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Keywords:

  • colon smooth muscle;
  • EP receptors;
  • ileal smooth muscle;
  • myenteric neurons;
  • prostaglandin E2

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments and Disclosures
  7. References
  8. Supporting Information

Background  Prostaglandin E2 (PGE2) is an inflammatory mediator implicated in several gastrointestinal pathologies that affect normal intestinal transit. The aim was to establish the contribution of the four EP receptor types (EP1–4), in human colon, that mediate PGE2-induced longitudinal smooth muscle contraction.

Methods  Changes in isometric muscle tension of human colon, mouse colon and mouse ileum were measured in organ baths in response to receptor-specific agonists and antagonists. In addition, lidocaine was used to block neurogenic activity to investigate whether EP receptors were pre- or post-junctional.

Key Results  PGE2 contracted longitudinal muscle from human and mouse colon and mouse ileum. These contractions were inhibited by the EP1 receptor antagonist, EP1A in human colon, whereas a combination of EP1A and the EP3 antagonist, L798106 inhibited agonist responses in both mouse preparations. The EP3 agonist, sulprostone also increased muscle tension in both mouse tissues, and these responses were inhibited by lidocaine in the colon but not in the ileum. Although PGE2 consistently contracted all three muscle preparations, butaprost decreased tension by activating smooth muscle EP2 receptors in both colonic tissues. Alternatively, in mouse ileum, butaprost responses were lidocaine-sensitive, suggesting that it was activating prejunctional EP2 receptors on inhibitory motor neurons. Conversely, EP4 receptors were not functional in all the intestinal muscle preparations tested.

Conclusions & Inferences  PGE2-induced contraction of longitudinal smooth muscle is mediated by EP1 receptors in human colon and by a combination of EP1 and EP3 receptors in mouse intestine, whereas EP2 receptors modulate relaxation in all three preparations.

Abbreviations:
AH6809

6-Isopropoxy-9-oxoxanthine-2-carboxylic acid

anova

analysis of variance

Ca2+

calcium

cAMP

cyclic adenosine monophosphate

Caps

capsaicin

CCh

carbachol

CCK-8

cholecystokinin octapeptide

COX

cyclooxygenase

DMSO

dimethylsulfoxide

EP1A

EP1 receptor antagonist

GW627368X

(N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydr-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulfonamide

g

grams

IBD

inflammatory bowel disease

IBS

irritable bowel syndrome

K+

potassium

KCl

potassium chloride

KH

Krebs Henseleit

L798106

5-Bromo-2-methoxy-N-[3-(2-naphthalen-2-yl-methylphenyl)-acryloyl]-benzenesulfonamide

Lid

lidocaine

lNNA

nitro-l-arginine

MEN10627

cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2β-5β)

LSM

longitudinal smooth muscle

Na+

sodium

NK

neurokinin

NO

nitric oxide

NOS

nitric oxide synthase

PG97269

[AcHis1,DPhe2,Lys15,Arg16,Leu27]VIP(1-7)/GRF(8-27)-amide

PGE1-OH

prostaglandin E1-alcohol

PGE2

prostaglandin E2

RP67580

(3aR,7aR)-Octahydro-2-[1-imino-2-(2-methoxyphenyl)ethyl]-7,7-diphenyl-4H-isoindol

SE mean

standard error of the mean

STC

slow transit constipation

sulp

sulprostone

VIP

vasoactive intestinal polypeptide

Inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and slow transit constipation (STC) are examples of diseases where normal colonic motility is affected. Prostaglandins, synthesized by the enzyme cyclooxygenase (COX), have been implicated as mediators of these intestinal diseases.1,2 Two COX isoforms (COX-1 and -2) are expressed in the gastrointestinal tract, particularly in smooth muscle, lamina propria mononuclear cells and myenteric neurons.3–5 COX-1 is constitutively active and produces prostaglandins that regulate normal physiological functions including cytoprotection,6 whereas COX-2 activity is predominantly induced by inflammation.5 COX-2 preferentially generates the key inflammatory mediator, prostaglandin E2 (PGE2),1 and increased levels of both have been observed in colonic specimens from patients with IBD and STC.1,4

PGE2 activates EP receptors and four different types (EP1, EP2, EP3 and EP4) have been cloned. EP receptors couple to G-proteins, triggering different signaling pathways. EP1 receptors preferentially couple to Gq and increase intracellular calcium (Ca2+) levels, whereas EP2 and EP4 receptors signal via Gs and increase cyclic adenosine monophosphate (cAMP) production.6 The EP3 receptor predominantly signals through Gi-coupling, reducing cAMP, although several splice variants exist (seven human and three murine) with different G-protein specificities, coupling to Gi, Gs and possibly Gq.7

