Signal Transduction

The coupling of prostanoid receptors to intracel-| lular signalling pathways has been examined by several methods using both recombinant systems and in vitro preparations to determine the Gproteins and second messengers involved.

3.4.1 DP Receptors. DP receptors couple through Gs to increases in intracellular cyclic AMP. Indirect evidence for this was obtained by Simon and colleagues (173), who showed that D, E, and I series prostaglandins all stim-

| ulated adenylate cyclase in human colonic mucosa. These early observations have been confirmed by studies using recombinant DP receptors, where it has been demonstrated that both PGD, and the DP receptor selective | agonist, BW245C, concentration dependently increase cyclic AMP levels in Chinese hamster ovary (CHO) cells expressing recombinant DP receptors (49, 112).

3.4.2 EP, Receptors. The G-protein involved in EPi receptor signaling has yet to be identified (1). Studies in recombinant cells suggest the involvement of Ca2+ mobilization. Watabe and colleagues (64) showed PGE, caused an increase in intracellular [Ca2+] in CHO cells expressing this receptor, an event

¿that was dependent on extracellular Ca2+ and accompanied by a very weak inositol triphosphate (IP3) response. This agrees with Creese and Denborough (174) who showed a similar extracellular Ca2+ effect in guinea pig trachea, a preparation known to contract through EP-l receptors (175).

3.4.3 EP, Receptors. EP2 receptors are coupled to Gs and mediate increases in intracellular cAMP concentrations. Evidence for his was first obtained by Hardcastle and co-workers, who reported a positive association between EP, and cAMP generation in entero-cytes (176) and more recently from recombinant systems.

3.4.4 B3, Receptors. EP3 receptors have Ibeen shown to couple to several different sig nal transduction systems. The major coupling is to Gi to reduce adenylate cyclase activity, but there are several reports of differential coupling of splice variants, including coupling to increases in intracellular calcium (139). This is consistent with responses observed in tissues, where EP3 receptor activation can inhibit gastric acid secretion (see below) and li-polysis, two actions classically mediated by decreases in cellular cAMP. In addition, it also seems that EP3 receptors can mediate smooth muscle contraction, consistent with increases in intracellular Ca2+ (27).In addition, theEP3 receptor seems to be involved in the inhibition of arginine vasopressin water reabsorption through a pertussis toxin-sensitive pathway, suggesting the involvement of Gj (177, 178). Likewise, the EP3-mediated inhibition of acid secretion in the stomach is also pertussis-sensitive (72).

Four EP3 receptor isoforms have been cloned from cDNA generated from a bovine adrenal gland, and these display coupling to multiple second messenger pathways. Thus, the bovine EPaA isoform couples to Gi? the EP3B and EP3C isoforms to G„ and the EPari isoform to G„ Gis andG, (179). Isoforms of the mouse EP3 receptor, EP3q, and EP3J3, differ in their responses to GTP7S and also in potency at inhibiting forskolin-induced cAMP accumulation, with EP3y requiring threefold lower concentrations of agonist than EP33 to evoke a 50% inhibition (119).

3.4.5 EP, Receptors. This receptor subtype, like the EP, receptor, is known to couple to G, and mediate increases in intracellular cAMP levels (116). There is some confusion in the early literature regarding the identity of the "true" EP4 receptor. The "EP2" subtype, originally cloned by Honda and colleagues (122) and Bastien and co-workers (121) has since been identified as the EP4 receptor, on the basis of its insensitivity to the EP2 receptor-selective agonist, butaprost (see Section 4), and sensitivity to the EP4 receptor antagonist, AH23848B. The EP2 receptor cloned by Regan and colleagues (117) is regarded as the "true" EP, receptor subtype.

3.4.6 FP Receptors. This receptor type mediates increases in inositol triphosphate for mation through Gq and phospholipase C activation. It has been known for some time that stimulation with PGF2q, is coupled to increases in phosphoinositide turnover and the elevation of intracellular calcium (180, 181). In mouse fibroblasts, the effect of PGF2ci to elevate intracellular calcium is pertussis toxin-insensitive, suggesting a lack of involvement of Gis and correlates with IP3 formation (182). Similar results have been obtained using recombinant FP receptors, where FP receptor activation resulted in a concentration-dependent increase in IP3 formation (97).

3.4.7 IP Receptors. IP receptors mediate an increase in cAMP levels through Gs (163), but recombinant studies in CHO cells have also revealed a phosphatidyl inositide response that is cholera- and pertussis toxin-insensitive, suggesting the involvement of Gq (126).

3.4.8 TP Receptors. TP receptors are generally regarded as signaling through Gq to cause an increase in the concentration of intracellular calcium (183). However, there are reports that TP receptors may also couple to Gn, G19, and G1S (184-186). Furthermore, there have also been reports that TP receptor splice variants, while both couplingto Gq, may interact differently with adenylate cyclase. Thus, the TPa receptor isoform induces increases in intracellular cAMP, whereas the TPj3 isoform induces decreases in intracellular cAMP levels (187).

4 PROSTANOID RECEPTOR-SELECTIVE LIGANDS AND STRUCTURE-ACTIVITY RELATIONSHIPS

From the desire to produce novel therapeutics, several ligands that are selective for subtypes of prostaglandin receptors have been developed, and these are detailed below.

4.1 DP Receptors and Selective Ligands

The structures of some DP receptor ligands are shown in Fig. 6.4. PGD, is not an inherently selective agonist; it also has FP and TP receptor activity (188). The dehydration product of PGD2, PGJ2 (9-deoxy-A9-PGD2), (189)

is equipotent to the natural agonist in producing vasodilation and inhibiting of platelet aggregation and more selective with respect to EP and FP receptor activity. PGJ2 has also been shown to be of similar potency to PGD2 in heterologous systems (190). Furthermore, PGJ2 and its derivative, 15-deoxy,12,14-PGJ2, are also agonists at the peroxisome prolifera-tor-activated receptor-gamma (PPARy) (191). The most widely described DP receptor agonist is the synthetic prostanoid, BW245C (192). This is a hydantoin prostanoid analog that is at least one order of magnitude more potent than PGD, as a DP receptor agonist, but is considerably less active at TP and FP receptors. It displaces [3H]-PGD2, but not [3H]-prostacyclin binding form in bovine platelets (152), and its inhibitory action on human platelets can be blocked by the DP receptor antagonist, AH6809. Furthermore, BW245C has been shown to potently inhibit the aggregation of human platelets and relax the rabbit transverse stomach strip (193). Another DP receptor selective agonist is the 9-chloro analog of PGE2, ZK110841 (194). SQ27986 has also been described as a potent and highly selective DP receptor agonist (195). Interestingly, introducing a 15-cyclohexyl group into the a-side chain of PGD2 and other prosta-glandins seems to enhance DP receptor-like selectivity. Finally, and most surprisingly, is RS93520. This is an analog of PGI„ but is far more potent as a DP receptor agonist, being only a modest IP receptor agonist on the human platelet (196). Recently, L-644698 has been described as a novel DP receptor selective agonist (190), and it has been reported to be at least 300-fold more selective for human DP receptors over human EP2 receptors and more than 4000-fold more selective over the other human recombinant prostanoid receptors. It also exhibits similar efficacy to PGD, and BW245C, as evaluated by the accumulation of cAMP in recombinant cells, suggesting this may prove a useful tool in DP receptor characterization in the future. The agonist and antagonist potencies of some ligands active at the DP receptor are shown in Table 6.3 and affinity constants at both mouse and human recombinant receptors in Table 6.4.

The first compounds with recognized DP receptor blocking activity were the phloretin

Agonists

ZK110841

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