1.3.4.1. Structure and localisation
Prostaglandin effects are mediated through the transmembrane, G-protein coupled prostanoid receptors, which were first classified in 1982 (Kennedy et al., 1982). There are five key members of the prostanoid receptor family, grouped by the ligand with which they bind to, these include DP, EP1-4, FP, IP and TP, which bind to PGD, PGE, PGF, PGI and thromboxane respectively (Ushikubi et al., 1995). Prostaglandins exhibit different biological functions, depending upon the receptor with which they bind, and effects may also differ depending on the cell type involved (Hata & Breyer, 2004). The prostanoid receptor gene structure has been shown to be similar between all receptors and across various species (Boie et al., 1995; Ogawa et al., 1995; Regan et al., 1994). The receptors consist of the seven hydrophobic, transmembrane domain, characteristic of G-protein coupled receptors and a putative extracellular-loop region (Breyer et al., 2001). As PGE2 is
considered to have the most important role in normal GI physiological processes, the focus of this review will be on the structure and function of the EP receptors to which PGE2 binds.
Four sub-types of the EP receptor exist, namely EP1, EP2, EP3 and EP4, each with varying structures and functional roles. The diverse biological effects of PGE2 may be attributed to the different signal transduction pathways that occur
upon activation of each receptor sub-type (Hata & Breyer, 2004). The EP receptors are encoded by different genes, but are well conserved throughout the mammalian species. All of the receptor sub-types are expressed on the plasma membrane, however EP3 and EP4 also localise at the nuclear envelope (Bhattacharya et al., 1999). EP3 is the only receptor that exhibits multiple alternatively spliced variants (Breyer et al., 1994), which can activate different second messenger signalling pathways (Pierce and Regan, 1998). Eight different EP3 isoforms have been recorded to date (Bilson et al., 2004).
EP receptor expression within the stomach varies greatly between species. For instance, in rat stomach tissue, EP1 mRNA was detected in the gastric muscle layers, while EP3 and EP4 mRNA was expressed primarily in the gastric mucosal layer (Ding et al., 1997). Within cultured gastric epithelial cells, EP3 and EP4 were expressed in parietal cells, while only EP4 was expressed in gastric mucous cells (Ding et al., 1997). In normal human gastric tissue, no EP1 protein was detected, EP2 was expressed on the luminal surface of the gastric epithelium, EP3 was expressed in the gastric epithelium only and was localised to the upper mucosal cells and intense EP4 expression was detected in the lamina propria mononuclear cells (Takafuji et al., 2002). Limited information is available with regards to the expression and localisation of the EP receptors in the normal canine gastric epithelium.
Out of the four EP receptor sub-types, EP1 has the lowest affinity for PGE2 with a
Kd of 16-25 nM (Dey et al., 2006). The affinity of EP2 for PGE2 differs
significantly between species, with the rat EP2 receptor having the highest affinity (Kd=5 nM) and the mouse receptor showing a much lower affinity (Kd=116 nM)
(Dey et al., 2006). The EP3 and EP4 receptors both have a relatively high affinity for PGE2, with Kd values of 0.33-2.9 nM and 0.59-1.27 nM respectively (Dey et
al., 2006). These variations in affinity between the receptor sub-types may be due to the degree of G-protein subunit coupling. It has been reported that a receptor that is coupled exhibits a lower affinity for PGE2 than an uncoupled receptor
1.3.4.2.Functions of the EP receptors
The roles of the specific EP receptors in gastric cytoprotection have been studied using both mouse „knockout‟ models and experiments utilising sub-type specific EP receptor agonists and antagonists. Pre-treating rats with either PGE2 or an EP1
specific agonist, dose-dependently prevented HCl/ethanol-induced gastric lesion development (Araki et al., 2000), thus it appears that PGE2 provides gastric
cytoprotection through activation of the EP1 receptor. This finding was confirmed using a mouse „knockout‟ model, which demonstrated that the protective effect of PGE2 disappeared in mice lacking the EP1 receptor (Araki et al., 2000).
Furthermore, PGE2 has been shown to dose-dependently protect the gastric
mucosa against NSAID-induced damage, via activation of the EP1 receptor (Suzuki et al., 2001).
Prostaglandins have gastric cytoprotective effects via the regulation of acid, bicarbonate and mucus secretion through activation of the EP3, EP1 and EP4 receptors respectively (Takeuchi et al., 2010). PGE2 has a biphasic effect on acid
secretion via the activation of different receptors; for instance, EP3 and EP4 activation cause the inhibition and stimulation of acid secretion respectively (Dey et al., 2006). PGE2-mediated inhibition of acid secretion via EP3 receptor
activation, involves both a direct effect on parietal cells and an indirect effect via the inhibition of histamine secretion from ECL cells (Takeuchi et al., 2010). Additionally, gastric mucosal blood flow is increased via activation of the EP2, EP3 and EP4 receptors, but not the EP1 receptor (Araki et al., 2000). Activation of EP2 and EP4 may also be important for gastric cytoprotection, in particular, inhibition of ethanol-induced rat gastric mucosal damage has been shown to occur via activation of EP2 and EP4 leading to inhibition of LTC4 production (Hattori et
al., 2008). LTC4 is considered to play an important role in the development of
ethanol-induced gastric damage via the promotion of vascular disturbances (Higa et al., 1991). COX-2-derived PGE2 has been shown to promote the healing of
gastric ulcers via EP4 receptor activation and the up-regulation of VEGF (Hatazawa et al., 2007) and EP4 receptor activation is associated with the modulation of cell migration in a variety of cell types (Kim et al., 2010; Ma et al., 2006).
1.3.4.3.EP receptor signalling
Each EP receptor is coupled to a different intracellular signalling pathway (Figure 1.7). The EP1 receptor activates PLC, causing mobilisation of intracellular calcium, EP2 and EP4 activation results in an increase in intracellular cAMP levels and EP3 activation leads to a reduction in cAMP (Hull et al., 2004). The EP3 receptors are able to couple to multiple G-proteins, upon coupling they activate the Gi subunits, leading to inhibition of adenylyl cyclase. They may also
activate Gs subunits causing an increase in cAMP production (Dey et al., 2006).
Figure 1.7- Diagram illustrating the signal transduction pathways induced via activation of the EP receptor sub-types
COX-1/-2 EP1 β ɣ Gα q PLC PIP2 DAG + IP3 Ca2+ EP2 β ɣ Gαs Adenylyl cyclase cAMP EP3 β ɣ Gαi EP4 β ɣ Gαs Adenylyl cyclase cAMP PGE2