Endothelin in the gastrointestinal tract

GASTROENTEROLOGY 1990;99:1660-1667

Endothelin in the Gastrointestinal

Tract

Presence of Endothelinlike Immunoreactivity, Endothelin-1 Messenger RNA, Endothelin Receptors, and Pharmacological Effect KAZUHIRO TAKAHASHI, PHILIP M. JONES, SANDIP M. KANSE, HING-CHUNG LAM, RACHEL A. SPOKES, MOHAMMAD A. GHATEI, and STEPHEN R. BLOOM Department of Medicine, London, England

Royal Postgraduate

The possible production and role of endothelin in the gastrointestinal tract was investigated in rats by radioimmunoassay, Northern-blot hybridization, receptor assay using membrane preparations, and pharmacological study using gut strips. Endothelinlike immunoreactivity was detected in all regions (from stomach to colon) of the rat gastrointestinal tract (13-48 fmol/g wet tissue) including the mucosal layer of the ileum and colon (8.4 f 2.0 fmol/g wet tissue and 18.4 rf- 2.1 fmol/g wet tissue, respectively, mean k SEM; n = 5). Fast protein liquid chromatographic analysis of the endothelinlike immunoreactivity in jejunum, ileum, colon, and colon mucosa extracts showed peaks in the positions of endothelin-l and endothelin-3. The presence of endothelin-1 messenger RNA was demonstrated by Northern-blot hybridization in the whole colon and pooled ileal and colonic mucosa, but not in the whole jejunum. Specific binding in the rat gastrointestinal tract was particularly high in the fundus of stomach, jejunum, ileum, and colon. In the ileum, many binding sites were found in the circular and longitudinal muscle layers, but few in the mucosal layer. Endothelin-1 and endothelin-5 caused contraction of rat stomach strips, rat colon, and guinea pig ileum. These findings indicate that endothelin is present in the rat gastrointestinal tract, perhaps produced by both vascular endothelial cells and mucosal epithelial cells, and can cause contraction of gastrointestinal smooth muscle. Thus, endothelin may have a physiological role in the control of gastrointestinal function. ndothelin-1 is a potent vasoconstrictor peptide which was originally isolated, and its structure determined, from the conditioned medium of cultured

E

Medical School, Hammersmith

Hospital,

porcine endothelial cells (1). Endothelin-3 was subsequently cloned and sequenced from a rat genomic library (2). It is now known that there are three endothelin genes (endothelin-1, -2, and -3 genes] in the human, porcine, and rat genome (3). Endothelin causes gut contraction (4,5), and local intraarterial infusion of endothelin causes hemorrhagic and necrotic damage in rat mucosa (6). Endothelin binding sites were shown to be widely distributed not only in the vascular tissues (7-9) but also in other tissues such as trachea (8), lung (91, kidney (lo), brain (11), and gastrointestinal tract (12). In the gastrointestinal tract, an autoradiographic study has shown that endothelin receptors are present in the mucosal layer of rat colon, intestine, and stomach (12). A vasoactive intestinal contractor peptide, which has a structure similar to endothelin, has been cloned and sequenced from the mouse genome (13). From these reports, endothelin or endothelinlike substances are thought to play a role in control of the digestive tract. However, the presence of endothelin and its exact physiological function in the gastrointestinal tract have not been established. To further clarify the role of endothelin, this study shows the presence of endothelinlike immunoreactivity (LI), endothelin-1 mRNA, and endothelin receptors in the rat gastrointestinal tract and its pharmacological effects using rat stomach strips, rat colon, and guinea pig ileum. Abbreviations used in this paper: B,, maximum binding capacity; BSA, bovine serum albumin; endothelin-LI, endothelinlike immunoreactivity; PPLC, fast protein liquid chromatography; Kd, dissociation constant; PCR, polymerase chain reaction: SDS, sodium dodecyl sulphate; SSC, standard saline citrate. 0 1990 by the American Gastroenterological Association 0016-5065/90/$3.00