The EP receptors can be grouped functionally according to their effects on smooth muscle and include: (i) the relaxant Gs-coupled EP2 and EP4 receptors, (ii) the contractile Gq-coupled EP1 receptor, and (iii) the EP3 receptor, which mediates contraction through Gi-signaling.8 Prostaglandin E2 contracts longitudinal and relaxes circular smooth muscle of human ileum and colon;9,10 albeit the receptor type(s) that mediate these responses have not been elucidated. Prostaglandin E2 also contracts longitudinal smooth muscle of rat11 and guinea-pig colon.12 A study using knockout mice, separately lacking each of the eight different prostanoid receptor types, suggested that PGE2-induced contraction of mouse ileum was mediated by EP1 and EP3 receptors with an additional non-selective effect on FP receptors.13 It has also been demonstrated that more than one EP receptor type can be expressed by the same muscle cells, for example EP1 or EP3 receptors mediate contraction of isolated circular smooth muscle from guinea-pig ileum or dog colon, respectively; whereas EP2 activation inhibits cholecystokinin octapeptide (CCK-8)-induced contractions in the same cells.14,15 Furthermore, there is evidence to suggest that pre-junctional EP receptors, whose activation indirectly alters motility by depolarizing myenteric neurons and inducing neurotransmitter release, are present in the intestine. For example, PGE2 has been shown to depolarize AH and S neurons in the myenteric plexus of the guinea-pig ileum and colon.16,17 Moreover, Das & Ganguly18 demonstrated that PGE2 stimulated acetylcholine release from excitatory motor neurons in the guinea-pig ileum.

The primary aim of this study was to elucidate the contribution of the four EP receptor types that mediate PGE2-induced contraction of longitudinal smooth muscle from human colon. In addition, species-specific differences and intestinal-regional differences were established by comparing human colon to mouse colon and ileum. Furthermore, as there is evidence to suggest that PGE2 depolarizes myenteric neurons as well as having direct effects on smooth muscle, the cellular locations of the expressed EP receptor types were investigated pharmacologically.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments and Disclosures
  7. References
  8. Supporting Information

Materials

6-Isopropoxy-9-oxoxanthine-2-carboxylic acid (AH6809), (N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydr-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulfonamide (GW627368X);19 EP1A11 and 5-Bromo-2-methoxy-N-[3-(2-naphthalen-2-yl-methylphenyl)-acryloyl]-benzenesulfonamide (L798106) were obtained from GlaxoSmithKline (Harlow, UK). Prostaglandin E2, PGE1-alcohol (PGE1-OH), butaprost free acid, and sulprostone were purchased from Cayman Europe (Tallinn, Estonia). [AcHis1,DPhe2,Lys15,Arg16,Leu27]VIP(1-7)/GRF(8-27)-amide (PG97269) was from Phoenix pharmaceuticals (Burlingame, CA, USA); (3aR,7aR)-Octahydro-2-1-[1-imino-2-(2-methoxyphenyl)ethyl]-7,7-diphenyl-4H-isoindol (RP67580) was purchased from Tocris Biosciences (Bristol, UK); cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2β-5β) (MEN10627) was a kind gift from Menarini Ricerche (Florence, Italy) and all other compounds were purchased from Sigma Chemical (Poole, Dorset, UK). Prostaglandin E2, PGE1-OH, butaprost, capsaicin were dissolved in 95% ethanol. GW627368X, EP1A, L798106, AH6809, sulprostone, RP67580 were dissolved in 100% dimethylsulfoxide (DMSO), whereas MEN10627 was dissolved in 50% DMSO and nitro-l-arginine (lNNA) was dissolved in 1N hydrochloric acid and all other compounds were made up into aqueous solutions. PGE1-OH, butaprost, GW627368X, EP1A, L798106, AH6809, sulprostone, PG97269, RP67580 and MEN10627 were stored at −20 °C and underwent a maximum of one freeze-thaw cycle and all other compounds were stored at 4 °C.

Preparation of human colon

Segments of human colon were obtained from 32 patients with a mean age of 67.1 ± 2.2 years, undergoing bowel resection surgery for primary carcinoma. There were no differences in PGE2 (300 nmol L−1) or carbachol (CCh, 10 μmol L−1) responses between either male or female patients or between anterior resection (providing tissue from the descending colon) and hemi-colectomy specimens (ascending and transverse colon; Table S1) and therefore the results from these specimens were pooled. Written consent was obtained from each patient according to the Declaration of Helsinki along with local Ethical Committee approval (Guy’s and St. Thomas’ Hospital Ethics Research Committee). Circumferential, full-thickness tissues were excised furthest from the tumor, and placed in fresh Krebs Henseleit (KH) buffer with the following composition (in mmol L−1): NaCl 118.0, KCl 4.7, NaHCO3 25.0, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5 and glucose 11.1 (pH 7.4). Specimens were cut along their longitudinal axis and the underlying mucosa was removed by sharp dissection. Inter-taenial strips of longitudinal smooth muscle (0.5 × 0.3 cm) were dissected further and mounted in organ baths.

Animal tissue collection and preparation

Intestinal preparations were taken from adult (≥10 weeks old) male mice that were a mixture of 129Sv and C57B16 strains. Studies using mouse tissue were performed in accordance with the Home Office (Scientific Procedures) Act 1986. Mice were culled by CO2 asphyxiation and the mid ileum and ascending colon were removed and placed in fresh KH buffer. Four adjacent, full-thickness, longitudinal strips of mid ileum (1 cm), or ascending colon (0.5 cm) were mounted in organ baths.

