Prostaglandin E₂ receptor distribution and function in the gastrointestinal tract

Abstract

Prostaglandin E₂ (PGE₂) is one of the most important biologically active prostanoids found throughout the gastrointestinal tract. Despite the fact that PGE₂ regulates many physiological functions of the gut including mucosal protection, gastrointestinal secretion and motility, it is implicated in the pathophysiology of inflammatory bowel diseases (IBD) and colorectal neoplasia. The varied biological functions exerted by PGE₂ are through the pharmacologically distinct, G-protein coupled plasma membrane receptors termed EP receptors. Disruptions of various prostanoid receptor genes have helped in unravelling the physiological functions of these receptors. To date, all four subtypes of EP receptors have been individually knocked out in mice and various phenotypes have been reported for each subtype. Similarly, in vitro and in vivo studies using EP receptor agonists and antagonists have helped in uncoupling the diverse functions of PGE₂ signalling involving distinct EP receptors in the gut. In this review, we will summarize and conceptualize the salient features of EP receptor subtypes, their regional functions in the gut and how expressions of EP receptors are altered during disease states.

Introduction

The interests in understanding prostaglandin (PG)-mediated functions of the gastrointestinal (GI) tract have grown steadily since the discovery of PGs in 1957. In recent years, technological advances in the field of molecular biology have helped researchers make substantial progress towards unraveling the complex functions of PGs in the gut. It is only now that the functional roles of the various PGs in the GI tract are established. Apparently, each of the bioactive PGs exhibits versatility and diversity in functions in various segments of the gut. However, the mechanisms by which they exert their biological functions are not clearly known. To discern the variable roles of PGs, it is imperative to understand thoroughly the mode of action of these mediators, including the pharmacology, expression and distribution of their respective receptors in the gut. Nevertheless, insights into PG receptor biology in the GI tract will be of fundamental importance in developing strategies for pharmacological intervention in various GI ailments.

PG biosynthesis

PGs are 20-carbon fatty acid derivatives that are found ubiquitously in all tissues and organs and mediate variety of physiological and pathological functions. They are synthesized in the cell from different essential fatty acid precursors, including arachidonic acid (AA). PGs derived from AA are termed series-2 PGs, which consist of prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂), prostaglandin I₂ (PGI₂), prostaglandin F_2α (PGF_2α) and thromboxane A₂ (TXA₂) (Calder, 2001). All of these PGs share a common initial biosynthetic pathway that begins with the hydrolysis of cell-membrane phospholipids, mediated by the enzyme phospholipase A₂ (PLA₂), which is found mostly in cellular membranes including the plasma membrane (Murakami et al., 1997). Diverse physiological and pathological stimuli can result in the activation of PLA₂ to liberate AA from membrane phospholipids into the cytoplasm (Murakami and Kudo, 2002). Upon release, AA is converted into unstable endoperoxide intermediates, PGG₂ and PGH₂ (Hamberg et al., 1974) by the action of cyclooxygenase (COX) in a rate limiting enzymatic reaction. Three isoforms of COX have been identified to date. The constitutively expressed COX-1 and the inducible COX-2 are the most important isoforms (Smith et al., 1994, 1996). COX-3 is a splice variant of COX-1 and is mostly expressed in the brain and the heart (Chandrasekharan et al., 2002). The oxygenated intermediate PGH₂ is in turn metabolized by cell-specific synthases and isomerases into PGD₂, PGE₂, PGF_2α, PGI₂, and TXA₂ (Vane et al., 1998; Figure 1). Prostanoids are released outside the cell immediately after their synthesis where they exert their biological functions through their interaction with the cell surface prostanoid receptors in an autocrine or paracrine fashion (Narumiya, 1994). Alternatively, the action of prostanoids are terminated when they are transported across the cell membrane into the cytoplasmic compartment with the help of PG transporters (Kanai et al., 1995) where they are acted upon by oxidizing and reducing enzymes, 15 hydroxy PG dehydrogenases and delta 13–15-ketoprostaglandin reductase, respectively (Tai et al., 2002).

Figure 1.

Biosynthesis of prostaglandins. Note that arachidonic acid liberated by the action of membrane bound phospholipase A2 and cyclooxygenase are the key rate-limiting steps for prostaglandin biosynthesis. Shown are the prostanoids, their respective receptors and their related pathophysiological functions.

Prostanoid receptors

Prostanoid receptors belong to the family of Rhodopsin-type receptors characterized by their seven transmembrane domains that are intracellularly coupled to different subunits of G proteins (Breyer et al., 2001). Five major types of prostanoid receptors, namely; D prostanoid (DP), E prostanoid (EP), F prostanoid (FP), I prostanoid (IP) and T prostanoid (TP), which include six subtypes namely; DP₁, DP₂, EP₁, EP₂, EP₃ and EP₄, have been described for the prostanoids PGD₂, PGE₂, PGF_2α, PGI₂ and TXA₂. The structures, properties and functions of most of these receptors have previously been reviewed (Narumiya et al., 1999). Investigation of the functional role of these receptors in gut physiology as well as pathophysiology is presently an important pursuit. For example, the role of DP, IP, EP and TP receptors in the propulsive peristaltic movement of the guinea-pig small intestine has been studied and the receptors responsible for differential peristaltic motor effect identified (Shahbazian et al., 2002). Similarly, the role of DP, FP, IP, TP and the EP receptor subtypes in the development of carcinogen-induced aberrant crypt foci in colon has been studied extensively (Watanabe et al., 1999; Mutoh et al., 2002).

