarrhoea of patients with colonic inflammatory lesions (Musch et al., 1982), but there is little information about the generation and functions of these compounds in the gastrointestinal tract. Preliminary experiments, some of which have been reported briefly (Capasso et al., 1984b; 1985a,b; Bennett et al., 1985) showed that various intestinal secretagogues stimulate the formation of arachidonic acid metabolites, probably by an effect on phospholipase A 2 (Capasso et al., 1986). We now describe further studies on the generation of lipoxygenase and cyclo-oxygenase products by intestinal tissues in vitro by drugs that affect gut function.
2. Materials and methods
2.1. Tissue source and experimental procedures
Segments of intestinal tissue were removed immediately from male Wistar rats, Swiss mice, guinea-pigs and rabbits after cervical dislocation or ether anaesthesia, and were used without separation of the different layers. Human colon was obtained from at operation for diverticular disease or carcinoma of the colon or rectum, and separated into the muscle and mucosal layers. After collection, the specimens were immediately washed with ice-cold Krebs solution and cut into small pieces. Weighed tissue samples (100 mg wet weight) were pre-incubated in 5 ml Krebs solution (37 ° C, 20 rain) without (control) or with a test drug (/~g/ml; mannitol 500, phenolphthalein 100, picosulphate 125, ricinoleic acid 100, sulfosuccinate 200). After adding [14C] arachidonic acid (0.1/LCi, 1.7 nM) the tissue was homogenised at room temperature for 30 sec using a Silverson homogeniser, and the samples were further incubated with shaking at 37°C for 60 min, as described by Capasso et al. (1984b). Human tissue was rinsed and incubated (100 mg) in a similar manner. In some experiments indometha-
cin (1 #g/ml) or the cyclo-oxygenase/lipoxygenase inhibitor BW755C (1 /zg/ml) were incubated with the tissue for 20 min at 37 °C before adding the arachidonic acid. Enzymic activity was terminated with methanol/formic acid (7.5 m1:145 /tl) and the fatty acids were extracted with chloroform (15 ml × 2). Following evaporation to dryness each extract was chromatographed on a 1 g column of silicic acid and eluted with hexane/diethyl ether (80: 20, 30 ml) followed by ethyl acetate (30 ml). The ethyl acetate fraction, which contained the arachidonic acid metabolites, was evaporated to dryness, redissolved in 75/tl of chloroform/methanol (1 : 1) and further chromatographed on silica gel thin layer plates (Eastman Kodak) developed in diethyl ether/hexane/acetic acid (40 : 60 : 3). In this system PGs and other prostanoids chromatograph together close to the origin (PGE z was therefore used as the standard for the group), but the lipoxygenase products are separated). In some experiments, the organic phase of ethyl acetate/ hexane/acetic acid/water (56 : 24 : 12 : 60) was also used to facilitate the separation of the individual prostanoids (PGE 2, PGF2~, PGD 2 and TXB2) from the lipoxygenase metabolites (LTB4, 5-HETE). Radioactive products were located using autoradiography (21 days contact with Kodak NS-2T photographic film). The silica gel zone corresponding to each radioactive band was removed, and the radioactivity determined by liquid scintillation counting (Packard scintillation counter). Arachidonic acid metabolites were characterised by co-chromatography with standard PGE 2, PGF2~ , PGD2, TXB2, LTB4 and 5-HETE, which were visualised by exposure of the thin layer chromatography plate to iodine. 2.2. Data analysis
The results were calculated as percentage of [14C]arachidonic acid conversion/100 mg tissue, and as the percentage increase compared to controls. Student's t-test for paired data was used to determine significance of the difference from controls (2-tailed tests).
109 [14C]arachidonic acid into p r o d u c t s w h i ch chrom a t o g r a p h e d with PGs, L T B 4 a n d 5 - H E T E (table 1). M e t a b o l i s m was greater in the large intestine than in the small intestine, and was greater via the cy cl o - o x y g en ase p a t h w a y than via lipoxygenase. T h e rank o r d e r of % c o n v e r s i o n was: colon (2.8 ± 0.6 S.E.), c a e c u m (2.6 ± 0.4), r e c t u m (2.6 _ 0.5), d u o d e n u m (1.5 ± 0.4), j e j u n u m (1.4 ± 0.4), i l e u m (1.2 + 0.4).