Measurement of changes in isometric tension in human and mouse intestine

Smooth muscle segments in 10 mL organ baths (containing oxygenated, 95% O2, 5% CO2, buffer and maintained at 37 °C) were connected to Grass FT03 force transducers (Harvard Apparatus, Edenbridge, UK), which were initially calibrated using a 1 g weight. Isometric changes in basal tension, measured in grams (g) were recorded continuously using a Powerlab 4/20 (AD Instruments, Oxford, UK). The signal was amplified using a quad-bridge amplifier and recorded on a computer using Labchart software (version 6.1.1; AD Instruments). Tissues were placed under 1 g of tension and allowed to equilibrate for 45 min, with three intermittent washes. Single additions of PGE2, sulprostone (EP3-preferring agonist), butaprost (EP2) or PGE1-OH (EP4) were added in the absence or presence of either vehicle (0.1% DMSO), or an EP receptor antagonist, namely EP1A (EP1 receptor antagonist), L798106 (EP3), GW627368X (EP4) or AH6809 (an EP1,2,3 receptor antagonist). The maximal effective concentration of each EP receptor antagonist, in the three different tissues tested here, were optimized in preliminary experiments and then used in this study (Figure S1).

To investigate whether PGE2 was activating prejunctional EP receptors, each agonist (single additions at the concentrations shown) was administered in the absence or presence of the neuronal sodium (Na+) channel blocker, lidocaine (1 mmol L−1 throughout). Agonist responses that were lidocaine-sensitive were investigated further to determine which neurotransmitters were involved. Initially tissues were pretreated with the transient receptor potential vanilloid 1 agonist, capsaicin (30 μmol L−1). This compound desensitizes primary afferent neurons (containing substance P and calcitonin-gene related peptide),20 and was used to investigate whether these afferent neurons were being targeted. Next, the contribution of inhibitory neurotransmitters were investigated by blocking nitric oxide (NO) synthesis, using the NO synthase (NOS) inhibitor, LNNA (100 μmol L−1),21 and by blocking VPAC1 receptors using PG97269 (100 nmol L−1).22 The involvement of excitatory neurotransmitters were investigated by blocking muscarinic and nicotinic receptors with atropine (1 μmol L−1) and hexamethonium (1 μmol L−1); and neurokinin receptors (NK1 and NK2) with RP67580 (2 μmol L−1)23 and MEN10627 (1 μmol L−1).24 Finally, CCh (10 μmol L−1) was used as an internal control (unless the tissue had been pretreated with atropine in which case KCl, 30 mmol L−1 was used).

Data analysis

Compound-induced changes in smooth muscle tension were measured in Labchart using the value parameter. The first value selected was the baseline tone immediately prior to compound addition and the second value was selected at the point of the maximal response within a predetermined time-point. Changes in tension (between these two points) were normalized to the maximal contractile response induced by CCh (10 μmol L−1). Data were expressed as a % of this maximal response; and were pooled as the mean ± 1 standard error (SE mean). Note that data have been pooled as absolute changes in baseline tension (measured in g) in Figure S2 (c and d) and Figure S4. Unpaired Student’s t-test was used to analyze two data groups and one-way analysis of variance (anova) with Dunnett’s post-test was used for multiple comparisons. A P-value ≤0.05 was considered statistically significant. Non-cumulative concentration-response curves were constructed from pooled data and EC50 values with 95% confidence limits are summarized in Table 1. GraphPad Prism (Version 5.0; GraphPad Software Inc., CA, USA) was used for all data analyzes.

Table 1.   EP receptor agonists: EC50 values and characteristics of EP receptor actions in human colon, mouse colon and mouse ileum
AgonistHuman colonMouse colonMouse ileum
EC50 values (with 95% confidence limits) and EP receptor actions on LSM
  1. EP1A, 6-[2-(5-chloro-2-{[(2,4-difluorophenyl)methyl]oxy}phenyl)-1-cyclopenten-1-yl]-2-pyridinecarboxylic acid; GW627368X, (N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydr-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulfonamide; L798106, 5-Bromo-2-methoxy-N-[3-(2-naphthalen-2-yl-methylphenyl)-acryloyl]-benzenesulfonamide; PGE1-OH, prostaglandin E1-alcohol; PGE2, prostaglandin E2; LSM, longitudinal smooth muscle; [UPWARDS ARROW], increase in tension; [DOWNWARDS ARROW], decrease in tension; direct, lidocaine-insensitive agonist responses; indirect, lidocaine-sensitive responses.

PGE2116.0 (1.4–8600) nmol L−1[UPWARDS ARROW] direct EP165.8 (18.6–232.6) nmol L−1[UPWARDS ARROW] direct EP1 and indirect EP3339.3 (102.9–1118) nmol L−1[UPWARDS ARROW] direct EP1 and EP3
Sulprostone2.7 (0.2–32.2) μmol L−1 non-EP receptor46.2 (2.3–916.1) nmol L−1[UPWARDS ARROW] indirect EP3187.2 (15.8–3,104) nmol L−1[UPWARDS ARROW] direct EP3
PGE1-OHNo activity87.5 (0.6–12 700) nmol L−1[UPWARDS ARROW] indirect EP3236.1 (21.6–2600) nmol L−1[UPWARDS ARROW] direct EP3
Butaprost8.3 (0.6–115.9) μmol L−1 [DOWNWARDS ARROW] direct EP23.9 (0.5–32.2) μmol L−1[DOWNWARDS ARROW] direct EP24.7 (0.5–44.5) μmol L−1 [DOWNWARDS ARROW] indirect EP2