ENDOTHELIN IN THE GASTROINTESTINAL TRACT

December 1990

Materials and Methods Tissues for Radioimmunoassay,

Hybridization,

Northern-Blot and Receptor Assay

Rat gastrointestinal tract tissues were obtained from male Wistar rats (250-350 g) for radioimmunoassay, Northernblot hybridization, and receptor assay immediately after the death of the rats. Rat thoracic aorta was also collected as a control for radioimmunoassay and receptor assay. The mucosal layers of ileum and colon were prepared by two procedures as previously described (14). In procedure A, rat ileum and colon were microdissected into mucosa, circular muscle layer, and longitudinal muscle layer. Procedure B was the modification of the methods previously reported (14-16)Rat ileum or colon was removed and inverted, and 20-30 mL of ice-cold NaCl 0.15 mol/L and EDTA 5 mmol/L, pH 7.4, was injected with a hypodermic needle between the mucosal and muscular layers in about twenty different places to produce a maximal distention of the mucosa. The tissue was then soaked in 30 mL of the same ice-cold medium and shaken by hand for 10 seconds. The suspension, after a first hand shaking, was discarded and replaced by fresh medium. Then the medium was collected after periods of shaking for 10 seconds (total four shakings) and replaced by fresh medium. The collected medium was centrifuged at 200 x g for 3 minutes and the resulting pellet was washed by resuspension and centrifugation at 200 x g for 3 minutes in the same medium. The resulting pellet was used for the extraction of endothelin-11 for radioimmunoassay, mRNA extraction for Northern-blot hybridization, and membrane preparation for receptor assay. Routine histological examinations of both mucosa and muscle preparations prepared by both procedures A and B showed no cross contamination [data not shown). This was confirmed by radioimmunoassay (17-20) of extracts of ileal and colonic muscle and mucosal preparations using standard peptide markers (14.21). More than 95% of the extracted substance P(SP) and vasoactive intestinal polypeptide [VIP) was found in the muscle layers with ~5% in the mucosal preparation. In contrast, over 90% of the enteroglucagon and 80% peptide YY (PYY) immunoreactivity was found in the mucosal preparation.

Extraction of Endothelinlike Immunoreactivity and Radioimmunoassay Tissues were extracted by boiling in a IO-fold volume of 0.5 mol/L acetic acid for 10 minutes (22). The supernatants were reextracted using Sep-Pak Cl8 cartridges (Waters Associates, Milford, MA) and endothelin-11 eluted with 2 mL of 60% (vol/vol) acetonitrile/water containing 0.026 mol/L ammonium acetate. The eluate was dried in a Savant vacuum centrifuge (Savant Instruments, Inc., Hicksville, NY), the resulting pellet reconstituted with assay buffer [60 mmol/L PO, buffer, pH 7.4, containing 10 mmol/L EDTA, 7 mmol/L sodium azide, and 0.3% (wt/vol) bovine serum albumin (BSA)], and the aliquot assayed in duplicate. The recovery of endothelin-1, -2, and -3, which were added to tissues before boiling, was >80% (n = 5). The radioimmunoassay for endothelin-1 was previously reported in detail

1661

(23). The antiserum to endothelin-1 used in this study crossreacted 40% with endothelin-2 and 60% with endothelin-3. Fractionation of the endothelin-11 in rat tissue extracts was performed by fast protein liquid chromatography (FPLC) using a high-resolution reverse-phase 5/5 (Pep Rpc HR 5/5) Cl8 column (Pharmacia, Uppsala, Sweden), with a gradient of acetonitrile from 15% to 35% (vol/vol) in water (both acetonitrile and water with 0.1% trifluoroacetic acid) over 1 hour at 1 mL per minute per fraction. Samples of each fraction were dried in a Savant vacuum centrifuge, reconstituted with assay buffer, and assayed.

