Physiological Actions of Prostanoids

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Prostaglandins have a wide range of actions in normal cell physiology, but also as pathophys-iological agents in, for example, inflammation and pain states. The roles of the natural pro-stanoids are reviewed in this section in relation to studies on receptor distribution, which provide useful information concerning potential pharmacological applications. Recent advances in molecular biology have also allowed the creation of prostanoid receptor "knockout" mice, which have proved valuable in helping to further elucidate the receptor types involved in mediating the various actions of prostanoids.

2.3.1 Prostaglandin D,. PGD2 acts predominantly at DP receptors, although it does have relatively high potency at FP and even TP receptors. DP receptors are probably the least widely distributed of all the prostanoid receptors and often co-exist with other prosta-receptors, making them difficult to study outside of recombinant systems. However, the availability of potent, selective agonists and antagonists (see Section 4) has proved pivotal in surmounting this problem.

DP receptors are present on vascular smooth muscle, platelets, and in nervous tissue, including the central nervous system (CNS). In the CNS, PGD2 has been associated with sleep induction, as well as body temperature regulation, hormone release, nociception, and olfactory function. DP receptors have also been localized to smooth muscle in the gut, uterus, and airways. Northern blot and in situ hybridization studies have reaffirmed this, with only low levels of expression being detected in human retina and small intestine (49) .The physiological actions of PGD2, mediated through DP receptors, are generally "inhibitory"; that is, smooth muscle relaxation and the inhibition of platelet aggregation. However, "excitatory" actions have also been reported: for example, in afferent sensory nerves PGD, seems to induce hyperalgesia (50) and can also stimulate the release of neuropeptides (51). PGD, has also been implicated in central roles, such as sleep-induction, in both rats and humans (52). DP receptors have been localized to the leptomeninges (53), and PGD, injected into the arachnoid space below the basal forebrain caused leptomenin-geal cellular activation, detected by immuno-histochemistry for the immediate early gene, fos (54). Furthermore, the brain-specific isoform of PGD synthase has recently been localized in the leptomeninges (55). DP receptors have also been recently detected in the mucous-secreting goblet cells in the rat gastrointestinal tract, suggesting a potentially novel role for PGD, in mucous secretion (56).

DP receptor localization also seems to vary considerably between species. For example, platelet DP receptors seem to be most abundant in humans and not in other animal species (57). PGD, is also the major prostaglandin produced by mast cells following immunological challenge (58). Recent studies using mice deficient in the DP receptor have suggested that PGD, production may be important in some aspects of asthma (59). In a model of asthma, when challenged with ovalbumin, DP_/_ mice showed reduced lymphocyte accumulation in the lung compared with wild-type controls, and in addition, failed to develop airway hyperreactivity. These data suggest that, in mice at least, DP receptors may play a role in airway hyperreactivity.

2.3.2 Prostaglandin £ PGE2 acts predominantly through EP receptors, which are abundantly distributed and are responsible for mediating the multitude of actions of PGE,. These include smooth muscle contraction and relaxation, stimulation and presyn-aptic inhibition of neurotransmitter release, neuronal sensitization, nociception, inhibition of lipolysis, and gastric acid secretion and modulation (positively and negatively) of water secretion. It was partly because of the sheer diversity of these responses and through the use of what are now known to be subtype specific ligands that it became clear that there was more than one subtype of EP receptor. It is now known that at least four receptor subtypes exist, and these are termed EPX, EP„ EP„ and EP, receptors (see Section 3).