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments and Disclosures
  7. References
  8. Supporting Information

EP1 receptors mediate PGE2-induced longitudinal smooth muscle contraction in human colon

In human colon, PGE2 contracted longitudinal smooth muscle. These responses peaked at 1–2 min and were sustained for up to 5 min (Fig. 1A; with EC50 values summarized in Table 1).

image

Figure 1.  Prostaglandin E2(PGE2)-induced contraction of human colon. Prostaglandin E2 increased muscle tension in longitudinal strips of human colon (A) and the insert illustrates a representative PGE2 response. In (B) PGE2 responses were significantly inhibited by the EP1 receptor antagonist, EP1A; but were unaffected by either the EP3 (L798106) or EP4 (GW627368X) receptor antagonists at the concentrations shown. Each point or bar represents the mean % increase in basal tension normalized to the maximal contractile response to CCh (10 μmol L−1),  ± 1 SE mean, and the n numbers are shown in parentheses. The significant difference between control PGE2 and PGE2 after 30 min pretreatment with EP1A is represented as *P < 0.05. The EC50 value for the concentration-response curve in (A) is quoted in Table 1.

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PGE2 (300 nM, ∼EC75) responses were significantly inhibited by EP1A (30 μmol L−1, Fig. 1B). In contrast, PGE2-induced contractions were partially, but not significantly, inhibited by the EP3 receptor antagonist and unaffected by the EP4 receptor antagonist (30 μmol L−1, Fig. 1B). The EP3-preferring agonist, sulprostone (10 nmol L−1–30 μmol L−1) also increased longitudinal smooth muscle tension but was approximately 20-fold less potent than PGE2 (Table 1). Furthermore, sulprostone (3 μmol L−1, ∼EC75), which increased basal tension by 19.4 ± 4.0% (n = 3), was unaffected by either the EP3 receptor antagonist, L798106 (30 μmol L−1, 17.4 ± 6.5%, n = 3) or EP1A (10 μmol L−1, 26.8 ± 8.1%, n = 3); implying that sulprostone-induced contractions were not mediated by an EP receptor in human colon.

Pretreatment with the local anesthetic, lidocaine was used to investigate whether the different EP receptor agonists were activating prejunctional receptors. Lidocaine was used instead of tetrodotoxin because there is evidence to suggest that myenteric neurons express Na+ channels that are insensitive to this toxin.24 Furthermore, in our assay, lidocaine (1 mmol L−1) abolished veratridine responses in mouse colon and ileum without any effect on CCh responses (Figure S2), suggesting that this pretreatment was inhibiting myenteric neurons selectively. In human colon, lidocaine had no effect on basal muscle tone per se (1.2 ± 1.2% c.f. 0.0, n = 4). Furthermore, PGE2 (300 nmol L−1) increased muscle tension by 8.6 ± 3.0% (n = 4) and these responses were partially inhibited by lidocaine (2.9 ± 0.8%, n = 4), although the results did not reach statistical significance (P = 0.1). Therefore, although these data suggest that PGE2 was activating EP1 receptors on smooth muscle to induce contraction directly, there is a possibility that these receptors may also be expressed by myenteric neurons. It is important to note that mucosa-denuded muscle strips were used here and therefore the activities of submucosal neurons and submucosal interstitial cells of Cajal should not be dismissed.

EP1 and EP3 receptors mediate PGE2-induced longitudinal smooth muscle contraction in mouse colon and ileum

PGE2 consistently increased longitudinal smooth muscle tension of mouse colon and ileum in a concentration-dependent manner (Fig. 2A,B; Table 1). The contractile effects of PGE2 in the two mouse tissues were similar to each other, but were more sustained than the responses observed in human colon. In both murine preparations, responses to PGE2 (100 nmol L−1 in colon and 1 μmol L−1 in ileum, used here and subsequently) were partially inhibited by either EP1A or L798106 (at 1 or 10 μmol L−1 in the colon or ileum, respectively), whereas a combination of these antagonists induced an inhibitory effect that significantly abolished agonist responses (Fig. 2C,D). Conversely, pretreatment with the EP4 receptor antagonist, GW627368X (up to 30 μmol L−1) had no effect on subsequent PGE2 responses (Figure S1). Taken together, these findings suggest that PGE2-induced contractions in mouse colon and ileum were mediated by a combination of EP1 and EP3 receptors.

image

Figure 2.  Prostaglandin E2(PGE2) and sulprostone responses in mouse colon and ileum. Single additions of either PGE2 or the EP3-preferring agonist, sulprostone (sulp) increased muscle tension in longitudinal strips of mouse (A) colon or (B) ileum. The inserts in (A) and (B) show representative PGE2 responses. In (C) and (D), PGE2 responses were partially inhibited by EP1A (+EP1) or L798106 (+EP3) at the concentrations shown; and a combination of the two antagonists induced a significant inhibitory effect on PGE2 responses in mouse (C) colon or (D) ileum. Furthermore, sulprostone responses were significantly inhibited by L798106 (EP3) and by a combination of L798106 and EP1A (+EP1 & EP3) but not by EP1A alone (E, F). Note the different PGE2 and sulprostone concentrations used in the two murine preparations, plus the different sensitivities used for the representative traces in each intestinal area. Each bar represents the mean % change in basal tension, normalized to the maximal contraction induced by CCh (10 μmol L−1), ± 1 SE mean, and the n numbers are shown in parentheses. Significant differences between PGE2 and sulprostone responses in the absence or presence of an EP receptor antagonist are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001. EC50 values for the concentration-response curves in (A) and (B) are quoted in Table 1