RNA Extraction

and Northern-Blot

Analysis

Extraction of total RNA from pooled ileal and coionic mucosa, whole jejunum, and whole colon was performed using the acid guanidinium thiocyanate-phenolchloroform extraction (AGPC) procedure (24). Poly(A) RNA was purified by oligo(d‘I’)-cellulose chromatography (25) before it was denatured and run on a MOPS [3-(Nmorpholino)propane-sulphonic acid]-formaldehyde-l% agarose-denaturing gel (26) and then transferred to Hybond-N membrane (Amersham International, Amersham, Buckinghamshire, England). Following transfer, RNA was fixed by baking at 80°C for 2 hours before being probed with a radioactively labeled RNA probe. A probe for endothelin-1 mRNA was synthesized by cloning a DNA fragment containing part of the human endothelin-1 coding sequence from human genomic DNA using the polymerase chain reaction (PCR) (27). A pair of oligonucleotides were synthesized using a Biosearch Cyclone DNA synthesizer [New Brunswick Scientific Co., San Rafael, CA], one of which was identical to nucleotides 132-153 of the published human endothelin-1 coding sequence (2). Thirty cycles of repeated denaturing, annealing, and DNA synthesis using Taq polymerase was then performed, the reaction products run out on an agarose gel, and a band of the expected size was observed, cut out, purified by electroelution, and ligated into the vector Bluescript (Vector Cloning Systems) to give the clone pBT-hET-1. The sequence and orientation of the cloned fragment were determined by direct dideoxy sequencing [28) of doublestranded plasmid DNA (29) and the cloned sequence was observed to be identical with that published. An RNA probe was prepared by in vitro transcription as previously described (30). For this purpose, the plasmid pBT-hET-1 was linearized by digestion with EcoRI for synthesis of labeled endothelin-1 antisense cRNA. The reaction mixture (10 rL total volume) contained 40 mmol/L Tris-HCl, pH 8.6; 25 mmol/L NaCI; 8 mmol/L MgCI,; 2 mmol/L spermidine; 10 mmol/L dithiothreitol; 100 pg/mL BSA; 1 pg linearized DNA template; 500 rmol/L each of ATP, GTP, and UTP with 3.7 mol/L Bq (100 PCi) of 3ZP-labeled CTP (Amersham; 800 Ci/mmol; 20 mmol/L), and 20 units of T7-RNA polymerase (Bethesda Research Laboratories; Gibco-BRL, Paisley, Scotland). Transcription occurred at 40°C for 60 minutes followed by digestion with I pm of RNase-free DNase (Pharmacia) at 37°C for 15 minutes and unincorporated nucleotides were removed by filtration through a column of Sephadex G-50 beads (Sigma, Poole, Dorset, England). Hybridization occurred for 16 hours at

1662 TAKAHASHI ET AL.

GASTROENTEROLOGY Vol. 99, No. 6

55°C in solution

of 50% formamide, 5 x Denharts solution saline citrate (SSC) (1 x SSC = 0.15 mol/L NaCVO.015 mol/L sodium citrate, pH 7.0) (31), 50 mmol/L sodium phosphate (pH 7.01, 200 pg/mL yeast RNA, 100 pg/mL denatured, sonicated salmon sperm DNA, 10% (wt/vol) dextran sulphate, and 20 ng/mL of probe. Filters were washed at room temperature three times in a solution of 2 x SSC/O.l% SDS for 5 minutes before being washed twice at 65°C in a solution of 0.1 x SSC/O.l% sodium dodecyl sulphate [SDS) for 60 minutes. After washing, filters were sealed into plastic bags and exposed to Kodak XAR-5 film at - 70°C with intensification screens. Following autoradiography, bound probe was removed by heating at 80°C for 20 minutes in 10 mmol/L Tris solution, pH 7.5, 1 mmol/L EDTA plus 0.5% SDS. Normalization was performed by rehybridization with a ubiquitin cDNA fragment. The ubiquitin cDNA was supplied by Professor K. Lund, School of Medicine, University of North Carolina at Chapel Hill (Chapel Hill, NC). (31),5 x standard

Receptor

Assay

Homogenization of tissues from Wistar rats was performed in a lo-fold volume of ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4) containing 0.25 mol/L sucrose using an Ultra-Turrax (Janke and Kunkel, Staufen, Federal Republic of Germany] homogenizer. The homogenate was centrifuged at 1000 x g for 20 minutes and the resulting supernatant was centrifuged at 50,000 x g for 20 minutes. The pellet was resuspended in 50 mmol/L Tris-HCl buffer (pH 7.4) at a final concentration of 1-5 mg/mL and stored as aliquots at -20°C. Protein content was estimated by the Coomassieblue dye method with BSA as standard (Pierce Chemical