It is clear that the different prostanoids synthesized and their interactions with respective cellular receptors are responsible for the varied biological actions of the host. As the cellular components of the GI tract are known to possess the machinery for the biosynthesis of PG, it is not surprising that PGs are produced throughout the gut. Among the different PGs that are produced, PGE₂ is considered to be the most important for normal physiological functions of the GI tract including gastric mucosal protection and motility and is also implicated in the pathology of various disease conditions such as inflammatory bowel disease (IBD) (Ahrenstedt et al., 1994), entero-invasive bacterial diseases (Reseta and Barrett, 2002) and colorectal cancers (Eberhart et al., 1994). Diversity in the cellular functions of PGE₂ is attributed to its binding to four different subtypes of EP receptors that in turn propagates signals through alteration in the intracellular calcium (Ca²⁺) or cyclic adenosine monophosphate (cAMP) levels. This results in the activation of an array of kinases modulating diverse cellular functions (Figure 2). Although, signalling through different EP receptor determines the various effects of PGE₂, regional and differential expression of EP receptors in the GI tract is critical for determining its biological functions. Based on this premise, we will summarize in this review the salient features of EP receptor subtypes, their regional functions in the gut and their differential expression during normal and disease states.

Figure 2.

PGE₂-EP receptor signaling pathways. Four major types of EP receptors are involved in the signaling pathway mediated via different G proteins (_{p/q, αs} and _αi) using different second messenger. PGE₂ induces intracellular Ca²⁺ or cAMP when it couples and signals through EP₁ or EP_2/4 receptors respectively. However, it reduces intracellular cAMP when it signals through EP₃ receptors. Several different kinases are involved in PGE₂ induced signaling pathways. PGE₂-EP₄ receptor coupling induces TNFα, cyclin D and angiogenesis using a specific pathway that is mediated via different MAP kinases. PGE₂ also promotes cancer cell proliferation and inhibits apoptosis. It also induces COX-2 gene transcription and induces cell proliferation. On the other hand, it could also inhibit cell cycle through phosphorylation of Akt, GSK-3β and β-catenin. Abbreviations: AC, adenylate cyclase; DAG, diacylglycerol; IP3, inositol triphosphate; PIP2, phosphatidylinositol diphosphate; PKA, protein kinase A; PKC, protein kinase C; PI3K, phosphoinositide-3-kinase; Akt, protein kinase B; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; EGR-1, early growth response 1; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; GSK-3, glycogen synthase kinase-3; ®, phosphorylation; ↑, up regulated; ↓, down regulated; X, inhibition.

EP receptors

The cellular membrane receptors for PGE₂ are termed the EP receptors that consist of four different subtypes namely EP₁, EP₂, EP₃ and EP₄. They are encoded by different genes and are well conserved throughout the mammalian system from mouse to human. Phylogenetic analysis of the amino-acid sequence of all the EP receptors indicates that they were all sequentially (EP₂, EP₄, EP₃ and EP₁) derived from a primitive PGE receptor by gene duplication (Toh et al., 1995). Isoforms of EP receptor subtypes generated by alternative splicing of their mRNA have been reported for EP₃ and EP₁ (Irie et al., 1993; Namba et al., 1993; An et al., 1994; Breyer et al., 1994; Regan et al., 1994b; Schmid et al., 1995; Okuda et al., 1996; Pierce and Regan, 1998; Oldfield et al., 2001). All EP receptor subtypes are expressed on the plasma membrane; additionally EP₃ and EP₄ also exhibit nuclear membrane localization (Bhattacharya et al., 1998, 1999).

EP₁ receptor

EP₁ is a 42-kDa protein that has been cloned and expressed from humans and rodents (Funk et al., 1993; Watabe et al., 1993; Okuda et al., 1996). Two splice variants of EP₁ receptor have been reported (Okuda et al., 1996). Among the four subtypes of EP receptors, EP₁ has the least affinity for PGE₂ (dissociation constant (K_D) of 16–25 nM, (Table 2)). It binds PGs in the rank order PGE₂>sulprostone, iloprost>PGE₁>misoprostol, M&B-28767>PGF_2α>PGD₂ (Abramovitz et al., 2000; Table 3). A few selective agonists for EP₁ like ONO-DI-004, 17-phenyl trinor PGE₂ are available, 17-phenyl trinor PGE₂ being the most well known of them. Other agonists including sulprostone, carbacyclin and enprostil exhibit the highest affinity for EP₁, but are also very potent EP₃ agonists. ONO-AE-829, ONO-8711, ONO-8713, SC-19220 and AH6809 are used as antagonists for the EP₁ receptor but AH6809 is a weak antagonist (Woodward et al., 1995, 2005).

Table 2.

Dissociation constants (K_d) of PGE₂ for EP receptor subtypes in various species

Receptor	Kd (nM) (human)	Kd (nM) (rat)	Kd (nM) (mouse)
EP1	25 (Abramovitz et al., 2000)	24 (Boie et al., 1997)	21 (Watabe et al., 1993)
	16 (Sharif and Davis 2002)
EP2	13 (Abramovitz et al., 2000)	5 (Boie et al., 1997)	116 (Nishigaki et al., 1996)
	12.9 (Stillman et al., 1998)
EP3	0.33 (Abramovitz et al., 2000)	1 (Boie et al., 1997)	2.9 (Sugimoto et al., 1992)
EP4	0.59 (Abramovitz et al., 2000)	1 (Boie et al., 1997)	1.27 (Nishigaki et al., 1996)
	0.72 (Davis and Sharif, 2000)
	1.12 (Marshall et al., 1997)

Abbreviations: EP, E prostanoid; PGE₂, prostaglandin E₂.