The first characterized EP receptor subtype, the EPj receptor, is probably the least widely distributed of all the EP receptors. It predominantly mediates the smooth muscle contraction actions of PGE, and is present in guinea pig trachea, gastrointestinal tract, bladder, and uterus. This receptor subtype has also been found in other animal species: for example, in the bovine iris sphincter (60), dog gastric fundus (61), and human myometrium (62). These receptors also seem to play a role in inflammatory pain, with EPX antagonists currently being investigated as potential an-tinociceptiveagents (63).In situ hybridization studies have detected EPX receptor transcripts in the kidney, lung, and stomach of the mouse (64, 65). Recently, EPX receptor knockout mice have been used to suggest that endogenous EPX receptors are important in the mediation of pain perception and also the regulation of blood pressure (66). EP2 receptor-deficient mice were both healthy and fertile but showed reduced pain sensitivity, similar to that obtained following inhibition of COX with an NSAID. Similar results have also been reported in mice deficient in the IP receptor (67) (see Section 2.3.4), thus suggesting that EPX and IP receptors may represent possible novel targets for the treatment of inflammatory pain.

EP, receptors, first recognized because of the lack of effect of EPt receptor antagonists in blocking the relaxant effects of PGE,, have a more widespread distribution than the EP, receptor subtype. This receptor subtype also mediates a more diverse array of actions. For example, smooth muscle EP2 receptors always mediate smooth muscle relaxation [through increases in cyclic adenosine monophosphate (cAMP) through Gs (see Section 3.3)]. Epithelial EP, receptors regulate non-acid secretion, and in mast cells and basophils, they mediate inhibition of mediator release. Furthermore, they may also mediate afferent sensory nerve activation, including neuropeptide release. Northern blotting in the mouse has suggested the presence of EP, receptor mRNA in the ileum, thymus, spleen, heart, and uterus (68). This receptor subtype has also been localized

Example Prostanoid Medication

Figure 2.8. JOM-13 (blue) in the 8-opioid receptor binding pocket (stereoview). [Taken from Fig. 2.9 in H. I. Mosberg, Biopolymers (Peptide Science), 51, 426 (1999). Reprinted by permission of John Wiley & Sons.]

Figure 7.15. Rhodopsin crystal structure. Three-dimensional structure of rhodopsin based on X-ray crystallography (186). Note that all-/rans-retinal is protected from the intradiscal side by multiple structural elements, including several ■ß strands. Carbohydrates are in blue, 11-cis-retinal is in green, and helices are-in gray. Red shadows are added for esthetic reasons. This figure was generated by C. Behnke (University of Washington) and is reprinted with permission from Prog. Retin. Eve Res., 20, 469-521 (2001).

Figure 2.8. JOM-13 (blue) in the 8-opioid receptor binding pocket (stereoview). [Taken from Fig. 2.9 in H. I. Mosberg, Biopolymers (Peptide Science), 51, 426 (1999). Reprinted by permission of John Wiley & Sons.]

Figure 7.15. Rhodopsin crystal structure. Three-dimensional structure of rhodopsin based on X-ray crystallography (186). Note that all-/rans-retinal is protected from the intradiscal side by multiple structural elements, including several ■ß strands. Carbohydrates are in blue, 11-cis-retinal is in green, and helices are-in gray. Red shadows are added for esthetic reasons. This figure was generated by C. Behnke (University of Washington) and is reprinted with permission from Prog. Retin. Eve Res., 20, 469-521 (2001).

Figure 7.21. Schematic representation of DNA binding domains. Structures of DNA-binding complexes involving RXR and their dimer interfaces. The overall structures are shown on the left, with close-up views of the protein-protein interactions shown on the right. Dotted blue lines indicate hydrogen bonds between proteins or between proteins and the DNA spacing. The dotted surface indicates complementary van der Waals interactions. DNA sequences (cyan) are shown with their 5' ends pointing up, with base pairs belonging to the spacing element of the DRs shown schematically in red. In each case, protein-protein contacts are formed directly over the minor groove of the spacing, with several protein-DNA phosphate contacts stabilizing the assembly. The interacting amino acid side-chains are shown in green, with nitrogen atoms indicated in blue and oxygen atoms indicated in red. (a and b) The RXR-TR DBD heterodimeric complex with DR4; (c and d) the RXR DBD homodimeric complex with DR1, and (e and f) the KXR-DBD heterodimeric complex with DR1. Reprinted with permission from Curr. Opin. Struct. Biol., 11, 33-38 (2001).