TABLE 1 [14C]Arachidonie acid metabolism by rat intestinal tissue in vitro. Results are expressed as % conversion, means ± S.E. Tissue
n
PGs
LTB4
5-HETE
Duodenum Jejunum Ileum Caecum Colon Rectum
7 7 7 7 7 7
0.87 ± 0.09 0.78 _+0.07 0.69 4-0.06 1.78 + 0.15 1.83 + 0.34 1.75 5:0.18
0.27 4-0.03 0.274- 0.03 0.21 5:0.03 0.39 ± 0.04 0.45 + 0.06 0.39_+0.07
0.34 _+0.03 0.30 :t:0.04 0.275:0.04 0.405:0.04 0.494-0.04 0.42 ± 0.06
3.2. Effects of intestinal secretagogues on [14C] arachidonic acid metabolism by rat and human colon
2.3. Drugs Mannitol, phenolphthalein, sulfosuccinate, r i c i n o l e i c acid, u n l a b e l l e d a r a c h i d o n i c acid (Sigma); [14C]arachidonic acid ( R a d i o c h e m i c a l Centre, A m e r s h a m ) . T h e o th e r substances were generously d o n a t e d : p i c o s u l p h a t e ( D e Angel±); P G D 2 , P G E 2 , PGF2~ a n d T X B 2 (Glaxo, U p j o h n ) ; L T B 4 an d 5 - H E T E ( M e r c k - F r o s s t ) ; B W 7 5 5 C (W e ll co m e) ; i n d o m e t h a c i n ( M e r c k Sharp a n d D o h m e ) . O t h e r chemicals were reagent grade.
As sh o w n in table 2, a r a c h i d o n i c acid m e t a b o lism via the cyclo-oxygenase an d lipoxygenase p a t h w a y s in rat w h o l e co l o n an d h u m a n co l o n muscle an d m u c o s a (studied separately) was stimulated b y several secretagogues. In b o t h species ricinoleic acid a n d p h e n o l p h t h a l e i n were the m o s t potent, p i c o s u l p h a t e an d sulfosuccinate usually caused s o m e stimulation, an d m a n n i t o l h ad little or no effect. C o n v e r s i o n of [14C]arachidonic acid to r a d i o a c tive p r o d u c t s by c o n t r o l h o m o g e n a t e s of rat c o l o n was 3.9 + 0.7% ( m e a n + S.E., n = 11) of the a d d e d substrate; the co n v er si o n s by h o m o g e n a t e s of hum a n colonic muscle an d m u c o s a were respectively 5.3 ___0.9% ( m ean + S.E., n = 14) and 2.9 + 0.7% (n = 11).
3. Results
3.1. Metabolism of exogenous [14Cjarachidonic acid by rat gastrointestinal tissues Segments of rat intestine (whole wall) inc u b a t e d in K r e b s solution c o n v e r t e d exogenous TABLE 2
Effect of laxatives on [14C]arachidonic acid metabolism by rat and human colon in vitro. Ricinoleic acid and phenolphthalein strongly stimulated the formation of prostaglandins and lipoxygenase products in rat and human colon. Picosulphate and sulfosuccinate were weaker and mannitol had little or no effect. Results are expressed as % increase and analysed by Student's t-test for paired data. n ~-8-11 for rat colon, 8-14 for human colon muscle, and 8-11 for human colon mucosa. P values: a < 0.1; b < 0.05; c < 0.02; d < 0.01.