directions to make one strip. This was then cut in half longitudinally to give two identical strips. Two adjacent sections of mid-colon, each 3-cm long, were also obtained. These were mounted for measurement of isotonic contractions under 1 g tension in lo-mL organ baths containing Krebs solution at 37“C and gassed with 95% O,, 5% CO,. Cumulative dose-response curves to endothelin-1 and -3 were obtained, testing one peptide in each of the two strips from the same rat. Male Dunkin-Hartley guinea pigs (300-350 g] were killed and sections of ileum set up as for rat colon. The responses for each tissue are given as the percentage of the maximal response to 10m7mol/L endothelin-1. Results Radioimmunoassay Endothelinlike immunoreactivity was detected by radioimmunoassay in all the regions of rat gastrointestinal tract and in the mucosal layers (Table 1). The concentration of endothelin-11 in these tissues was much greater than in the rat thoracic aorta. Fast protein liquid chromatography showed that the endothelin-11 in whole jejunum [Figure lA), whole ileum (Figure lB], whole colon (Figure lC), and colon mucosa extracts [Figure 1D) eluted just after the void volume in the positions of endothelin-3 and endothelin-1. A peak in the position of endothelin-2 was found in FPLC of whole j ej unum and colon mucosa extracts. Several other peaks were found in FPLC of ileum extract.

Co., Rockford, IL) (32). ‘251-endothelin-l was prepared by the chloramine-T method (33) and purified by high-performance liquid chromatography as described previously (9). Under standard conditions, the binding assays were performed in a final volume of 0.5 mL of 50 mmol/L Tris-HCI buffer (pH 7.4) containing BSA (0.3% wt/vol), 5 mmol/L EDTA, membrane (30 pg], ‘251-endothelin-l (20-40 fmol), and unlabeled competing peptide as specified. Membranes were incubated at 37’C for 180 minutes and the separation of bound and free radioactivity was performed by centrifugation at 12,000 x g for 3 minutes in a microfuge. The supernatant was removed and the radioactivity in the pellet counted in a gamma counter. Specific binding was calculated as the difference between the amount of ‘9-endothelin-1 bound in the absence [total binding) and in the presence of 0.1 rmol/L unlabeled endothelin-1 (unsaturable binding). Scatchard plots were analyzed by linear regression to determine dissociation constant (Kd) and the maximum binding capacity (B,,).

Pharmacological

Study

of Endothelin

Male Wistar rats were killed and the stomach and the colon were removed. The fundus was separated and cut open into a flat sheet. Two cuts were made approximately two-thirds of the way through the stomach from opposite

Table 1. Distribution of Endothelinlike Immunoreactivity and Endothelin Receptors in the Rat Gastrointestinal Tract

Region Fundus of stomach Antrum of stomach Duodenum jejunum Ileum Mucosal layer Procedure A Procedure B Circular muscle layer Longitudinal muscle layer Colon Mucosal layer Procedure A Procedure B Thoracic aorta

Endothelin-LI UmW wet tissue) 19 * 2 13 f 3 48 f 10 35 f 2 37 f 4 15.2* 1.5 8.4f 2.0 63.3zt20.0 8.4f 1.0 33 f 2 16.8-L0.8 18.4+ 2.1 7.0(n = 1) and nd (~2.5) [n = 4)

Specific binding (% of total 1z5rendothelin-1 added] 14.3* 3.1 0.8f 0.5 0.1f 0.06 11.3* 3.1 11.0* 3.1 0.4* 1.0* 17.7* 18.2A 12.3*

0.2 0.1 3.5 4.3 3.6

2.9* 0.2 0.4f 0.1 10.4f 2.2

NOTE. Mean f SEM, n = 5.Binding is expressed as percent of tracer that is specificallybound to 30 pg of protein. nd, not detectable.

December

ENDOTHELIN

1999

IN THE GASTROINTESTINAL

TRACT

1663

Receptor Assay

a 60

= E c ;

46

36

z 7 &

24

12

50

40

30

20

10

0 60

50

= E c 5 E z

46

40

36

30

?

24

20

12

10

I;

n.