Arg-48

Arg-48

Figure 7.22. Schematic representation of apo- and holo-RXR ligand binding domain. Helices 1-12 (H1-H12) are indicated. Helices indicated in yellow and red represents the apo- and holo-forms of the receptor, respectively, whereas helices indicated in blue and green are positioned similarly in both forms of the receptor. The arrows represent movement of the helices to accommodate 9-cis-RA in the binding pocket (indicated). This figure was kindly supplied by Dr. Pascal Egea.

Figure 7.22. Schematic representation of apo- and holo-RXR ligand binding domain. Helices 1-12 (H1-H12) are indicated. Helices indicated in yellow and red represents the apo- and holo-forms of the receptor, respectively, whereas helices indicated in blue and green are positioned similarly in both forms of the receptor. The arrows represent movement of the helices to accommodate 9-cis-RA in the binding pocket (indicated). This figure was kindly supplied by Dr. Pascal Egea.

Figure 11.2. Photolithographic process for on-chip synthesis of oligonucleotides. A: the steps in this process in two cycles of nucleotide addition. A lithographic mask and light source are shown in panel a that results in exposure of specific spots on the microarray chip. The light activates groups on the chip such that nucleotide coupling can occur only on the specific spots as shown in panel h. The nucleotide added is shown by green blocks and is thymidine for this exam-

of new spots to light in a second round of light activation of reactive groups. Panel d shows the next round of nucleotide addition in which the red blocks represent the next nucleotide, for exam adenosine. This process is repeated until all spots have the desired DNA sequence.

Figure 11.2. Photolithographic process for on-chip synthesis of oligonucleotides. A: the steps in this process in two cycles of nucleotide addition. A lithographic mask and light source are shown in panel a that results in exposure of specific spots on the microarray chip. The light activates groups on the chip such that nucleotide coupling can occur only on the specific spots as shown in panel h. The nucleotide added is shown by green blocks and is thymidine for this exam-

of new spots to light in a second round of light activation of reactive groups. Panel d shows the next round of nucleotide addition in which the red blocks represent the next nucleotide, for exam adenosine. This process is repeated until all spots have the desired DNA sequence.

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Figure 11.3. Hybridization of two differentially labeled cDNA sequences to arrayed DNA. This am shows a single array spot with probe DNA strands (in gray) bound to the chip. cDNA sequences from two different cell samples, one labeled with Cy3 (green) and one labeled with Cy5 ¡red), and hybridized to the spotted DNA.

Figure 11.3. Hybridization of two differentially labeled cDNA sequences to arrayed DNA. This am shows a single array spot with probe DNA strands (in gray) bound to the chip. cDNA sequences from two different cell samples, one labeled with Cy3 (green) and one labeled with Cy5 ¡red), and hybridized to the spotted DNA.

Figure 11.4. A spotted DNA array with two-color detection of hybridization.An example of a spotted DNA array (16 X 20 elements) is shown after hybridizaton with two differentially labeled cDNA preparations, Cy3 (pseudo-coloredgreen) and Cy5 (pseudo-colored red). The overlaying of the green and red images produces the image shown. The hue of each spot, ranging from green to red, indicates the relative expression level for the gene specific for each spot (image courtesy Packard Biochip Technologies).

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MDA-MB-435 MDA-N UACC-62 M14 SK-MEL-28 UACC-257 MALME-3M SK-MEL-2 SK-MEL-5 SNB-19 U251 HS 578T SF-539 SF-268 SNB-75 BT-549 SF-295 HOP-92 MDA-MB-231 ADR RES OVCAR-8 H0P-62 sn12c

Figure 11.5. Cluster analysis of the NCI 60 cell lines. Gene expression data from the Ross et al. (12) study was filtered to contain only genes with complete data for all cell lines and genes with the highest variance across cell lines. Only 548 genes were selected for this analysis based on genes with variance of greater than twice the variance for the entire dataset. The cluster distance or similarity calculation was based on the Pearson correlation coefficient, and clusters were linked by average linkage using the software SPSS. Most cell lines can be seen to cluster based on tissue of origin, shown by the labels on the left and the fraction of the total number of cell lines for each tissue. The abbreviations for tissue types are as follows: BRE, breast cancer; CNS, central nervous system cancers; COL, colon cancers; LEU, leukemias; MEL, melanomas; NSC, non-small cell lung cancers; OVA, ovarian cancers; PRO, prostate cancers; REN, renal cancers.

