EP3 Subtype Research Articles

Spinal inflammatory hyperalgesia is mediated by prostaglandin E receptors of the EP2 subtype

Blockade of prostaglandin (PG) production by COX inhibitors is the treatment of choice for inflammatory pain but is also prone to severe side effects. Identification of signaling elements downstream of COX inhibition, particularly of PG receptor subtypes responsible for pain sensitization (hyperalgesia), provides a strategy for better-tolerated analgesics. Here, we have identified PGE2 receptors of the EP2 receptor subtype as key signaling elements in spinal inflammatory hyperalgesia. Mice deficient in EP2 receptors (EP2-/- mice) completely lack spinal PGE2-evoked hyperalgesia. After a peripheral inflammatory stimulus, EP2-/- mice exhibit only short-lasting peripheral hyperalgesia but lack a second sustained hyperalgesic phase of spinal origin. Electrophysiological recordings identify diminished synaptic inhibition of excitatory dorsal horn neurons as the dominant source of EP2 receptor-dependent hyperalgesia. Our results thus demonstrate that inflammatory hyperalgesia can be treated by targeting of a single PG receptor subtype and provide a rational basis for new analgesic strategies going beyond COX inhibition.

Spinal inflammatory hyperalgesia is mediated by prostaglandin E receptors of the EP2 subtype

Blockade of prostaglandin (PG) production by COX inhibitors is the treatment of choice for inflammatory pain but is also prone to severe side effects. Identification of signaling elements downstream of COX inhibition, particularly of PG receptor subtypes responsible for pain sensitization (hyperalgesia), provides a strategy for better-tolerated analgesics. Here, we have identified PGE2 receptors of the EP2 receptor subtype as key signaling elements in spinal inflammatory hyperalgesia. Mice deficient in EP2 receptors (EP2–/– mice) completely lack spinal PGE2-evoked hyperalgesia. After a peripheral inflammatory stimulus, EP2–/– mice exhibit only short-lasting peripheral hyperalgesia but lack a second sustained hyperalgesic phase of spinal origin. Electrophysiological recordings identify diminished synaptic inhibition of excitatory dorsal horn neurons as the dominant source of EP2 receptor–dependent hyperalgesia. Our results thus demonstrate that inflammatory hyperalgesia can be treated by targeting of a single PG receptor subtype and provide a rational basis for new analgesic strategies going beyond COX inhibition.

Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1beta of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP(2), EP(3), and EP(4) receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1beta promoted the elaboration of G-CSF, which was augmented by PGE(2). Cicaprost (IP receptor agonist) was approximately equiactive with PGE(2), whereas PGD(2), PGF(2alpha), and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP(1) receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE(2), the EP receptor agonists 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3) selective), 17-phenyl-omega-trinor PGE(2) (EP(1) selective), ONO-AE1-259, and butaprost (both EP(2) selective) were full agonists at enhancing G-CSF release. AH 6809 (10 microM) and L-161,982 (2 microM), which can be used in HASMC as selective EP(2) and EP(4) receptor antagonists, respectively, failed to displace to the right the PGE(2) concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE(2)-induced G-CSF release. Augmentation of G-CSF release by PGE(2) was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE(2) facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP(2) and EP(4) prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.

Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in cultured rat hepatic stellate cells: Yoshimasa Kobayashi, Shigeki Koide, Hirotoshi Nakamura, Hamamatsu University School of Medicine, Hamamatsu Japan

Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in cultured rat hepatic stellate cells: Yoshimasa Kobayashi, Shigeki Koide, Hirotoshi Nakamura, Hamamatsu University School of Medicine, Hamamatsu Japan

Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP.

cAMP has been found to play a role in mediating the negative regulation of cell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese-hamster ovary) cells that express the EP2 subtype of PGE2 (prostaglandin E2) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE2 dose-dependently inhibited chemotaxis towards IGF-I (insulin-like growth factor-I), which is a Rac-dependent process, with the maximal 75% inhibition observed at 10(-8) M PGE2. EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositide 3-kinase or Akt in response to IGF-I, but potently and dose-dependently inhibited IGF-I-induced activation of cellular Rac activity and membrane ruffling. However, PGE2 failed to inhibit Val12-Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE2 inhibited PDGF (platelet-derived growth factor)-induced Rac activation and chemotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE2 on IGF-I-induced chemotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10(-8) M PGE2, and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3',5'-cyclic monophosphorothioate reduced PGE2 inhibition of Rac activation and chemotaxis. These results indicate that EP2 mediates Rac inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chemotaxis.