Drug
#g/ml
Rat whole colon PGs
LTB4
5-HETE
PGs
Mannitol
500
Picosulphate
125
Sulfosuccinate
200
Phenolphthalein
100
Ricinoleic acid
100
2.2 +11.8 76.5 b _+15.1 44.7 a 5:22.2 104.6 ¢ +22.3 118.0 ¢ + 18.9
9.3 -1-11.2 71.6 a +16.4 37.3 +12.6 75.8 b 5:20.0 85.5 b 5:15.2
2.6 5:5.1 65.2 5:26.1 50.6 a 5-17.4 68.6 a 5:11.0 88.1 b ± 12.4
8.9 4.0 +8.8 +7.3 77.7 a 70.4 +19.1 _+30.0 59.8 a 18.7 +10.3 +10.3 155.2 e 87.5 b ±36.8 ±18.3 197.1 a 131.2e ± 44.7 ~ + 33.8
Human colon muscle LTB4
Human colon mucosa 5-HETE
PGs
LTB4
5-HETE
2.0 +0.7 57.3 _+15.5 51.5 ±13.4 96.0 b +20.8 116.0b + 36.1
- 13.2 5:13.1 20.0 5:8.1 85.7 a ±36.8 139.2 d +26.8 150.9 d -t-38.6
7.6 5:6.8 25.3 5:6.8 39.6 ±7.0 52.1 a 5:10.8 69.5 b 5:13.1
14.2 5:10.0 71.4 a ±33.1 8().4 b ±30.0 66.6 b +11.0 94.4 c 5:24.6
110 TABLE 3 Stimulation by ricinoleic acid (100/~g/ml) of [14C]arachidonic acid metabolism by rat colon and human colon muscle in vitro. Effect of indomethacin (1/tg/ml) or BW755C (1 /~g/ml). Results are expressed as % increase and analysed by Student's t-test for paired data. n = 6 in both cases. P values: a < 0.05; b < 0.02; c < 0.01 compared to controls; d < 0.01 compared to ricinoleic acid alone. Drug
Whole rat colon
Indomethacin BW 755C Ricinoleic acid Ricinoleic acid + indomethacin Ricinoleic acid + BW 755C
Human colon muscle
PGs
LTB4
5-HETE
PGs
LTB4
5-HETE
- 55.3 c -+4.0 - 47.2 c -+4.7 93.9 c -+11.8 26.1 d -+ 11.2 15.6 d -+ 18.3
- 2.4 -+3.1 - 41.1 c _+5.0 62.2 b -+8.2 54.0 _+6.5 -- 31.2 d -+7.0
18.3 -+7.6 - 46.3 ~ _+2.7 118.6 a -+17.0 75.0 _+24.7 15.9 d -+ 13.8
- 64.9 b -+4.5 - 59.7 b _+3.3 115.0 b -+2.1 69.3 ~ -+ 8.7 71.8 ~ -+ 7.1
-- 12.1 -+6.6 -- 53.2 b _+4.0 103.7 a -+10.4 52.6 -+ 10.6 10.7 ~ _+6.4
14.4 _+6.6 - - 62.0 b _+2.7 112.9 a +14.0 57.2 ___5.3 12.0 d -+7.8
3.3. Effects of indomethacin and BW755C on [:4C]arachidonic acid metabolism stimulated by ricinoleic acid in human and rat colon
cyclo-oxygenase/lipoxygenase inhibitor BW755C 1 t t g / m l ( t a b l e 3). T h e l a t t e r d r u g , b u t n o t i n d o m e t h a c i n , also i n h i b i t e d the f o r m a t i o n of the lipoxygenase products.