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Unlike human samples, sulprostone increased basal tension in mouse tissues with similar potency as PGE2 (Fig. 2A,B; Table 1). Furthermore, sulprostone (100 nmol L−1 in colon and 1 μmol L−1 in ileum used here and subsequently) responses were significantly inhibited following L798106 (10 μmol L−1) pretreatment (Fig. 2E,F), indicating that this agonist was activating EP3 receptors in mouse colon and ileum to induce contraction.

In contrast to human tissue, lidocaine had significant effects on basal muscle tone per se in preparations of mouse colon (45.2 ± 15.9% c.f. 0.0, n = 5, P < 0.05) or mouse ileum (14.4 ± 5.4%, n = 8, P < 0.05). In addition, PGE2-induced contractions were not significantly inhibited by lidocaine pretreatment in either mouse colon (15.6 ± 6.9% c.f.19.5 ± 3.6%, n = 5) or mouse ileum (51.8 ± 11.9 c.f. 30.1 ± 5.4%, n = 6), suggesting that PGE2 was predominantly activating postjunctional EP receptors in both preparations.

Neuronal EP3 receptors mediate contraction indirectly in mouse colon

Sulprostone increased smooth muscle tension of mouse colon by 25.5 ± 3.1% (n = 21); and these responses were significantly inhibited by lidocaine (2.4 ± 1.6%, n = 5, P < 0.01). In contrast, sulprostone responses were unaffected by the neuronal blocker in the ileum (35.0 ± 6.1% c.f. 27.4 ± 4.3%, n = 8). Taken together these data indicate that sulprostone activates prejunctional EP3 receptors in mouse colon and EP3 receptors on ileal smooth muscle. Colonic neurogenic mechanisms were investigated further, but sulprostone responses were not inhibited by optimized combinations (Figure S3–5) of either: (i) capsaicin (23.1 ± 3.6%, n = 6), (ii) LNNA and PG97269 (38.7 ± 7.7%, n = 4), or by (iii) atropine, hexamethonium, RP67580 and MEN10627 (201.9 ± 101.1% c.f. 102.5 ± 17.1%n = 6; the latter data set was normalized to KCl, 30 mmol L−1). Note that the combined administration of LNNA and PG97269 increased basal muscle tension per se by 25.2 ± 14.5% (n = 4), whereas the combined treatment of atropine, hexamethonium, RP67580 and MEN10627 increased tension by 14.4 ± 5.4% (n = 4, the latter being normalized to KCl, 30 mM). Although we have provided functional evidence to suggest that EP3 receptors are prejunctional, we have been unable to identify which neurotransmitters were modulating these responses.

PGE1-OH induced contraction by activating EP3 receptors in mouse colon and ileum

The EP4-preferring receptor agonist, PGE1-OH (up to 100 μmol L−1) did not induce a response in human colon, but this agonist concentration-dependently increased basal tension in the two mouse preparations (Table 1). Although PGE1-OH (100 nmol L−1 in colon and 3 μmol L−1 in ileum, used here and subsequently) increased tension, these responses were unaffected by the EP4 receptor antagonist, GW627368X in both mouse tissues (Fig. 3). In contrast, PGE1-OH responses were significantly inhibited by the EP3 receptor antagonist, L798106 (Fig. 3), indicating that this agonist was activating EP3 receptors (in the absence of functional EP4 receptors) to increase longitudinal muscle tension.

image

Figure 3.  PGE1-OH increased longitudinal smooth muscle tension of mouse (A) colon and (B) ileum and these responses were unaffected by the EP4 receptor antagonist, GW627368X (+EP4), but were significantly inhibited by the EP3 receptor antagonist, L798106 (+EP3). Each bar shows the mean % increase in basal tension, normalized to the maximal contraction induced by CCh (10 μmol L−1), ± 1 SE mean, and the n numbers are shown in parentheses. The significant difference between control PGE1-OH and PGE1-OH following L798106 is represented as *P < 0.05, ***P < 0.001.

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EP2 receptors mediate butaprost-induced relaxation of longitudinal smooth muscle from human and mouse colon and mouse ileum

The EP2 receptor agonist, butaprost induced a sustained decrease in longitudinal smooth muscle tension of human and mouse colon, (Fig. 4) and mouse ileum (Fig. 5; Table 1). Single (∼EC75) butaprost concentrations (10 μmol L−1 for human colon, 3 μmol L−1 for mouse colon, and 5 μmol L−1 for mouse ileum) were used for subsequent investigation. Butaprost responses were inhibited by the non-selective EP1,2,3 receptor antagonist, AH6809 (30 μmol L−1) in human colon (−6.1 ± 1.9%, n = 5 c.f. 0.0%, n = 3, P = 0.05); mouse colon (−9.3 ± 2.0% c.f. −2.0 ± 0.6%, n = 6, P < 0.01); and mouse ileum (−11.9 ± 2.5% c.f. −1.25 ± 1.25%, n = 4, P < 0.01) suggesting that this agonist was activating an EP receptor, probably the EP2 type. Furthermore, butaprost responses were unaffected by lidocaine in human or mouse colon indicating that butaprost-induced relaxations were mediated by a direct effect on smooth muscle in these colonic preparations.