Prostaglandin E and F Receptor Expression and Myometrial Sensitivity at Labor Onset in the Sheep1

Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE(2) and PGF(2alpha) acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1-4) expression. Thus, the relative expression of FP and EP1-4 may determine the responsiveness to PGE(2) and PGF(2alpha). The aims of this study were to characterize the expression of EP1-4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE(2) and PGF(2alpha). This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.

Lactams as EP4Prostanoid Receptor Agonists. 3. Discovery ofN-Ethylbenzoic Acid 2-Pyrrolidinones as Subtype Selective Agents

Two distinct synthetic schemes were applied to access heteroatom-containing alpha-chain lactams or lactams terminated as aryl acids. The latter lactams were devised using a pharmacophore for EP(4) receptor activity. gamma-Lactams were characterized for their prostanoid EP receptor affinities and EP(4) activity and found to be selective for the EP(2) and EP(4) receptors or selective for the EP(4) subtype. Benzoic acid 17 displayed enhanced in vivo exposure relative to 3.

Microarray analysis in the HSV-1 latently infected mouse trigeminal ganglion.

To review our previous studies regarding alterations in gene expression in HSV-1 latently infected mouse trigeminal ganglia (TGs) following treatment with immunosuppressants and hyperthermia. Uninfected and HSV-1 latently infected mice were treated with immunosuppressants or heat stressed (43 degrees C for 10 minutes). In the immunosuppressant study, 4 groups of animals were examined: (1) uninfected, not treated; (2) uninfected, drug-treated; (3) latently infected, not treated; and (4) latently infected, drug-treated. In the hyperthermia study, TG from 6 groups of mice were studied: (1) uninfected, not stressed; (2) uninfected, heat-stressed; killed at 6 hours after hyperthermia; (3) uninfected, heat-stressed, killed at 24 hours after hyperthermia; (4) latently infected, not stressed; (5) latently infected, heat-stressed, killed at 6 hours after hyperthermia; and (6) latently infected, heat-stressed, killed at 24 hours after hyperthermia. PolyA mRNA from the TGs of each group was reverse-transcribed, labeled with P, incubated on a gene array membrane, and analyzed by phosphorimaging. As a comparison and to confirm microarray results, semiquantitative RT-PCR for selected genes was also performed. The immunosuppressive drugs significantly increased expression of two genes--calpactin 1 light chain and guanine nucleotide-binding protein alpha stimulating activity polypeptide (GNAS)--in the ganglia of uninfected mice compared with untreated, uninfected mice. Ten genes were shown to be significantly increased in the latent TGs from mice treated with the immunosuppressants compared with latently infected untreated mice. These genes were prostaglandin E2 receptor EP4 subtype (PTGER4), insulin promoter factor 1 (IPF1), glutathione S-transferase mu2, cyclin D2, peripherin, plasma glutathione peroxidase, methyl CpG-binding protein 2, retinal S-antigen, ErbB2 protooncogene, and GNAS. Eight genes were shown to be significantly decreased in the HSV-1 latent TGs treated with the drugs compared with untreated latent mice. These genes were peripheral myelin protein 22, decorin, transcription factor AP-1, dystroglycan 1, myelin protein zero, mitogen-activated protein kinase 3, prothymosin beta4, and brain lipid-binding protein. The results obtained by semiquantitative RT-PCR results were similar to those obtained by microarray analysis. Six hours after heat stress, the genes whose expression was altered included the FK506-binding protein gene (decreased), the T-complex protein 1alpha subunit gene (increased), and the 94-kDa glucose-regulated protein gene (increased in uninfected TG, decreased in infected TG). Heat stress increased expression of the DNA excision repair protein ERCC5 gene 24 hours after the treatment. Genes previously reported to exhibit increased transcription 1 hour after heat stress did not continue to show significant transcriptional activation at 6 or 24 hours. Those genes whose expression is altered by immunosuppressive drug treatment may play an important role in ocular HSV-1 recurrence. Changes in gene expression in the prostaglandin pathway, a transcription factor, and an enzyme in the cell cycle are considered of special importance for HSV-1 reactivation by immunosuppression. Altered gene expression at 6 and 24 hours after heat stress was different from previously reported changes in gene expression 1 hour after hyperthermia in HSV-1 latently infected mice.