Table 3.

Competition for radioligand binding to HEK 293(EBNA) cell membranes expressing recombinant prostanoid receptors

Ligands	EP1	EP2	EP3	EP4
PGE2	9.1	4.9	0.33	0.79
Butaprost	27 721	91	1643	19 104
AH-6809	1217	1150	1597	100 000
Sulprostone	107	100 000	0.35	7740
M&B-28767	419	988	0.14	10
Iloprost	11	1870	56	284
Misoprostol	11 935	34	7.9	23
PGD2	5820	2973	421	1483
PGF2α	547	964	38	288
SC-51322	13.8	>100 000	698	14 032
Enprostil	82	>10 000	12	>10 000
Carbacyclin	23	942	14	352
Cicaprost	>1340	>1340	255	44

Abbreviations: EP, E prostanoid; PGD₂, prostaglandin D₂; PGE₂, prostaglandin; PGF_2α, prostaglandin F_2α.

Inhibitory constant (K_i) values are expressed in nM.

Adapted from Abramovitz et al. (2000).

EP₂ receptor

Before the discovery of the EP₂ receptor in 1994, the cloned EP₄ receptor was referred to as EP₂ (Bastien et al., 1994; Regan et al., 1994a). The EP₂ receptor is a 53 kDa protein that has been cloned and expressed from humans, bovine, rabbits, rodents and several other species (An et al., 1993; Katsuyama et al., 1995; Guan et al., 1996; Nemoto et al., 1997; Arosh et al., 2003). The affinity of PGE₂ for EP₂ receptor differs greatly across species. The rat EP₂ receptor has a significantly higher affinity for PGE₂ than human or mouse. The mouse EP₂ receptor shows the least affinity (Table 2). The relative affinity of binding of various EP ligands for the mouse EP₂ receptor was PGE₁, PGE₂>16, 16-dimethyl-PGE₂>11-deoxy-PGE₁>butaprost>AH13205, misoprostol> AH-6809. Similarly, the human EP₂ receptor binds PGE₂ and PGE analogues with a rank order of PGE₂>PGE₁>16, 16-dimethyl-PGE₂>11-deoxy-PGE₁>butaprost, 1-OH-PGE₁, M&B-28767> sulprostone (Table 3). Butaprost and ONO-AE1-259-01 are highly selective EP₂ receptor agonists, whereas AH-6809 is a well-known EP2 antagonist.

EP₃ receptor

The EP₃ receptor of humans and rodents has been successfully cloned and expressed (Sugimoto et al., 1992; Yang et al., 1994). The EP₃ is unique among the prostanoid family of receptors in the sense that multiple alternatively spliced variants of EP₃ exist that can activate contrasting second messenger signalling (Pierce and Regan, 1998). To date, eight different isoforms for human EP₃ have been reported. In general, the EP₃ receptor has relatively higher affinity for PGE₂ reflected by a very low K_D value of 0.33–2.9 (Table 2). The rank order of affinity of PGE₂ to the mouse EP₃ receptor is sulprostone, M&B-28767, PGE₂, PGE₁, 11-deoxy-PGE₁, 16,16-dimethyl-PGE₂, misoprostol>1-OH-PGE₁. Different PGE analogs, SC-46275, ONO-AE-248, sulprostone, GR 63799X and 11-deoxy-PGE₁ are used as agonists for EP₃ with SC-46275 and ONO-AE-248 being highly selective. L826266 is an EP₃ antagonist.

EP₄ receptor

The EP₄ receptor cDNA encodes a 487–513 amino-acid polypeptide that has been cloned and expressed in vitro from human, rodents, rabbit, bovine and several other species (An et al., 1993; Honda et al., 1993; Breyer et al., 1996; Boie et al., 1997). They have very high affinity for PGE₂ with a K_D value of 0.59–1.27 (Table 2). The rank order of affinity of PGE ligands for the mouse EP₄ receptor was PGE₂, PGE₁>11-deoxy-PGE₁, 16, 16-dimethyl-PGE₂, misoprostol>1-OH-PGE₁ and M&B-28767 (Table 3). At present, there are few selective agonists or antagonists available for EP₄. ONO-AE1-329 is a selective EP₄ agonist whereas L161982 and ONO-AE3-208 are selective antagonists.