image

Figure 4.  Butaprost responses in human and mouse colon. The EP2 receptor agonist, butaprost decreased muscle tension of longitudinal strips from (A) human and (B) mouse colon; and the inserts in (A) and (B) show representative agonist responses. Each point represents the mean % decrease in basal tension, normalized to the maximal response induced by CCh (10 μmol L−1), ± 1 SE mean, and the n numbers are in parentheses. Note the different time scales used for the representative traces. The EC50 values calculated from the concentration-response curves are summarized in Table 1.

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image

Figure 5.  Butaprost-induced relaxation in mouse ileum. Butaprost decreased longitudinal smooth muscle tension of mouse ileum (A) and the insert is a representative response. Furthermore, butaprost responses were significantly inhibited by lidocaine (+Lid, 1 mmol L−1) but were unaffected by capsaicin (+Caps, 30 μmol L−1) pretreatment. In addition, butaprost responses were inhibited after a combination of LNNA (100 μmol L−1) and the VPAC1 antagonist, PG97269 (100 nmol L−1; +inhibit blockade). Each bar is the mean % reduction in basal tension, normalized to the maximal response induced by CCh (10 μmol L−1), ± 1 SE mean, and the n numbers are shown in parentheses. Significant differences between control butaprost and pre-treated agonist responses are represented as follows: *P < 0.05.

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In contrast, butaprost responses (Fig. 5A) were significantly inhibited by lidocaine but not by capsaicin in mouse ileum (Fig. 5B), suggesting that this agonist was activating prejunctional receptors but not primary afferents. A combination of the NOS inhibitor, LNNA, and the VPAC1 receptor antagonist, PG97269 were tested next. This pretreatment significantly increased tone per se (15.7 ± 0.8% c.f. −1.9 ± 2.7%, P < 0.001, n = 4), and significantly inhibited responses to subsequent additions of butaprost (Fig. 5B). Taken together these findings indicate that, in mouse ileum, butaprost activates an EP receptor, most likely EP2, on inhibitory motor neurons, which increases the release of inhibitory neurotransmitters and thus induces relaxation indirectly.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments and Disclosures
  7. References
  8. Supporting Information

This study demonstrates that EP1 receptors mediate PGE2-induced contractions in human colon, mouse colon and mouse ileum. In addition, EP3 receptors mediate PGE2-induced contractions in both mouse tissues but not in human colon. Although PGE2 contracted the three muscle preparations, butaprost decreased tension by specifically activating EP2 receptors. Furthermore, we have provided functional evidence that EP3 receptors are expressed by postjunctional cells in mouse ileum and by myenteric neurons in mouse colon. Similarly, we have shown that butaprost activates smooth muscle EP2 receptors in human and mouse colon; and neuronal EP2 receptors in mouse ileum (Table 1).

PGE2 induced a sustained contraction of longitudinal muscle from human colon, mouse colon and mouse ileum. These data support the findings of several studies, which have shown that PGE2 consistently increased longitudinal muscle tension of human ileum and sigmoid colon9,25 as well as mouse,13 guinea-pig,26 and rabbit ileum.27 In all three tissues, PGE2 responses were sensitive to the EP1 receptor antagonist, EP1A. Prostaglandin E2 responses were also significantly inhibited by the EP3 receptor antagonist, L798106, in both mouse preparations but not in human colon. Furthermore, the EP3-preferring agonist, sulprostone increased tension in all three preparations, however, these responses were not sensitive to EP antagonism in human colon. Conversely, sulprostone responses were significantly inhibited by L798106 but not by EP1A in both mouse tissues. We tested the effects of EP1A on sulprostone responses in the mouse because this agonist also binds to murine EP1 receptors but with a lower binding affinity than the EP3 type.26 Our results demonstrate that EP1A had, at most, a partial inhibitory effect on subsequent sulprostone responses, confirming the presence of EP3 receptors in both murine preparations. Taken together, our data indicates that EP1 receptors alone mediate PGE2-induced contractions in human colon, whereas a combination of EP1 and EP3 receptors mediate PGE2 contractions in mouse intestine. Our data support the findings of a previous study, using knockout mice lacking each of the eight different prostanoid receptor types, which showed that EP1, EP3 and FP receptors mediated PGE2-induced contractions of longitudinal muscle from mouse ileum.13 Our functional data also supports a molecular study that demonstrated the presence of EP3 mRNA in myenteric neurons and longitudinal smooth muscle from mouse intestine.28 Other studies using both expression and/or functional approaches have implicated the involvement of both EP1 and EP3 receptors in PGE2-induced contractions of guinea-pig (EP1 only), pig (EP3 only)14,26 and rabbit ileum.27

In human colon, our findings demonstrate that EP3 receptors do not mediate either PGE2 or sulprostone responses, as L798106 failed to inhibit responses induced by either agonist. We conclude that PGE2 activates EP1 receptors to increase muscle tension in human colon, however, sulprostone responses are mediated by a non-EP receptor mechanism. Unlike mouse colon and ileum, where sulprostone and PGE2 were equipotent, sulprostone responses were 20-fold less potent than PGE2 in human tissue. We suggest that this agonist was having an ‘off’-target contractile effect at these high concentrations.