Prostaglandin E 2 receptor subtype EP-2 is not involved in the induction of non-pregnant guinea pig uterine contractions associated with terminal pregnancy

Prostaglandin E 2 (PGE 2) exerts its biological effects through 4 different receptor subtypes, EP-1, EP-2, EP-3, and EP-4. Recently we have demonstrated the importance of the prostaglandin E 2 receptor subtype EP-2 in the healing of bone defects and fractures. This discovery led to the identification of CP-533,536, an EP-2 selective agonist, a promising therapeutic alternative for the enhancement of bone healing and the treatment of fractures (J Bone Miner Res 18 (2003) 2033). PGE 2 has a myriad of effects throughout the body including the induction of uterine contractions, which results in termination of pregnancies. Our objective in this study was to determine the role of the EP-2 receptor and specifically that of CP-533,536, an EP-2 specific agonist, to induce uterine contractions and terminate pregnancy in guinea pigs, an animal model of human pregnancy. Preliminary experiments confirmed earlier reports that the guinea pig uterus was more sensitive than that of the rat. The guinea pig uterus contains the four PGE 2 receptor subtypes, and ex vivo treatment of the uterus with PGE 2 as expected causes profound uterine contractions. However, using receptor selective prostaglandin agonists including CP-533,536 we showed that the EP-1 and 3 receptors not the EP-2 receptor is responsible for the induction of uterine contractions of PGE 2. Further, CP-533,536 did not antagonize the ability of PGE 2 to induce uterine contractions in this model.

DNA microarray analysis of gene expression profiles in deep endometriosis using laser capture microdissection.

Endometriosis, a common gynecological disorder that causes infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites. However, despite extensive studies its etiology and pathogenesis are not completely understood. Differentially expressed genes were investigated in epithelial and stromal cells from deep endometriosis and matched eutopic endometrium using cDNA microarrays and laser capture microdissection. Validation of results of several up- and down-regulated genes was performed by quantitative real-time RT-PCR. Our data showed that platelet-derived growth factor receptor alpha (PDGFRA), protein kinase C beta1 (PKC beta1) and janus kinase 1 (JAK1) were upregulated, and Sprouty2 and mitogen-activated protein kinase kinase 7 (MKK7) were downregulated in endometriosis stromal cells, suggesting the involvement of the RAS/RAF/MAPK signaling pathway through PDGFRA in endometriosis pathophysiology. In addition, two potential negative regulators of aromatase expression, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 receptor subtype EP3 (PGE2EP3), were downregulated in endometriosis epithelial cells, which might result in increased local production of estrogen in endometriosis epithelial cells. Furthermore, three potential candidate genes that might be involved in endometriosis related pain were identified: tyrosine kinase receptor B (TRkB) in endometriosis epithelial cells, and serotonin transporter (5HTT) and mu opioid receptor (MOR) in endometriosis stromal cells were all upregulated. One of the candidate genes, MOR, may be involved in a defective immune system in endometriosis. This study has provided new insights into endometriosis pathophysiology.