Diversity in EP receptor signalling and functions

Diverse factors govern the outcome of EP receptor signalling. Basically, structural, pharmacological and functional differences that exist between the subtypes of EP receptors determine the biological effects of PGE₂. Structurally, differences observed in the length and composition of amino acids of the N and the C-termini, the second and the third extracellular as well as the intracellular domains are associated with variations in receptor signalling. In fact, differences in the length of C-termini of EP₃ isoforms are attributed for their differential activation of varied second messenger pathways (Pierce and Regan, 1998). Likewise, the extracellular sequence of the EP₂ receptor is a critical determinant of its structure and function (Stillman et al., 1999). Similarly, a cluster of hydrophobic aromatic amino acids in the second intracellular loop of EP₂ but not EP₃ isoform β is absolutely essential for activation of G_αs subunit (Sugimoto et al., 2003, 2004). Pharmacologically, EP receptors exhibit differences in binding to various ligands (Table 1) and surprisingly, the affinity of these receptors to its principal ligand, PGE₂, greatly varies between receptor subtypes (Tables 2 and 3). In fact, affinity of PGE₂ to its receptor may depend on the state of coupling of G protein subunits. A G-protein coupled receptor displays more affinity for PGE₂ than a free uncoupled receptor. Apart from differences in the ligand affinity to EP receptors, the phenomena of receptor desensitization and internalization may regulate preferential signalling through certain EP receptor subtypes. Agonist-induced desensitization is a common phenomenon in G-protein coupled receptor that is characterized by the loss of receptor signalling. The EP receptors have been shown to undergo PGE₂-induced desensitization. Interestingly, a differential desensitization of EP receptor subtypes has been observed wherein, the EP₄ receptor is highly desensitized in contrast to the EP₂ receptor which is not (Nishigaki et al., 1996). Similarly, differences in ligand-induced internalization of EP receptors have been reported. EP₄ and EP₃ isoform-I are readily internalized when activated by PGE₂ whereas EP₂ as well as EP₃ isoform-III and IV are not. Interestingly, the C-terminus of EP₄ has been implicated in its ligand-induced sequestration (Desai et al., 2000; Bilson et al., 2004). Functionally, differences among EP receptors directly correlate with the type of signal that it transduces. For example, receptor activation leading to intracellular Ca²⁺ mobilization is associated with the contraction of smooth muscle cells, whereas increase in cytoplasmic cAMP levels is associated with its relaxation. Evidently, signalling through EP₁ receptors increases intracellular Ca²⁺ levels whereas, EP₂ and EP₄ have been shown to increase cytoplasmic cAMP. Signalling via EP₃ receptors is unique wherein cAMP levels are decreased (Narumiya et al., 1999). Clearly, diverse factors govern the fate of signalling through EP receptors and all these may play a critical role in determining the differential biological effect of PGE₂ in the GI tract.

Table 1.

K_i values of PGE₂ for heterologously expressed human and mouse EP receptors

Species	Heterologous system	EP1	EP2	EP3	EP4	References
Humans	Human embryonic kidney cells	9.1	4.9	0.33	0.79	Abramovitz et al. (2000)
Mouse	Chinese hamster ovary cells	20	12	0.85	1.9	Kiriyama et al. (1997)

Abbreviations: EP, E prostanoid; PGE₂, prostaglandin.

Inhibitory constant (K_i) values are expressed in nM.

Downstream signalling pathways affected by EP receptors

Coupling of PGE₂ or the specific receptor agonists to EP receptors results in their activation and induces signalling cascade inside the cell (Figure 2). Interestingly, signalling through different subtypes of EP receptors seems to alternate and sometimes overlap; yet they are unique in terms of their signalling outcomes. EP₁ receptors mediate signalling events by activation of phospholipase C and elevation of cytoplasmic signalling intermediates including inositol triphosphate, diacylglycerol and Ca²⁺. Coupling of PGE₂ to EP₁ activates protein kinase Cα and c-Src. In addition to directly activating downstream kinases, signalling through EP₁ can also transactivate HER's-2/Neu tyrosine kinase receptor, which is mediated by c-Src resulting in upregulation of vegetative endothelial growth factor-C (Su et al., 2004). Recently, it was reported (Han and Wu, 2005) that c-Src mediated the transactivation of epidermal growth factor receptor by the EP₁ receptors through activation of protein kinase B (Akt) that promotes cell proliferation and invasion. These data corroborate with those obtained from studies in EP₁ receptor knockout mice, thus implicating a role of EP₁ receptors in colon carcinogenesis (Watanabe et al., 1999).

The EP₂ and EP₄ receptors are linked to the stimulation of cAMP/protein kinase A (PKA) signalling through the sequential activation of G_αs and adenylate cyclase. Contradictory reports associate this signalling pathway to growth and proliferation. Increased cAMP production, upon activation of EP₂ and EP₄ has been shown to have an antiproliferative effect in human gastric carcinoma cell lines (Okuyama et al., 2002). In contrast, activation of PKA is increasingly being linked to proliferation of various epithelial cell types. Phosphorylation of PKA is coupled to the regulation of glycogen synthase kinase-3 (GSK-3) and Akt (Filippa et al. 1999; Li et al., 2000). PKA phosphorylates and activates Akt kinase, which indirectly inhibits GSK-3. Inhibition of GSK-3 decreases the inhibitory phosphorylation of cytosolic β-catenin that promotes the translocation of β-catenin to the nucleus resulting in cellular proliferation (Cadigan and Nusse, 1997). A recent study has directly linked EP₂ receptor activation to cellular proliferation. Upon activation the G_αs subunit of EP₂ receptors can directly associate with the regulator of G protein signalling domain of Axin, which inactivates and releases GSK-3β from the Axin complex causing β-catenin activation and nuclear translocation (Castellone et al., 2005). Until recently, it was believed that the phosphoinositide-3-kinase (PI3K) signaling pathway was activated only by the EP₄ receptor. However, recent studies have shown that PGE₂ signalling through EP₂ receptors can activate both PI3K and Akt by using the free βγ subunit of G-protein (Castellone et al., 2005).