The EP4-preferring agonist, PGE1-OH, increased tension in mouse colon and ileum, with no effect in human colon. These contractile responses were initially unexpected as EP4 receptors mediate vasodilatation of other smooth muscle, including vascular smooth muscle.8 Importantly though, PGE1-OH responses were unaffected by EP4 antagonism. Kiriyama et al.,29 demonstrated that PGE1-OH binds to mEP3 receptors (Ki = 330 nmol L−1), with similar affinity to mEP4 receptors (Ki = 190 nmol L−1), and therefore it was hypothesized that PGE1-OH was activating EP3 receptors. We accept this hypothesis as PGE1-OH responses were significantly inhibited by L798106, suggesting that this agonist was activating EP3 receptors and not EP4 to induce contraction. Our data indicates that there are no functional EP4 receptors in longitudinal smooth muscle. Similarly, Grasa et al.,27 demonstrated that EP4 protein was not present in either longitudinal or circular smooth muscle of the rabbit small intestine.

Unfortunately there are no commercially available agonists that selectively activate the EP1 receptor and the agents currently used (iloprost, carbacyclin and enprostil) bind to either human or murine EP3 or IP receptors with equivalent affinities.29,30 Therefore, we were unable to determine the cellular location of EP1 receptors functionally. Previous studies using in situ hybridization or immunohistochemistry have demonstrated different EP1 expression patterns depending on the species; for example, EP1 mRNA is expressed in longitudinal smooth muscle from rat small intestine and colon;31 whereas EP1 receptors are expressed by myenteric neurons of the rabbit small intestine.27 These molecular studies combined with our functional data highlight the significant species differences that exist in terms of EP receptor localization.

We have also demonstrated that sulprostone responses were lidocaine-sensitive in mouse colon, suggesting that EP3-mediated contractions induced by PGE2 or sulprostone are neurogenic. Conversely, sulprostone responses were unaffected by lidocaine in mouse ileum, implying that EP3 receptors are postjunctional, probably expressed by smooth muscle cells. We investigated the lidocaine-sensitive component observed in mouse colon further, but sulprostone responses were not inhibited by any of the pretreatments used. Initially, capsaicin was used to desensitize sensory afferent neurons but had no effect on subsequent sulprostone responses in mouse colon, suggesting that these afferents, with AH electrophysiology, were not being targeted. This is in contrast to the results of Dekkers et al.,16 who demonstrated that PGE2 depolarized both AH and S neurons in the myenteric plexus of guinea-pig intestine. Next, a combination of LNNA and PG97269 (used to block NO production and VPAC1 receptors) was tested and although this pretreatment had significant effects on basal tone, no inhibitory effects on sulprostone responses were observed; indicating that the inhibitory neurotransmitters, NO and VIP were not modulating sulprostone responses. Finally, sulprostone was administered in the absence or presence of atropine, hexamethonium, RP67580 and MEN10627 (used to antagonize muscarinic, nicotinic and NK receptors); however, this pretreatment also had no effect, indicating that sulprostone was not activating excitatory motor neurons. We were therefore unable to identify the neurotransmitters that modulated sulprostone-induced contractions in mouse colon. One explanation for this is that only a VPAC1 receptor antagonist was used to inhibit VIP responses, but VPAC2 and PAC1 receptors are expressed in colonic smooth muscle from rodents.32,33 In addition, the presence of several EP3 isoforms, which couple to different G-proteins and initiate various signaling pathways in the same cell types, including human small intestinal cells,6,34 makes this receptor type difficult to study in a functional setting.

Our results also demonstrate that butaprost relaxed smooth muscle in all three preparations. Butaprost responses were significantly inhibited by AH6809, providing further evidence that these responses were specifically mediated by EP2 receptors. Previous studies have demonstrated that EP2 activation inhibits CCK-8-induced contractions of circular smooth muscle cells from guinea-pig ileum, pig ileum,14 and dog colon.15 Moreover, the activation of EP2 receptors mediates relaxation of other smooth muscle preparations including human cerebral arteries.35 However, in contrast, Morimoto et al.,28 demonstrated using in situ hybridization that only low levels of EP2 mRNA were detected throughout the mouse intestine. Butaprost responses were unaffected by lidocaine in human and mouse colon, implying that the agonist was activating postjunctional EP2 receptors. Conversely, in mouse ileum, butaprost responses were abolished by lidocaine or by a combination of LNNA and PG97269, suggesting that the EP2 agonist was inducing relaxation indirectly, most likely by activating EP2 receptors on inhibitory motor neurons. In contrast to our findings, Grasa et al.,27 showed that butaprost had no effect on longitudinal muscle from the rabbit small intestine. Furthermore, these authors showed, using immunohistochemistry, that low levels of EP2 receptor expression were detected in myenteric neurons, whereas no EP2 expression was observed in smooth muscle. These differences are most likely due to species variations. Our findings also demonstrate that EP4 receptors do not mediate PGE2 or PGE1-OH responses in intestinal longitudinal smooth muscle, as responses to both agonists were insensitive to the EP4 receptor antagonist in all three tissues tested.