Regulation of the Na,K-ATPase in MDCK cells by prostaglandin E 1: a role for calcium as well as cAMP

Prostaglandins (PGs) play a significant role in the regulation of sodium reabsorption by the kidney, in addition to accumulating during inflammation as well as in several solid tumors. Previously, we presented evidence indicating that prostaglandin E 1 (PGE 1), a supplement in the serum-free medium for MDCK cells, increases the activity of the Na,K-ATPase in MDCK cells, in addition to its growth stimulatory effect [J. Cell. Physiol. 151 (1992) 337]. This report defines the molecular mechanisms, and signaling pathways responsible for the increased Na,K-ATPase activity. Our results indicate that the increased activity of the Na,K-ATPase in MDCK monolayers treated with either PGE 1 or 8Bromocyclic AMP (8Br-cAMP) can be attributed to an increase in the rate of biosynthesis of the Na,K-ATPase, and an increase in the levels of Na,K-ATPase α and β subunit mRNAs. As β subunit mRNA increased to a larger extent than α subunit mRNA, transient transfection studies were conducted using a human β1 promoter/luciferase construct [Nucleic Acids Res. 21 (1993) 2619]. While an 8Br-cAMP stimulation was observed (suggesting the involvement of cAMP), our results also suggest that the observed PGE 1 stimulation could be explained by the involvement of Ca 2+ as well protein kinase C (PKC). Consistent with the involvement of Ca 2+, TMB-8 (which inhibits Ca 2+ efflux from intracellular stores) inhibited the PGE 1 stimulation. Moreover, PGE 1 was observed to stimulate the translocation of PKC β1 from the soluble to the particulate fraction. The translocation of PKC, the PGE 1 stimulation of transcription, and the PGE 1-mediated increase in the β subunit mRNA level were all inhibited by the PKC inhibitor Gö6989. These results can be explained by the involvement of two classes of cell surface receptors in mediating the PGE 1 stimulation, including the EP1subtype (which activates phospholipase C), as well as the EP2 subtype (which activates adenylate cyclase).

Gene mutations and altered gene expression in azoxymethane-induced colon carcinogenesis in rodents.

Studies of colon carcinogenesis in animal models are very useful to elucidate mechanisms and provide pointers to potential prevention approaches in the human situation. In the rat colon carcinogenesis model induced by azoxymethane (AOM), we have documented frequent mutations of specific genes. K-ras mutations at codon 12 were found to be frequent in hyperplastic aberrant crypt foci (ACF) and large adenocarcinomas. In addition, mutations of the beta-catenin gene in its GSK-3beta phosphorylation consensus motif could also be identified in many adenomas and adenocarcinomas, and altered cellular localization of beta-catenin protein was observed in all of the dysplastic ACF, adenomas and adenocarcinomas examined, indicating that activation of Wnt signaling by accumulation of beta-catenin is a major mechanism in the AOM-induced colon carcinogenesis model. Frequent gene mutations of beta-catenin and altered cellular localization of the protein are also features of AOM-induced colon tumors in mice. Expression of enzymes associated with inflammation, such as inducible nitric oxide synthase (iNOS) and the inducible type of cyclooxygenase (COX), COX-2, is increased in AOM-induced rat colon carcinogenesis, and overproduction of nitric oxide (NO) and prostaglandins is considered to be involved in colon tumor development. We have demonstrated that increased expression of iNOS is an early and important event occurring in step with beta-catenin alteration in rat colon carcinogenesis. Activation of K-ras was also found to be involved in up-regulation of iNOS in the presence of inflammatory stimuli. In addition, expression levels of prostaglandin E(2) (PGE(2)) receptors may be altered in colon cancers. For example, the EP(1) and EP(2) subtypes have been shown to be up-regulated and EP(3) down-regulated in AOM-induced colon cancers in rats and mice. EP(1) and EP(4) appear to be involved in ACF formation, while alteration in EP(2) and EP(3) is considered to contribute to later steps in colon carcinogenesis. Increased expression of some other gene products, such as the targets of Wnt/beta-catenin signaling, have also been reported. The further accumulation of data with this chemically-induced animal colon carcinogenesis model should provide useful information for understanding colorectal neoplasia in man.

Prostaglandin E2 protects the heart from ischemia-reperfusion injury via its receptor subtype EP4.