Although EP₂ and EP₄ receptors are capable of stimulating both PKA and PI3K signalling pathway, a receptor-specific signalling outcome has always been observed. For example, a study showed that PGE₂ stimulation of EP₄, but not EP₂ receptor that are stably expressed in HEK cells lead to the phosphorylation of the extracellular signal-regulated kinases (ERKs) by a PI3K-dependent mechanism and induced the expression of early growth response factor-1 (Fujino et al., 2003). The differences in the signalling potential of both EP₂ and EP₄ receptors are corroborated by the fact that both receptors mediate the phosphorylation of cAMP responsive element binding protein (CREB) by different signalling pathways (Fujino et al., 2005). Signalling through EP₂ receptors can decrease the inhibitory tyrosine phosphorylation of PTEN, a phosphatase that can act as a PI3K pathway inhibitor (White et al., 2005). A novel EP₂ receptor-signalling pathway, which can transactivate the EGF receptor leading to increased migration and invasion of colon cancer cells, has also been reported (Pai et al., 2002; Buchanan et al., 2003).

The EP₃ receptors are unique in their ability to couple to multiple G proteins. They couple and activate the G_i subunits, which results in the inhibition of adenylyl cyclase. Apart from activation of G_i subunits, signalling through EP₃ receptors can also activate G_s resulting in cAMP production. Evidence also indicates that the EP₃ receptor can activate the small G protein Rho and its target p160 Rho-A binding kinase ROK α (Katoh et al., 1998; Tamma et al., 2003). EP₃ receptors have also been shown to activate the Ras signalling pathway leading to cancer (Yano et al., 2002).

Distribution of EP receptors in the gut

Although PGE₂ is produced by a variety of cells in the GI mucosa and is found throughout the gut, its cellular targets in the mucosa and the resulting physiological changes in the GI tract are predominantly determined by the presence and the distribution of EP receptors. Pharmacological as well as cellular localization studies have been aimed towards identifying the type and pattern of EP receptor distribution and their density in major cell types in the GI mucosa. However, very little is known of the regional distribution or the temporal changes in the pattern of EP receptor expression and function under normal and disease states in the gut.

EP₁ receptor distribution

EP₁ receptor expression has been reported in the GI tract of various species. In rat, EP₁ mRNA expression is detected at the gastric, small intestinal and colonic muscle layers (Ding et al., 1997). A non-radioactive in situ hybridization technique used to study the pattern of localization of EP receptors in the rat GI tract revealed the presence of EP₁ receptors in gastric chief cells that secrete pepsinogen, parietal cells that secrete hydrochloric acid and in mucus secreting gastric epithelial cells. In the small intestine, EP₁ receptor expression was noticed only in goblet cells whereas in the large intestine, EP₁ receptor expression was seen in goblet cells and in other epithelial cell types. Expression of EP₁ receptors has been reported in enteric glial cells (Northey et al., 2000). In mice, EP₁ mRNA expression was noticed in the muscularis mucosa (Morimoto et al., 1997). In rabbits, EP₁ receptors are highly expressed on the intestinal brush border membranes of intestinal villi. Goblet cells of the jejunum and ileum also show considerable expression. In contrast, goblet cells of the duodenum show a lack of expression of EP₁ receptors. Interestingly, EP₁ receptors are also highly expressed in the neurons of the myenteric and submucosal ganglia throughout the rabbit intestinal tract (Grasa et al., 2006).

EP₂ receptor distribution

EP₂ receptors are normally expressed in rat gastric mucous cells, in the goblet cells of the small intestine and also in various epithelial cells of the large intestine (Northey et al., 2000). In mice, an abundant expression of EP₂ receptors in the stomach and ileum has been detected by Northern blot analysis (Katsuyama et al., 1995). A recent report suggested a relatively uniform expression of EP₂ receptors throughout the mouse GI tract except for the ileum and caecum, where it is weakly expressed. Interestingly, expression of EP₂ receptors depends on the state of differentiation of the epithelial cells. Undifferentiated crypt epithelial cells predominantly express EP₂ receptors on their nuclear membranes whereas the highly differentiated epithelial cells at the apex of the villi express these receptors on their plasma membrane (Houchen et al., 2003). In rabbits, EP₂ receptors are localized on the intestinal brush border membrane of the villi and are also strongly expressed in goblet cells throughout the small intestine. EP₂ receptors are weakly expressed in neurons but are completely absent in the smooth muscle cells of the small intestine (Grasa et al., 2006). In humans, EP₂ receptor expression is restricted to the luminal surface of the gastric epithelium (Takafuji et al., 2002) and at the apex of the colonic mucosa (Takafuji et al., 2000). In guinea-pigs, the presence of EP₂ receptors in the neurons of the enteric nerve plexus in the ileum was detected through pharmacological studies (Lawrence et al., 1992).

EP₃ receptor distribution

EP₃ receptor gene expression appears to be localized to the muscular parts of the rat intestine whereas in the stomach EP₃ mRNA expression is seen in the mucosal layer especially in parietal cells (Ding et al., 1997). In most rodents, EP₃ receptor expression is predominantly found in gastric parietal and small intestinal goblet cells. They are also highly expressed in enteric glial and myenteric neuronal cells (Morimoto et al., 1997; Takahashi et al., 1999; Northey et al., 2000). EP₃ receptors are highly expressed in rabbit intestinal circular and longitudinal smooth muscles of the duodenum and in the circular muscles of the ileum (Grasa et al., 2006). In humans, EP₃ receptor expressions are observed throughout the gastric epithelium (Takafuji et al., 2002) and in the apex of colonic mucosa (Takafuji et al., 2000).