0 0

10

20

30

40

60

In a preliminary receptor-assay experiment, we observed that addition of 5 mmol/L EDTA or EGTA to the incubation medium with rat-gut membrane increased specific binding significantly (5-6 times increase in specific binding was found when rat-gut membrane with 80 pg protein was incubated with lz51-endothelin-1 in the medium with EDTA or EGTA), whereas addition of phosphoramidon, aprotinin, bacitracin, or soya bean trypsin inhibitor did not affect specific binding significantly. As a result of these findings, we added 5 mmol/L EDTA to the incubation medium in all the experiments. The binding of ‘251-endothelin-l to rat-gut membrane was linear at low protein concentrations and reached saturation at higher concentration (loo-150 pg protein/O.5 mL). All experiments were done at protein concentrations in the linear range of binding (30 pg protein/O.5 mL]. Specific binding of ‘251-endothelin-l to rat-gut membranes increased time-dependently to 300 minutes [Figure 3). Addition of 0.1 pmol/L cold peptide to membrane at 180 minutes caused a very small dissociation of the hormone receptor complex. A survey of the regional distribution of lz51endothelin-1 binding sites in the rat gastrointestinal tract was performed [Table 1). These values represent determinations at a single ligand concentration (20-40

A

0 60

70

minutes

Figure I. Fast protein liquid chromatography of endothelin-Ll in extract, (B) whole ileum extract, (C) whole colon extract, and (D) colon mucesa extract. El’l, JTR&and ET3 indicate the elution pusitions of endothelin-1, -2, and -3, respectively.

(A) whole jejunum

Northern-Blot

Hybridization

Endothelin-1 mRNA was detectable in both whole colon and in pooled ileal and colonic mucosa poly(A) RNA but not in whole jejunum poly(A) RNA (Figure 2). Northern-blot analysis showed that mature rat preproendothelin mRNA was present as a single form of approximately 2.3-2.4 Kb. The signal from the track containing whole colon poly(A) RNA was approximately twofold more intense than that in the colonic mucosa poly(A) track. The size of rat preproendothelin mRNA was similar to that of the porcine and human prohormone encoding mRNAs (1,34).

Figure 2. Northern-blot hybridi2ation. A. Pooled ileal and colonic mucosa poly(A) RNA (10 pg). B. Whole-colon (19 4. C. Whole-jejunum (16 ml.

poly(A) RNA polylA) RNA

B

C

1664 TAKAHASHI ET AL.

OY 0

GASTROENTEROLOGY Vol. 99, No. 6

’ SO

60

90

120

lime

of incubation

160

160

210

240

270

200 -11

(minutes)

Figure 3. Time cour6e of ‘261-endothelln-l e66ociation (01 and dissociation (0) to rat ileum membranes. Diseociation we6 initiated by addition of synthetic endothelln-1 to give a final concentration of 0.1 pmol/L (arrow].

fmol) rather than B,,. High-specific binding in the rat gastrointestinal tract, which was comparable to that in thoracic aorta, was observed in the fundus of stomach, jejunum, ileum, and colon, whereas the antrum of stomach and duodenum showed negligible binding. In the ileum, high-specific binding was found in the longitudinal and circular muscle layers, whereas specific binding in the mucosal layer prepared by two different procedures was negligible. The binding of 1251-endothelin-l to gut membrane was pH dependent and maximal binding was observed at pH 6.0. To determine the specificity of endothelin binding sites, a variety of peptides were tested to inhibit ‘251-endothelin-l binding with rat-gut membranes. Inclusion of up to 1 pmol/L calcitonin gene-related peptide, neuropeptide Y, SP, VIP, peptide histidine methionine, glucagon, glucagonlike peptide-l (7-36), oxytomodulin, human atria1 natriuretic peptide, and angiotensin II did not affect binding in rat-gut membranes. Endothelin-1 was more active in displacing 1251-endothelin-l than endothelin-2 or -3 [Figure 4). IC,, of endothelin-1, -2, and -3 in colon were 0.075 * 0.020 nmol/L, 0.22 +- 0.01 nmol/L, and 0.61 + 0.08 nmol/L (mean f SEM, n = 4), respectively. IC,, in fundus of stomach and ileum were also similar to these values (data not shown]. The concentration dependence of endothelin binding to rat whole-gut membrane was determined by adding increasing concentrations of labelled endothelin. Scatchard analysis of these data showed that Kd and B,, in the fundus of stomach were 0.19+ 0.01 nmol/L and 995 f 117 fmol/mg protein; in the ileum, 0.26 f 0.03 nmol/L and 787 f 149 fmol/mg protein; and in the colon, 0.28 + 0.10 nmol/L and 710 f 126 fmol/mg protein (mean + SEM, n = 4), respectively (Figure 5).