The identity of the EP receptor types that mediate smooth muscle contraction had not been elucidated in human colon at the start of this study; however, the recent availability of selective tools that preferentially activate or block the different EP receptors has enabled a detailed characterization of the receptor types involved. Specifically, we have shown that PGE2 contracts longitudinal smooth muscle of human colon by activating EP1 receptors, whereas this prostanoid activates a combination of EP1 and EP3 receptors in mouse colon and ileum. In all three tissues, selective EP2 activation induced relaxation and there appears to be no functional role for EP4 receptors in any of the tissues tested. The characterization of EP receptors using human tissue is particularly important as these results provide evidence of both species and regional differences in terms of EP receptor pharmacology. This characterization is significant because targeting EP receptors may be a potential therapy for gastrointestinal pathologies where muscle contractility is altered. Such pathologies include postinfectious IBS, where chronic intestinal hypercontractility has been shown to be associated with an increase in COX-2 expression and PGE2 release.36,37,38 Our findings indicate that an EP1 receptor antagonist may alleviate hypercontractility, whereas an EP1 agonist or an EP2 receptor antagonist may improve symptoms of gastrointestinal paresis.

Acknowledgments and Disclosures

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments and Disclosures
  7. References
  8. Supporting Information

This research was funded by a CASE award with GSK and the Biotechnology and Biological Sciences Research Council, UK. We thank the surgical team in the Department of General Surgery, St Thomas’ Hospital for their help in obtaining the specimens used in this study. We thank Miriam Basma for her assistance during her final year research project.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments and Disclosures
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments and Disclosures
  7. References
  8. Supporting Information

Figure S1. The effect of the EP1 receptor antagonist, EP1A (histograms to the left); the EP3 antagonist, L798106 (middle) or the EP4 receptor antagonist, GW627368X (right) on PGE2 responses in human colon (a), mouse colon (b), and mouse ileum (c). Each bar represents the mean ± 1 SE mean % change in basal tension, normalized to the maximal contraction induced by CCh (10 μmol L−1), and the n numbers are shown in parentheses. Significant differences between control PGE2 ± an EP receptor antagonist are as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure S2. Pooled responses to veratridine and veratridine after 15 min pretreatment with lidocaine in mouse (a) colon and (b) ileum. Note that veratridine decreased longitudinal smooth muscle tension of mouse colon, whereas the depolarizing agent induced a biphasic response with a predominant increase in tension in the ileum; and the separate phases in the latter are pooled as separate bars. Lidocaine abolished veratridine responses, without any effect on subsequent CCh (10 μmol L−1)-induced contractions, in mouse (c) colon and (d) ileum. In (a) and (b), each bar represents the mean ± 1 SE mean % change in basal tension, normalized to the maximal contraction induced by CCh (10 μmol L−1). In (c) and (d), each bar is the mean ± 1 SE mean absolute change in basal tension. The n numbers throughout are shown in parentheses. Significant differences between control veratridine and veratridine after lidocaine are represented as follows: *P < 0.05, ***P < 0.001.

Figure S3. Representative responses to capsaicin (Caps) in mouse (a) colon and (b) ileum. The first addition of capsaicin induced a sustained decrease in tension, which was re-adjusted manually back to 1 g after 10 min. A subsequent addition of capsaicin, 5 min later, did not induce a response in either tissue.

Figure S4. Single additions of CCh, nicotine (Nic) and substance P (SP) in the absence or presence of the following combined antagonist pretreatment, hexamethonium (1 μmol L−1); atropine (10 μmol L−1); RP67580 (2 μmol L−1) and MEN10627 (1 μmol L−1; +antagonists) in mouse colon. Each bar shows the mean ± 1 SE mean absolute change in basal tension and the n numbers are shown in parentheses. Significant differences between CCh and SP responses in the absence or presence of the combined antagonist pretreatment are as follows: *P < 0.05, ***P < 0.001.

Figure S5. VIP decreased longitudinal smooth muscle tension of mouse ileum and these responses were inhibited in a concentration-dependent manner by the VPACI receptor antagonist, PG97269. Each bar shows the mean ± 1 SE mean % change in basal tension, normalized to the maximal response induced by CCh (10 μmol L−1), and the n numbers are shown in parentheses. Significant differences between control VIP and VIP following PG97269 pretreatment are represented as follows: *P < 0.05, **P < 0.01.

Table S1. Comparison of PGE2 (300 nmol L−1) and CCh (10 μmol L−1) responses in longitudinal smooth muscle preparations from male and female patients and from either descending colon or ascending colon specimens. Descending colon was obtained after anterior resected tissue and ascending colon from hemi-colectomies. Values are the mean ± 1 SE mean (with n numbers) and there were no significant differences between data groups with either agonist.

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NMO_1727_sm_FigS1.tif24KSupporting info item
NMO_1727_sm_FigS2.tif10KSupporting info item
NMO_1727_sm_FigS3.tif78KSupporting info item
NMO_1727_sm_FigS4.tif7KSupporting info item
NMO_1727_sm_FigS5.tif6KSupporting info item
NMO_1727_sm_TableS1.doc32KSupporting info item

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