EP₄ receptor distribution

EP₄ receptor mRNA are mainly expressed in the rodent intestinal mucosal layers and in the parietal as well as the mucosal epithelial cells of the gastric mucosa (Ding et al., 1997; Northey et al., 2000). In the ileum, EP₄ receptor expression was demonstrated on mature enterocytes of the villi (Morimoto et al., 1997). Normal EP₄ receptor mRNA expression was observed in rabbit gastric epithelial cells (Takahashi et al., 1999). However, EP₄ receptor proteins were not detected in any of the segments (Grasa et al., 2006). In humans, EP₄ expression was modest in the gastric epithelium but intense expression of EP₄ was detected in lamina propria mononuclear cells (Takafuji et al., 2002). In the colon EP₄ receptors are strongly expressed in the lateral crypt epithelia (Takafuji et al., 2000) and lamina propria mononuclear cells of CD+ T lymphocytes (Cosme et al., 2000).

Physiological role of EP receptors in the GI tract

The role of PGE₂ in GI physiology has been distinctly proved in various animal models. Presently, agonist/antagonist-based approaches along with EP receptor knockout strategies are used to determine the role that each of the EP receptors plays in GI physiology. Using these approaches, the role that EP receptor subtype(s) play in gastric acid secretion, GI motility and mucosal barrier functions including bicarbonate secretion, mucus secretion and epithelial cytoprotection have been elucidated.

Gastric acid secretion

Gastric acid is the main secretion of the stomach. A dual action of PGE₂ on gastric acid secretion was observed in rats, wherein, lower concentrations inhibited and higher concentration stimulated secretion (Ding et al., 1997). The inhibitory action of PGE₂ on acid secretion in rats was mediated by the EP₃ receptor whereas the stimulatory effect was due to the EP₄ receptor. It is interesting to note that both EP₃ and EP₄ receptors are present in parietal cells and acid-producing chief cells in the gastric mucosa. Parietal cells can respond to histamine in releasing acid into the gastric lumen. An EP₄ agonist, ONO-AE1-329, was shown to stimulate gastric acid secretion through histamine released from entero chromaffin cells (Kato et al., 2005).

GI motility

The peristaltic movement of the GI tract is maintained through coordinated contraction and relaxation of GI smooth muscle and PGE₂ has been reported to play a major role in GI motility. In rabbits, a complex mechanism involving activation of both EP₃ and EP₁ receptors was shown to be responsible for the contraction of the small intestine. A direct effect of EP₃-mediated activation of intestinal smooth muscles and an indirect effect of EP₁-mediated stimulation of myenteric neurons acts together during PGE₂-induced intestinal contractions (Grasa et al., 2006). In rats, inhibition of small intestinal hypermotility was mediated through EP₄ receptors (Kunikata et al., 2002). Studies conducted in EP receptor knockout mice clearly underscore the importance of EP₁ and EP₃ receptors in the contraction of longitudinal smooth muscles of gastric fundus and ileum and EP₄ receptors in the relaxation (Okada et al., 2000). In guinea-pig ileum, pharmacological investigation revealed that EP₃ receptor stimulation mediates contraction of circular smooth muscles and increases peristalisis, whereas EP₂ mediates relaxation and decreases peristalisis (Botella et al., 1993; Shahbazian et al., 2002).

Bicarbonate secretion

GI bicarbonate secretion is one of the first lines of defense of the host epithelial cells against the harsh acidic environment of the stomach and the duodenum. Bicarbonates help in maintaining a narrow zone of neutral pH just above the mucosal lining that offers a protective barrier against diffusing acid (Takeuchi et al., 1997). PGE₂ has a stimulatory effect on the GI bicarbonate secretion. In fact, low physiological concentrations of PGE₂ in the duodenum would induce an acid-provoked bicarbonate secretion (Hirokawa et al., 2004). An agonist–antagonist-based approach in rats, as well as studies involving EP receptor knockout mice have proved the involvement of different EP receptor subtypes in bicarbonate secretion in different segments of the gut. In the stomach of rats and mice, EP₁ receptors are responsible for bicarbonate secretion whereas, the same effect was mediated through EP₃ receptors in the duodenum (Takeuchi et al., 1999a). In fact, the presence of EP₃ receptors in the duodenum is essential for duodenal bicarbonate secretion to counter luminal acid-induced mucosal damage (Takeuchi et al., 1999b). In addition to EP₃ receptors, EP₄ receptors have also been proved to be involved in duodenal bicarbonate secretion (Aoi et al., 2004). In humans, EP₄ receptors are solely responsible for duodenal bicarbonate secretion, which is in contrast to that seen in rats and mice, where EP₃ receptors mediate duodenal bicarbonate secretion (Larsen et al., 2005). This study suggests that species differences occur with regard to EP receptor function(s) and therefore caution should be taken in ascribing a particular function to a particular EP receptor.

Mucus secretion

Mucins are polymers made of glycoproteins that are secreted by the mucous cells of the stomach and the goblet cells of the intestine that form a protective covering over the lining of the mucosa. They form the major component of the innate immune response of the GI mucosa. PGE₂ is reported to be a potent mucin secretagogue. It strongly stimulates mucin secretion from rat gastric epithelial cells (Tani et al., 1997). More recently, the molecular mechanism of PGE₂-induced-mucin gene transcription via the ERK MAPK/RSK1/CREB pathway has been elucidated (Cho et al., 2005). PGE₂-induced mucus secretion from the gastric epithelial cells of rabbit are mainly mediated through EP₄ receptors in a cAMP/PKA-dependent manner (Takahashi et al., 1999). Mucin exocytosis from the antral mucous cells of guinea-pigs is also mediated through EP₄ but activated EP₁ receptors are shown to potentiate the effect of mucus secretion mediated by EP₄ (Ohnishi et al., 2001). An agonist-based approach in identifying the EP receptors responsible for mucin exocytosis in the rat colon and in a human colonic epithelial cell line LS174T revealed that coupling of PGE₂ with EP₄ receptors was essential for mucin secretion (Belley and Chadee, 1999).