Both the normal and the stimulated formation of cyclo-oxygenase products by rat and human colon were i n h i b i t e d by the c y c l o - o x y g e n a s e inhibitor indomethacin
3.4. Metabolism of [:4C]arachidonic acid by colon and ileum tissue from different species
1 /~g/ml and by the dual Arachidonic
acid
metabolism
was
markedly
g r e a t e r in t h e c o l o n t h a n i n t h e i l e u m o f g u i n e a - p i g s TABLE 4
a n d rabbits, but m a i n l y similar in these 2 regions
Effect of ricinoleic acid (100/~g/ml) on [14C]arachidonic acid metabolism by intestinal tissue from different species. Results are expressed as % increase and analysed by Student's t-test for paired data. n = 5-8; P values: a < 0.1; b < 0 . 0 5 ; c < 0.02; d < 0.01. Control values: guinea-pig ileum (PGs 1.80-+0.17; LTB4 0.30_+0.06, 5-HETE 0.39+0.05) and colon (PGs 2.50-+ 0.29; LTB4 0.42 _+0.08; 5-HETE 0.36 + 0.04; mouse ileum (PGs 1.26-+0.16; LTB4 0.32-+0.11; 5-HETE 0.30+0.07; and colon (PG 1.52 + 0.14; LTB4 0.28 + 0.06; 5-HETE 0.38 -+0.06); rabbit ileum (PGs 2.60+0.40; LTB4 0.40+0.2; 5-HETE (0.40-+0.11) and colon (PGs 3.60+0.61; LTB4 0.58-+0.20; 5-HETE 0.68-+ 0.18).
o f t h e m o u s e ( t a b l e 4). I n all c a s e s r i c i n o l e i c a c i d or phenolphthalein stimulated the conversion of
Tissue
PGs
LTB4
100.3_+16.2b 159.0+16.3 c
67.5_+ 7.7 a 75.5_+15.4a
55.5_+12.9b 109.0+24.7 b
u e s b e i n g T X B 2 0.18, P G E 2 0.23, P G F 2 , 0.30, P G D 2 0.41. T h e a m o u n t s o f c y c l o - o x y g e n a s e p r o d u c t s c h r o m a t o g r a p h i n g at t h e a p p r o p r i a t e R e
162.7+69.8 c 235.2+48.5 d
84.8+41.0 b 154.1_+32.9 c
72.2+11.1 b 91.8+28.1 b
v a l u e s w e r e : P G E 2 > P G F 2 , > T X B 2 > P G D 2. Ricinoleic acid enhanced their formation, whereas
76.9_+19.7a 118.3-+18.8 b
105.2_+28.7 b 103.4-+22.3 b
90.0--+23.1 a 73.5-+10.8 a
i n d o m e t h a c i n or B W 7 5 5 C 1 / ~ g / m l i n h i b i t e d their b a s a l a n d r i c i n o l e i c a c i d - s t i m u l a t e d s y n t h e s i s (tab l e 5).
Rabbit Ileum Colon
Table 5 shows the formation of cyclo-oxygenase p r o d u c t s f r o m e x o g e n o u s a r a c h i d o n i c acid. P G E 2, PGFz,, PGD 2 and TXB z were characterised by c o m p a r i s o n w i t h a u t h e n t i c s t a n d a r d s , t h e R e val-
Mouse Ileum Colon
3.5. Effects of ricinoleic acid on the formation of different prostanoids by rat colon
5-HETE
Guinea-pig Ileum Colon
exogenous arachidonic acid into cyclo-oxygenase and lipoxygenase products.