In the heart with acute myocardial infarction, production of prostaglandin (PG) E2 increases significantly. In addition, several subtypes of PGE2 receptors (EPs) have been reported to be expressed in the heart. The role of PGE2 in cardiac ischemia-reperfusion (I/R) injury, however, remains unknown. We intended to clarify the role of PGE2 via EP4, an EP subtype, in I/R injury using mice lacking EP4 (EP4-/- mice). In murine cardiac ventricle, competitive reverse transcription-polymerase chain reaction revealed the highest expression level of EP4 mRNA among EP mRNAs. EP4-/- mice had larger infarct size than wild-type mice in a model of I/R; the left anterior descending coronary artery was occluded for 1 hour, followed by 24 hours of reperfusion. In addition, isolated EP4-/- hearts perfused according to the Langendorff technique had greater functional and biochemical derangements in response to I/R than wild-type hearts. In vitro, AE1-329, an EP4 agonist, raised cAMP concentration remarkably in noncardiomyocytes, whereas the action was weak in cardiomyocytes. When 4819-CD, another EP4 agonist, was administered 1 hour before coronary occlusion, it reduced infarct size significantly in wild-type mice. Notably, a similar cardioprotective effect was observed even when it was administered 50 minutes after coronary occlusion. Both endogenous PGE2 and an exogenous EP4 agonist protect the heart from I/R injury via EP4. The potent cardioprotective effects of 4819-CD suggest that the compound would be useful for treatment of acute myocardial infarction.

Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site.

hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.

PGE2 stimulates Cl- secretion in murine M-1 cortical collecting duct cells in an autocrine manner.

Prostaglandin E2 (PGE2) is thought to be an important modulator of renal ion and water transport, but its effects remain complex and incompletely understood. Here we examined the effects of PGE2 on transepithelial ion transport of M-1 mouse cortical collecting duct cells using short-circuit current (ISC) measurements. Basolateral addition of PGE2 (1 microM) produced a transient peak increase in ISC of 6.3+/-0.8 microA cm(-2) (n=11), followed by a sustained plateau. The PGE2-evoked response was preserved in the presence of 100 micro M apical amiloride with an average peak increase of 10.6+/-1.0 microA cm(-2) (n=23). However, it was greatly diminished in both the presence of apical diphenylamine-2-carboxylic acid (DPC, 1 mM) and the absence of extracellular Cl-, indicating that Cl- secretion had been stimulated. Basolateral PGE2 induced a concentration dependent response, with an EC50 of about 8 nM. Apical addition of PGE2 elicited an ISC response similar to that observed with basolateral PGE2. Furthermore, apical exposure to arachidonic acid (AA) produced a similar increase in ISC, which could be prevented by the cyclooxygenase inhibitor indomethacin, while AA failed to exert an additional effect in the presence of PGE2. Using RT-PCR, we confirmed the expression of the PGE2 (EP) receptor subtypes EP1, EP3 and EP4 but not of EP2 in cultured M-1 CCD cells. We conclude that M-1 cells express functional cyclooxygenase activity and can generate PGE2 which acts in an autocrine manner, causing Cl- secretion.

EP1- and FP-mediated cross-desensitization of the alpha (alpha) and beta (beta) isoforms of the human thromboxane A2 receptor.

1. Heterologous desensitization or intermolecular cross-talk plays a critical role in regulating intracellular signalling by diverse members of the G-protein-coupled receptor superfamily. We have previously established that the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) undergo differential desensitization of signalling in response to 17 phenyl trinor prostaglandin (PG)E(2), an agonist of the EP(1) subtype of the PGE(2) receptor (EP) family. 2. Herein, we investigated the molecular basis of TPalpha and TPbeta desensitization in human embryonic kidney (HEK) 293 cells and in renal mesangial cells in response to 17 phenyl trinor PGE(2) and in response to the PGF(2alpha) receptor (FP) agonist PGF(2alpha), and sought to identify the target site(s) of those desensitizations. 3. Our results demonstrated that TPalpha and TPbeta receptors are subject to desensitization in response to both EP(1) and FP receptor activation and that these effects are mediated by direct protein kinase (PK)C phosphorylation of the individual TP isoforms within their unique carboxyl-terminal (C)-tail domains. 4. Moreover, deletion/site-directed mutagenesis and metabolic labelling studies identified Thr(337), within TPalpha, and Thr(399), within TPbeta, as the specific target residues for PKC phosphorylation and EP(1)- and FP-mediated desensitization of TPalpha and TPbeta signalling, respectively. 5. Hence, in conclusion, while the TPalpha and TPbeta diverge within their C-tail domains, they have evolved to share a similar mechanism of PKC-induced phosphorylation and desensitization in response to EP(1) and FP receptor activation, though it occurs at sites unique to the individual TP isoforms.