Figure 11.5. Cluster analysis of the NCI 60 cell lines. Gene expression data from the Ross et al. (12) study was filtered to contain only genes with complete data for all cell lines and genes with the highest variance across cell lines. Only 548 genes were selected for this analysis based on genes with variance of greater than twice the variance for the entire dataset. The cluster distance or similarity calculation was based on the Pearson correlation coefficient, and clusters were linked by average linkage using the software SPSS. Most cell lines can be seen to cluster based on tissue of origin, shown by the labels on the left and the fraction of the total number of cell lines for each tissue. The abbreviations for tissue types are as follows: BRE, breast cancer; CNS, central nervous system cancers; COL, colon cancers; LEU, leukemias; MEL, melanomas; NSC, non-small cell lung cancers; OVA, ovarian cancers; PRO, prostate cancers; REN, renal cancers.

to mouse glomeruli of the kidney (65). Furthermore, it seems that this receptor may be upregulated in response to cell stimulation. This has been demonstrated in a macrophage cell line in response to lipopolysaccharide (69). EP, receptor knockout mice have also been used! to further highlight the functions of PGE, in the cardiovascular system. For example, Kennedy and colleagues (70) examined the effects of PGE2 and receptor selective an-

alq^ in both wild-type and EP2 mice. In wild-type animals, the EP2 receptor selective agonist, butaprost, evoked a transient hypotension, which was not observed in mice deficient in the EP, receptor. However, the hypertensive effects of sulprostone were similar in r both groups of animals. Hence, the EP2 receptor may be important, in the mouse vascula-| ture at least, in mediating some of the vasodi-? latatory responses to PGE2.

EP3 receptors are probably the best studied |of all the EP receptors. They are present in ¡gastrointestinal, uterine, and vascular smooth jpuscle, where they cause smooth muscle contraction. In autonomic nerves they cause inhibition cf neurotransmitter release, and in adies, they inhibit lipolysis (71). In gastric tucosal cells, they inhibit gastric acid secre-n (72), and E series prostaglandins are well-piaracterized stimulators of mucus and bicar-ate secretion. Several PGE analogs have ieen developed to exploit this role as it was thought that EP3 receptor agonists might be Iseful in the treatment of gastric ulceration and also prophylaxis of the side effects associ-th long-term use of aspirin and other pSAIDs (see Section 4.2.4). In renal medulla ¡they regulate the inhibition of water reabsorp-ion (73). Distribution studies using Northern g and in situ hybridization reveal ¡aRNA expression in the kidney and uterus, as 11 as the stomach, spleen, brain, and lung 74). Furthermore, this receptor subtype has localized to the tubules in the medulla so the macula densa and distal tubules ithin the kidney itself (75). The receptor dis-ution has also been examined in neuronal ue, and has been reported in both dorsal ganglion and trigeminal ganglion tissue also within the brain in areas including hypothalamus, hippocampus, locus coer-leus, and raphe nuclei (75, 76). Within the hypothalamus, EP3 receptor transcripts have been detected in cells surrounding the orga-num vasculosalamina terminalis (OVLT), and receptor activation here has been associated with fever generation. E series prosta-glandins are potent fever inducers when injected into the brain, and their involvement in the central mediation of fever was proposed as early as 1970 (77). At the spinal cord level, it has been suggested that PGE, may induce thermal hyperalgesia through EP, and EP3 receptors (78). Furthermore, it has been proposed that brain-derived PGE,, at lower doses, produces hyperalgesia through its actions on EP3 receptors, whereas at higher doses, hypoalgesia is produced through actions on EP, receptors (79-81). Further evidence for involvement of EP3 in mediating the hyperalgesic effects of PGE2 comes from Xin and colleagues (82).They report that the in-tracerebroventricular injection of GR63799X (anEP3 receptor agonist) caused fever and hyperalgesia in rats, which could be blocked by an anti-sense, but not a mis-sense oligonucle-otide directed against the EP3 receptor. In addition, the use of EP knockout mice has suggested that only mice lacking the EP3 receptor failed to exhibit febrile responses to PGE2, in-terleukin-lj3, and lipopolysaccharide injections, thus providing further evidence for EP3 receptor involvement in mediating the febrile response (83).