111
TABLE 5 S t i m u l a t i o n b y ricinoleic a c i d (100 ~ t g / m l ) o f [ 1 4 C ] a r a c h i d o n i c a c i d m e t a b o l i s m b y r a t c o l o n in vitro. E f f e c t o f i n d o m e t h a c i n (1 / ~ g / m l ) o r B W 7 5 5 C (1 ~ g / m l ) . R e s u l t s a r e e x p r e s s e d as % i n c r e a s e a n d a n a l y s e d b y S t u d e n t ' s t-test f o r p a i r e d d a t a , n = 5; P values: a < 0.1; b < 0.05; c < 0.02; d < 0.01. Drug
PGE 2
PGF2a
PGD 2
TXB 2
Indomethacin BW755C Ricinoleic acid Ricinoleic acid + indomethacin Ricinoleic acid + BW755C
-58.6+ 6.8 0 -41.8+ 7.0 d 147.0 + 30.0 c
-38.4+ 6.5 a - - 3 5 . 2 + 6.6 a 105.4 + 18.8 ¢
-36.2+ 7.1 a - - 4 1 . 6 + 5.7 111.1 + 36.6
-39.2+ 9.1 a - - 4 0 . 0 + 4.7 a 90.7 -t- 15.7 b
56.4 _ 21.7 d
36.4 + 20.7 e
36.1 + 17.4 b
33.8 + 15.1 a
54.1 + 20.7 d
32.4 + 18.8 ¢
27.7 _+ 18.7 a
29.2 + 10.0 a
4. Discussion
Our results confirm that arachidonic acid cyclo-oxygenase and lipoxygenase products can be formed by rat, guinea-pig, mouse, rabbit and human intestine. The products have been characterised by their chromatographic mobility and by the inhibitory effect of indomethacin and the dual cyclo-oxygenase/lipoxygenase inhibitor BW755C. In the rat we found that PGE 2 is the major product. Ahlquist et al. (1982) and BoughtonSmith et al. (1983) found that human colonic mucosa also produced mostly PGE2, whereas Bennett et al. (1981, 1987) found that PGI 2 was the most abundant metabolite formed when the human colonic mucosa or muscles were homogenised in Krebs solution. Arachidonic acid metabolism via the cyclooxygenase pathway was quantitatively greater than via the lipoxygenase pathway, and the large intestine was more active than the small intestine except in the mouse where the tissues were mainly equi-active. Other investigators have also observed regional metabolic variation in PG generation, possibly due to regional differences in muscle cyclo-oxygenase activity (Ahlquist et al., 1982; Ali and McDonald, 1980). We have found a similar profile for the lipoxygenase products LTB4 and 5-HETE, presumably because of regional differences in lipoxygenase activity. Our results also demonstrate that the conversion of exogenous [14C]arachidonic acid was stimulated by all the laxatives except mannitol,
with rinicoleic acid being the most potent. The effect of sulfosuccinate and picosulphate were at most weak, particularly on the formation of lipoxygenase products. It is not clear from the experiments of Beubler and Juan (1979) whether the laxative effect of sulfosuccinate is mediated by PGs, but our results with this drug indicate that PGs are unlikely to be important. This is in contrast to phenolphthalein whose laxative effect is suppressed by PG synthesis inhibitors (Capasso et al., 1984a). The activity of the laxatives varied in different tissues. Cohen (1982) showed regional differences in the effect of laxatives on PG synthesis by rat gut. We found that ricinoleic acid stimulated arachidonic acid metabolism more in the large intestine than in the small intestine. With phenolphthalein a similar difference occurred in the rat (Capasso et al., 1984b) and rabbit (unpublished), but the reverse occurred in the mouse and guineapig (unpublished). Hypotheses on the mechanisms by which secretagogues cause laxation include stimulation of cAMP (see Beubler and Juan, 1979) or inhibition of Na/K-ATPase (see Gaginella and Bass, 1978). In rats, laxatives increase the luminal amount of PGs in the colon (Autore et al., 1984; Beubler and Juan, 1979; Cohen, 1982; Capasso et al., 1983, 1986) and the net secretion of water and electrolytes into the small intestine (Beubler and Juan, 1979). Increased intestinal formation of histamine and 5-hydroxytryptamine by laxatives (Autore et al., 1984; Capasso et al., 1986), possibly due to mucosal injury (Beubler and Juan, 1979),
112
might also contribute to the cathartic effect. Histamine and 5-HT increase the availability of calcium to phospholipase A 2 and consequently increase PG production (see Capasso et al., 1986). Some laxatives require calcium for their effects on electrolyte transport (Donowitz et al., 1984) and arachidonic acid metabolism (Capasso et al., 1985a,b). Since lipoxygenase and cyclo-oxygenase metabolites can stimulate intestinal secretion, these products are likely to play an important part in the effect of some laxatives. The stimulation of eicosanoid formation by the muscle may also make a substantial primary contribution to the cathartic effect.
Acknowledgements This research was supported by Wellcome Trust, N.A.T.O. and Consiglio Nazionale delle Ricerche.
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