-10 Log

[peptide

-9

-8

concentrationl

-7

M

Figure 4. Displacement of ‘Z51-endothelln-l binding in rat colon membrane6 by endothelin-1 (O),endothelin-3 (A), and endothelin-3 (A)

Pharmacological

Study

The effects of endothelin-1 and -3 on rat stomach, rat colon, and guinea pig ileum are shown in Figure 6A-C. Both peptides were equipotent at contracting the rat stomach strip. On the guinea pig ileum, endothelin-3 was 12 times less potent than endothelin-1, and on the rat colon, endothelin-3 was less potent than endothelin-1 with a lower maximal response. The rat colon was the most sensitive tissue to endothelin-1, being approximately five times more sensitive than the guinea pig ileum and 10 times more sensitive than the rat stomach strip. Discussion We have shown the presence of endothelin-11, endothelin-1 mRNA, and endothelin-binding sites in the rat gastrointestinal tract and a potent pharmacolog-

E

0.24

a

0.16

c z

m

0.08 0.00 Bound

(fmd

par

mg of

Protein)

Figure 5. Scatchard analysis of “%endothelin-1 binding in the fundue of the stomach (O),ileum (A), and colon (0). Increasing quantities of lZSI-endothelin-l were incubated with membrane (30 I.cgprotein).

December

-12

a E

ENDOTHELIN

1990

100

-

40

-

-12

-11

-10

-9

-8

-7

-11

-10

-9

-8

-7

log

Iendothelin] 64

Figure 6. Dose-response curves of the &ect of endothelin-1 (0) and endothelin-3 (0) on (A] rat stomach strip, (II)rat colon, and (C) guinea pig Ileum. Results are means fiwm six animals in each case. Vertical bars indicate SRM.

ical effect of endothelin on the gut. Particularly interesting is the presence of endothelin-11 and endothelin-1 mRNA in the mucosal epithelial layers. These results suggest that endothelin is produced not only by vascular endothelial cells but also by mucosal epithelial cells in the gastrointestinal tract and extend the previously reported findings that cultured porcine and canine tracheal epithelial cells (35) and cultured renal epithelial cells (36) produce endothelin. However, endothelin-1 mRNA was present at a higher level in whole gut poly(A) RNA than in mucosal poly(A) RNA and quantitatively the mucosa forms only a minor part of the mass of whole-gut

IN THE GASTROINTESTINAL

TRACT

1665

tissue. Thus, endothelin-1 mRNA synthesized in the mucosa could contribute to only a small part of the signal detected in whole colon. In addition, higher levels of endothelin-11 were found in the circular muscle layer of ileum than the mucosal layer. These results would indicate that most of endothelin-1 is synthesized in other sites throughout the gut and the most obvious site is the gut vasculature. However, endothelin has been found to be widespread throughout the central nervous system and in particular is found in the neurons of the dorsal root ganglia (37). It is thus probable that endothelin is also synthesized in the neurons of the entire neural plexus. The levels of endothelin-11 in the gastrointestinal tract were low compared with the levels of gut hormones and neuropeptides, for example somatostatin, VIP, or SP (13). However, surprisingly, the level of endothelin-11 in rat thoracic aorta was much lower than the levels in the gastrointestinal tract and this may reflect the fact that endothelin produced in aortic endothelial cells is not stored (1). Fast protein liquid chromatography showed that endothelin-1 and +-like materials were present in the whole ileum and colon extracts, and endothelin-1, -2, and &like materials were found in the whole jejunum and colon mucosa extracts. Material eluting just after the void volume on FPLC was found in all these four samples and its hydrophilic@ may indicate a larger molecule, perhaps a precursor of endothelin (1). We could not show the presence of endothelin-1 mRNA in whole jejunum, although the presence of endothelin-1, -2, and -&like materials in the whole jejunum were shown by FPLC. This may be due to the relatively low sensitivity of the technique used in Northern-blot analysis. The interaction of endothelin-1 with rat gastrointestinal tract membranes showed high affinity and specificity, which are some of the criteria for a receptor ligand interaction. Dissociation of receptor bound endothelin was minimal, as shown in rat lung or porcine aorta membranes (9). This suggests that the binding of endothelin-1 with rat gastrointestinal tract membrane is similar. Contrary to a previous report by others (u), we found very low binding sites in the mucosal layer of ileum, but high binding sites in both longitudinal and circular muscle layers of ileum. The findings of the contraction of gut by endothelin confirmed the previous reports (4,5). High endothelin binding sites in muscular layer may mediate this effect. However, endothelin may also bind with vascular smooth muscle cells or entire nervous plexi in the gastrointestinal tract as there is high binding of endothelin with cultured rat aortic vascular smooth muscle cells (7) and rat brain (11).