Cytoprotection

PGE₂ plays an important role in protecting the GI mucosa from injuries caused by the harsh environment of the lumen. The mechanism by which PGE₂ exerts its protective effects on GI mucosa from noxious agents is termed cytoprotection. For example, PGE₂ exerts a potent cytoprotective effect on gastric glandular cells against indomethacin-induced injury, which is independent of neural, vascular and hormonal factors (Brzozowski et al., 2005). Current studies are underway to identify EP receptor subtype that mediates this protective action. It has been reported that, in an acute rat esophagitis model, low doses of PGE₂ confer a protective effect that is mediated through EP₁ receptors (Yamato et al., 2005). Similarly, in the stomach of rat, EP₁ receptors are essential for offering protection against indomethacin or ethanol-induced injury (Araki et al., 2000; Suzuki et al., 2001). In rodents, EP₁ receptors also mediate adaptive gastric cytoprotection, under conditions in which initial exposure to mild irritants prevents subsequent damage by more severe irritants (Takeuchi et al., 2001). Activation of the EP₁ receptors helps in maintaining gastric mucosal integrity (Takeuchi et al., 2002). In the small intestine of rats, EP₃ and EP₄ receptors offer cytoprotection against indomethacin-induced injury possibly through increased mucus secretion, enteropooling and through inhibition of hypermotility mediated by EP₄ alone (Kunikata et al., 2002). Activation of both EP₂ and EP₄ receptors in the guinea-pig gastric mucosal cells offers cytoprotection against ethanol-induced apoptosis (Hoshino et al., 2003). In mice, EP₂ receptors are essential for preventing radiation-induced apoptosis of crypt epithelial cells at the jejunum (Houchen et al., 2003).

Differential expression of EP receptors during pathological conditions in the gut

This area is perhaps the least studied aspect of EP receptor biology in the gut. To complicate the issue, the limited data that are available does not always discriminate whether EP receptor expressions are altered in individual cells, mucosa or submucosa or the full thickness of the gut. Nevertheless, these data on differential expression of EP receptors during various pathological conditions such as radiation injury, tumorigenesis and inflammation may explain the modus operandi of PGE₂ in exerting its pathological functions as against its well-known physiological roles.

Radiation-induced injury

A regional and temporal difference in the expression of EP receptors following radiation-induced injury in mice has been observed. Significant changes in EP₂ and EP₄ mRNA and protein levels were noticed in the jejunum and colon after radiation-induced injury. EP₂ receptor expression increased steeply whereas a decrease in EP₄ expression was observed. Interestingly, EP₂ receptor expression corresponded with epithelial restitution and early crypt morphogenesis of the injured tissues (Houchen et al., 2003).

GI tumorigenesis

A differential expression of EP receptors in the colon during early stages of tumorigenesis is observed in rodents and humans. The most notable changes reflect an increase in the expression of EP₁ and EP₂ receptors and a marked decrease in EP₃ receptors. However, EP₄ receptor levels remained constant (Shoji et al., 2004). Upregulation of EP₁ receptors is clearly observed during early tumorigenesis that helps in cellular proliferation and in the antiapoptotic effects observed in cancer cells, indicating a major role played by EP₁ receptors in cancer (Kawamori et al., 2005). A few studies involving RT–PCR analysis have revealed that as well as EP₁, EP₂ and EP₄ receptor mRNAs expression are increased in azoxymethane-induced colorectal cancer tissues (Mutoh et al., 2002; Kawamori et al., 2003). Similarly, EP₂ and EP₄ mRNA levels are also increased in ApcΔ716 mouse small intestinal and colonic polyps (Sonoshita et al., 2001). These studies implicate EP₂ and EP₄ receptors in tumorigenesis. The signalling mediated by EP_1/2/4 receptors that lead to cellular proliferation and tumorigenesis was briefly discussed under the previous section ‘Downstream signalling pathways affected by EP receptors.'' Apart from the upregulation and activation of certain EP receptors during cancer, it is interesting to note that EP₃ receptor is downregulated. Downregulation of the EP₃ receptor is linked to the hypermethylation of the EP₃ receptor gene, thus inhibiting the suppressive role of EP₃ in colon cancer development. Increased expression of EP₄ receptors on CD3⁺ lymphocytes adjacent to the tumour was also evident in human gastric mucosal cancers but the role of T lymphocyte EP₄ signalling in cancer is not clear (Takafuji et al., 2002). An increased expression of EP₂, EP₃ and EP₄ receptor mRNA levels was observed in rat oesophageal squamous cell dysplasia and Barrett's metaplasia induced by duodenal contents reflux; however, no changes in the mRNA levels of the EP₁ receptor was observed (Jang et al., 2004). This study suggests that regional differences may exist for the role of EP receptor subtypes in GI tumorigenesis.