The most recently characterized EP receptor, the EP, receptor, also shows a widespread distribution and is found in most tissues examined. It has been shown to exist in piglet saphenous vein (29) and also in the jugular vein of the rabbit, hamster uterus, and rat trachea (84-86).In the kidney, it is expressed in the glomerulus, mediating the effects of PGE2 on glomerular filtration. Within the brain, EP4 receptor mRNA has been found in the hypothalamus and lower brain stem. It has also been suggested that the EP4 receptor may be involved in bone remodeling and the induction of osteoclasts that are involved in bone resorption. For example, Sakuma and colleagues (87) found that osteoclast formation was stimulated most potently by PGE, analogs that display agonist activity at EP4 receptors. Although it has been suggested that EP4 receptors are important in maintaining a patent ductus arteriosus (88), EP4_/_ mice died from heart failure within 3 days of birth of pulmonary congestion and a fully patent ductus (89). This result remains to be explained, given the previously demonstrated di-latatory effects in the ductus of EP4 receptor activation.

It is also interesting to note that, given the clinical use of exogenous PGE, (dinoprostone) in cervical ripening and labor induction (see Section 5), the expression of the different EP receptors has been shown to alter with time in mice undergoing pseudopregnancy (90).

A role for E (and I) series prostaglandins in inflammatory pain is well established, not least because of the anti-nociceptive effects of NSAIDS, but also because of observations that exogenous prostaglandins can induce allo-dynia and hyperalgesia (91). In the periphery, sensitizing effects of PGE, have been recorded on several aspects of sensory nerve function, including ion channels and neuropeptide release. The EP receptors involved in mediating these effects remain poorly characterized, but these responses have been correlated with increases in cyclic AMP, which would be suggestive of EP,, EP4, or possibly, EP, receptor involvement (see Section 3.4). Further studies will hopefully address these issues and may aid in the design of future anti-nociceptive medicines.

2.3.3 Prostaglandin F2tt. Although PGF„ can act through EP and TP receptors, it is particularly potent at FP receptors. FP receptors are mostly concentrated in corpora lutea, where, in domestic animals, they mediate lu-teolysis stimulated by PGF,,. It has also been shown that, like uterine EP receptors, FP receptor expression changes during the oestrus cycle, becoming most abundant just before the luteal cells undergo apoptosis (92). In addition, FP receptor-deficient mice do not undergo parturition even after the administration of oxytocin. Therefore, it seems that FP receptor activation is required for normal parturition and labor in mice (93). This function of PGF,, is used widely in veterinary practice, and the receptors also seem to be present in this capacity in humans. However, initial hopes for FP receptor-specific agonists being useful in controlling human fertility (94) were dashed, because, unlike in animals, prosta-glandins are not central in the destruction of the corpus luteum. Hence, PGF2ct and its analogs have proved ineffective as human luteo-lytics. However these agents are used successfully in animal husbandry to help synchronize the oestrus cycles of farm animals.

Functional FP receptors have also been demonstrated in the myometrium of some rodents and also humans (95, 96). However, in dogs, FP receptor agonists are lethal—probably as a result of the presence of contractile FP receptors on airway smooth muscle (61). This has yet to be reported in any other species. FP receptors are also present in iris sphincter muscles, and the ocular actions mediated by these receptors have been exploited to lower intraocular pressure and in glaucoma treatment in humans.