1666 TAKAHASHI

ET AL.

Local intraarterial infusion of endothelin was reported to cause hemorrhagic and necrotic damage in rat-stomach mucosa and endothelin is thought to have a potential proulcerogenic role (6). But the results of our receptor study showed that there were few binding sites for endothelin-1 in the antrum of stomach and duodenum, where ulcers are likely to occur, although endothelin-11 is present in both stomach and duodenum. The physiological significance of the low bindingsite density for endothelin-1 in the antrum of stomach and duodenum requires further investigation. In conclusion, this study showed the presence of endothelin-11, endothelin-1 mRNA, and high-specificity binding sites for endothelin-1 in the rat gastrointestinal tract and a potent pharmacological effect on gut smooth muscle. Endothelin may be produced by a variety of cells, including mucosal epithelial cells, in the gastrointestinal tract. Its release and influence may form a hitherto unknown physiological control system in the gastrointestinal tract. References 1. Yanagisawa

M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-415. 2. Yanagisawa M, Inoue A, Ishikawa T, Kasuya Y, Kimura S, Kumagaye S, Nakajima K, Watanabe TX, Sakakibara S, Goto K, Masaki T. Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci USA 1988;85:8964-6967. 3. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 1989;86:2863-2867. 4. Walder C, Warner TD, Vane JR. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endotheliumderived relaxing factor. Proc Nat1 Acad Sci USA 1988;85:97979800. 5. Spokes RA, Ghatei MA, Bloom SR. Studies with endothelin-3 and endothelin-1 on rat blood pressure and isolated tissues: evidence for multiple endothelin receptor subtypes. J Cardiovast Pharmacol1988;95:S191-S192. 6. Whittle BJR. Esphtgues JV. Induction of rat gastric damage by the endothelium-derived peptide, endothelin. Br J Pharmacol 1988;95:1011-1013. 7. Hirata Y, Yoshimi H, Takata S, Watanabe TX, Kumagai S, Nakajima K, Sakakibara S. Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commun 1988154868-875. 8. Power RF, Wharton J, Zhao Y, Bloom SR, Polak JM. Autoradiographic localization of endothelin-1 binding sites in the cardiovascular and respiratory systems. J Cardiovasc Pharmacoll989; 13:S50-S56. 9. Kanse SM. Ghatei MA, Bloom SR. Endothelin binding sites in porcine aortic and rat lung membrane. Eur J Biochem 1989;182: 175-179. 10. Orita Y, Fujiwara Y, Ochi S, Takama T, Fukunaga M, Yokoyama K. Endothelin-1 receptors in rat renal glomeruli. J Cardiovasc Pharmacol1989;13:S159-S181. 11. Jones CR, Hiley CR, Pelton JT, Mohi M. Autoradiographic