GI inflammation

A differential expression of EP receptors on the GI mucosa may occur during inflammation. One interesting study showed defined patterns and identified striking differences of mucosal expression of the EP₄ receptors between cells in normal human colonic lamina propria and those from the inflamed human colon (Cosme et al., 2000). In particular, mucosal EP₄ receptor expression was upregulated in T lymphocytes. In ulcerative colitis, apart from an increase in EP₄ receptor expression in lamina propria T lymphocytes, an increase in the levels of EP₂ and EP₃ receptors was apparent in epithelial cells (Takafuji et al., 2000). At present, there are no reports on EP receptor expression levels in the gut during microbial or parasitic infections.

Role of EP₄ receptors in colitis

Even though PGE₂ levels are significantly increased during IBD, the functional role PGE₂ and individual EP receptors play in the pathogenesis of IBD remains undefined. PGE₂–EP receptor coupling may have a critical role in homeostasis or in the onset of GI inflammation and/or tissue repair. The limited data to date suggest that signalling via EP receptors can predetermine whether PGE₂ exerts a proinflammatory or anti-inflammatory effect in the gut. For example, studies in vitro, to address early responses of PGE₂ in a variety of colonic epithelial cell lines clearly demonstrate that PGE₂ couples via high-affinity EP₄ receptors to upregulate IL-8 mRNA expression and protein secretion confirming a proinflammatory role for PGE₂ (Yu and Chadee, 1999). IL-8 is a potent chemokine that can attract and activate neutrophils to cause nonspecific tissue damage important in the onset of colonic inflammation. In contrast, in EP₄ receptor knockout mice, a 7-day regime of dextran sodium sulphate-induced colitis was reported to be more severe than that in wild-type controls. This suggests that signalling via EP4 receptors may play a critical role in maintaining normal mucosal integrity and/or to promote healing (Kabashima et al., 2002). Unfortunately, the role of EP₄ signaling in either epithelial cells or the submucosal cells that induce an anti-inflammatory effect has not been elucidated. Moreover, it is not clear whether epithelial barrier functions or cytokine productions are altered in EP₄ knockout animals. Similarly, studies in rats using an EP₄ receptor agonist reported the suppression of colitis caused by extended DSS treatment through upregulation of an anti-inflammatory cytokine, IL-10 (Nitta et al., 2002). Suppression of Th1-type response, coupled with evidence of increased expression of EP₄ receptors on the mucosal T lymphocytes in inflamed colonic mucosa (Cosme et al., 2000), suggests a fundamental role for PGE₂-EP₄ signalling in T lymphocytes in repair and restitution of damaged tissues. It appears that EP₄ signalling outcomes differ between the early onset (pro-inflammatory on mucosal epithelial cells) and the late progressive stages of colitis (anti-inflammatory on immune cells in the lamina propria). Collectively, these studies emphasize the importance of PGE₂-EP₄ receptor signalling events in the cell types present in colonic mucosa as a fundamental determinant of colitis. More studies are needed to define the diverse functions of PGE₂-EP receptor coupling on various cell types before we can model their role in normal and disease states.

Concluding remarks

Recent evidence clearly suggests that PGE₂-EP receptor signalling plays an important role in the GI tract. Importantly, PGE₂-EP receptor coupling on different cell types may exert either a pro-inflammatory or anti-inflammatory response. In general, it appears that EP₁, EP₂ and EP₄ receptors are major determinants in the early stages of intestinal tumorigenesis and inflammation, whereas EP₄ receptors on immune cells may promote the restitution of colitis/inflammation. There maybe redundancy of EP receptor signalling and EP receptor functions may vary from onset to different stages of disease (initiation versus progression). However, it appears that all receptors are needed for various normal functions of the GI tract. As regional, temporal and species differences exist in EP receptor expression and distribution in the GI tract, future research should aim at identifying EP receptor expression in various cellular components of the mucosa across the length and the breath of the gut. As regional and species differences are apparent with regards to EP receptor functions, it is dangerous to ascribe specific functions for EP receptors in the gut. A priority is to define the distinct EP receptor signalling events in various cell types in the gut. Knowledge of receptor expression coupled with insights into specific signalling events in specific cell types may help the cause of understanding distinct roles that EP receptors play in the GI tract. More research is also needed to define the activity and the toxicity of single/combination EP receptor functions under normal and inflamed/diseased conditions. The long-term goal of future research in EP receptor biology should aim at identifying the Achilles' heel at the receptor level as well as at the signalling that it propagates. This would help in effective pharmacological intervention of various GI ailments whose pathologies are mediated by PGE₂.

Abbreviations

AA

arachidonic acid

Akt

protein kinase B

Ca²⁺

calcium

cAMP

cyclic adenosine monophosphate

COX

cyclooxygenase

CREB

cAMP responsive element binding protein

DP

D prostanoid

EP

E prostanoid

EP₁, EP₂, EP₃ and EP₄

E prostanoid receptor subtypes 1, 2, 3 and 4

ERK

extracellular signal-regulated protein kinase

FP

F prostanoid

G (_{p/q, αs} and _αi)

G protein subunits

GI

gastrointestinal

GSK-3

glycogen synthase kinase-3

IBD

inflammatory bowel disease

IP

I prostanoid

K_D

dissociation constant

PG

prostaglandin

PGD₂

prostaglandin D₂

PGE₂

prostaglandin E₂

PGF_2α

prostaglandin F_2α

PGI₂

prostaglandin I₂

PI3K

phosphoinositide-3-kinase

PKA

protein kinase A

PLA₂

phospholipase A₂

TP

T prostanoid

TXA₂

thromboxane A₂

Conflict of interest

The authors state no conflict of interest.