Messenger RNA distribution studies have identified FP receptor transcripts in the uterus, further confirming the role of PGF2q. in this tissue. In situ hybridization has also revealed that expression in the ovary is confined to the large luteal cells of the corpus luteum (97). No labeling was observed in the ovarian follicle. Receptor mRNA was also found in the kidney, lung, heart, and stomach, and these studies are entirely consistent with the actions of PGF2q, in mediating luteolysis and mesangial cell contraction in the glomer-ulus of the kidney (98).

2.3.4 Prostaglandin I,. The primary role of prostacyclin is thought to be in the local control of vascular tone and also the inhibition of platelet aggregation (99). It is synthesized mostly by vascular endothelial cells. In agreement with this, IP receptors have been located in both platelets and vascular smooth muscle cells (100). IP receptor knockout mice, generated by homologous recombination, did not display a hypotensive response to the potent, selective IP receptor agonist, cicaprost (see Section 4), but their basal blood pressure was unaltered when compared with controls. The mice survived normally, but following endothelial damage, an increase in thrombosis was observed, lending weight to assertions that PGI2 acts as an antithrombotic agent (67).

IP receptors have also been found on nervous tissue. PGI, is a potent hyperalgesic, act ing on peripheral sensory neurones and may be more potent than PGE2 in this respect, at least in rodents (101). IP receptors have been localized to specific subsets of dorsal root ganglion (DRG) neurons (102).In situ hybridization signals were detected in 40% of DRG neurons (60% small; 15-25 jam) but not in glia. Seventy percent of IP-positive neurons also contained mRNA for preprotachykinin A (PPTA), a substance P precursor. In addition, around 25, 41, and 24% of IP-positive neurons co-expressed EP,, EP,, and EP, transcripts, respectively, suggesting a possible overlapping role of IP and EP receptors in mediating pain sensation.

Mice lacking IP receptors have also been used to suggest a role for PGI2 in inflammation. For example, following carageenan administration, IP_/_ mice exhibited paw swell-dig that was similar to that observed in wildtype mice treated with indomethacin (67). Similar studies have also further suggested a role for prostacyclin in mediating peripheral hyperalgesia (67).

2.3.5 Thromboxane A As far as is known, TXA2 acts exclusively through TP receptors. TP receptors exhibit a widespread distribu-in platelets and vascular smooth muscle, where they mediate platelet aggregation and also smooth muscle contraction (103,104). TP receptors are also present in airway smooth muscle (105), where they mediate broncho-constriction. Several studies have examined |the distribution of TP receptor mRNA. In the mouse, TP receptor transcripts have been detected abundantly in the thymus, spleen, and pung (106). In human tissues, TP receptor ¡transcripts were found in the placenta and the ihirg (107). Therefore, it seems that TP recep-rs are present in immune related organs ;(e.g., spleen and thymus) as well as those rich In smooth muscle, such as the lung. Ushikubi |md colleagues (108) have further investigated the TP receptor population in the thymus. Us-radioligand binding, they found TP recep-expression was greatest in immature D4-8- and CD4+8+ cells, and that recep-expression decreased as cells matured. ey also suggested the presence of functional receptors on immature thymocytes, as the addition of a TP receptor agonist induced ap-optosis in this cell population that was sensitive to TP receptor antagonism. Hence, in addition to roles in the cardiovascular and respiratory systems, TP receptors may also act in thymocyte development.

Recently, mice deficient in TP receptors have been generated to further elucidate potential roles of thromboxane A■ These TP_/_ mice exhibited an increased tendency to bleed and were unresponsive to the intravenous injection of the TP receptor specific agonist, U46619 (109). The increase in bleeding tendency has also been reported in human patients with a point mutation (Arg-60 to Leu) in the first cytoplasmic loop of their TP receptor (110). These patients display a defective platelet aggregatory response to TXA2, which further suggests a role for TP receptors in hemo-stasis.

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