GASTROENTEROLOGY

Vol. 99, No. 6

visualization of the binding sites for [“‘I]endothelin in rat and human brain. Neurosci Lett 1989;97:278-279. 12. Koseki C, Imai M, Hirata Y, Yanagisawa M, Masaki T. Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide? Am J Physiol1989;256:R858-R868. 13. Ishida N, Tsujioka K, Tomoi M, Saida K, Matsui Y. Differential activities of two distinct endothelin family peptides on ileum and coronary artery. FEBS Lett 1989;247:337-340. 14. Ferri G-L, Adrian TE, Ghatei MA, O’Shaughnessy DJ, Probert L, Lee YC, Buchan AM, Polak JM, Bloom SR. Tissue localization and relative distribution of regulatory peptides in separated layers from the human bowel. Gastroenterology 1983;84:777786. 15. Harrison DD, Webster HL. The preparation of isolated intestinal crypt cells. Exp Cell Res 1969;55:257-260. 16. DuPont C, Laburthe M, Broyart JP, Bataille D, Rosselin G. Cyclic AMP production in isolated colonic epithelial crypts: a highly sensitive model for the evaluation of vasoactive intestinal peptide action in human intestine. Eur J Clin Invest 1980;10:6776. 17. McGregor GP. Substance P. In: Bloom SR, Long RG, eds. Radioimmunoassay of gut regulatory peptides. London: Saunders, 1982:154-163. 18. Mitchell SJ, Bloom SR. Measurement of fasting and postprandial VIP in man. Gut 1978;19:1043-1048. 19. Ghatei MA, Bloom SR. Enteroglucagon in man. In: Bloom SR, Polak JM, eds. Gut hormones. 2nd ed. Edinbourgh: ChurchillLivingstone, 1981:332-338. 20. Adrian TE, Ferri G-L, Bacarese-Hamilton AJ, Fuessl HS. Polak JM, Bloom SR. Human distribution of a putative new gut hormone, peptide YY. Gastroenterology 1985;89:1070-1077. 21. Domin J, Ghatei MA, Chohan P, Bloom SR. Neuromedin U-a study of its distribution in the rat. Peptides 1987;8:779-784. 22. Bryant MG, Bloom SR. Measurement in tissues. In: Bloom SR, Long RG, eds. Radioimmunoassay of gut regulatory peptides. London: Saunders, 1982:38-41. 23. Takahashi K, Brooks RA, Kanse SM. Ghatei MA, Kohner EM, Bloom SR. Production of endothelin-1 by cultured bovine retinal endothelial cells and presence of endothelin receptors on associated pericytes. Diabetes 1989;38:1200-1202. 24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Analyt Biochem 1987;162:156-159. 25. Jacobson A. Purification and fractionation of poly(A)+ RNA. In: Berger SL, Kimmel AR, eds. Methods in enzymology 152: guide to molecular cloning techniques. Chapter 25. London: Academic, 1987. 26. Maniatis T. Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Chapter 6. Cold Spring Harbour, NY: Cold Spring Harbour Lab, 1982. 27. Saiki R, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of B-globulin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985:230:1350-1356. 28. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain terminating inhibitors. Proc Nat1 Acad Sci USA 1977;74:54635468. 29. Mierendorf RC, Pfeffer D. Direct sequencing of denatured plasmid DNA. In: Berger SL, Kimmel AR, eds. Methods in enzymology 152: guide to molecular cloning techniques. Chapter 58. London: Academic, 1987. 30. Melton DA, Krieg PA, Rebagliati MR. Maniatis T, Zinn K, Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 1982;12:70537056.

December

1990

31. Maniatis T, Fritsch EF, Sambrook 1. Molecular cloning: a laboratory manual. Appendix A. Cold Spring Ha&our, NY: Cold Spring Harbour Lab, 1982. 32. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-260. 33. Hunter WM, Greenwood FC. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 1962;194:495-497. 34. Itoh Y, Yanagisawa M, Ohkubo S, Kimura C, Kosaka T, Inoue A, Ishida N, Mitsui Y, Onda H. Fujio M, Masaki T. Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. FEBS Lett 1988;231:440444. 35. Black PN, Ghatei MA, Takahashi K, Bretherton-Watt D. Krausz T, Dollery CT, Bloom SR. Formation of endothelin by cultured airway epithelial cells. FEBS Lett 1989;255:129-132.

ENDOTHELIN

IN THE GASTROINTESTINAL

TRACT

1667

38. Shichiri M, Hirata Y, Emori T, Ohta K, Nakajima T, Sato K, Sato A. Marumo F. Secretion of endothelin and related peptides from renal epithelial cell lines. FEBS Lett 1989;253:203-296. 37. Giaid A, Gibson S J, Ibrahim NBN, Legon S, Bloom SR, Yanagisawa M, Masaki T, Varndell IM, Polak JM. Endothelin 1. an endothelium-derived peptide is expressed in neurons of the human spinal cord and dorsal root ganglia. Proc Nat1 Acad Sci USA 1989;86:7634-7638.

Received October 27.1989. Accepted May 24.1990. Address requests for reprints to: Professor Stephen R. Bloom, Department of Medicine, Francis Fraser Laboratory, 2nd Floor, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London Wl2 ONN, England. The authors are grateful to Drs. J. J. Calvo, R. Gonzalez, and L. F. de Carvalho for their kind technical assistance and K. Davies for her critical reading and helpful discussions.