Role of prostaglandin receptor subtype EP 1 in prostaglandin E 2-induced nociceptive transmission in the rat spinal dorsal horn

It has been indicated that prostaglandin E 2 (PGE 2) and the receptor for PGE 2 (EP receptor) are key factors contributing to the facilitated generation of nociception. This study was designed to investigate the roles of PGE 2 and EP 1 receptors in the spinal cord in the nociceptive transmission, using behavioral and intracellular calcium ion concentration ([Ca 2+] i) assays and in situ hybridization. Experiments were conducted on Sprague–Dawley rats. In behavioral assays, withdrawal thresholds to mechanical stimuli were evaluated using von Frey filament. The effect of an intrathecally administered selective EP 1 antagonist, 6-[(2 S,3 S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo[2.2.2]octan-2-yl]-5 Z-hexenoic acid (ONO-8711), on the intrathecal PGE 2-induced hyperalgesia was examined. In [Ca 2+] i assays, we measured [Ca 2+] i in the dorsal horn of spinal cord slices and examined the effects of PGE 2 and ONO-8711 perfusion on the [Ca 2+] i changes. In situ hybridization using EP 1 digoxigenin probe was performed on the slice sections of the lumbar spinal cord and bilateral L 4 and L 5 dorsal root ganglions (DRGs). Mechanical hyperalgesia was observed after intrathecal PGE 2 administration. Intrathecal administration of ONO-8711 attenuated the PGE 2-induced mechanical hyperalgesia in a dose- and time-dependent manner. Perfusion of ONO-8711 markedly suppressed PGE 2-induced [Ca 2+] i increment in laminae II–VI in dorsal horn of the spinal cord slice. Moreover, in situ hybridization revealed EP 1 hybridization signals in the DRG neurons, but not in the spinal cord. The results of this study suggested that spinal PGE 2 activates the EP 1 receptors existing on the central terminals of primary afferents, subsequently increasing in [Ca 2+] i in the spinal dorsal horn, which are involved in the mechanisms of spinal PGE 2-induced nociceptive transmission.

Characterization of the PGI2/IP system in cultured rat mesangial cells

Mesangial cells play an important role in glomerular function. They are an important source of cyclooxygenase (COX)-derived arachidonic acid metabolites, including prostaglandin E 2 and prostacyclin. Prostacyclin receptor (IP) mRNA was amplified from cultured mesangial cell total RNA by RT-PCR. While the prostaglandin E 2 receptor subtype EP 2 was not detected, EP 1,3,4 mRNA was amplified. Also, IP protein was noted in mesangial cells, proximal tubules, inner medullary collecting ducts, and the inner and outer medulla. But no protein was detected in whole cortex preparations. Prostacyclin analogues: cicaprost and iloprost, increased cAMP levels in mesangial cells. On the other hand, arginine-vasopressin and angiotensin II increased intracellular calcium in mesangial cells, but cicaprost, iloprost and prostaglandin E 2 had no effect. Moreover, a 50% inhibition of cicaprost- and iloprost-cAMP stimulation was observed upon mesangial cell exposure to 25 and 35 mM glucose for 5 days. But no change in IP mRNA was observed at any glucose concentration or time exposure. Although 25 mM glucose had no effect on COX-1 protein levels, COX-2 was increased up to 50%. In contrast, PGIS levels were reduced by 50%. Thus, we conclude that the prostacyclin/IP system is present in cultured rat mesangial cells, coupling to a cAMP stimulatory pathway. High glucose altered both enzymes in the PGI 2 synthesis pathway, increasing COX-2 but reducing PGIS. In addition, glucose diminished the cAMP response to prostacyclin analogues. Therefore, glucose attenuates the PGI 2/IP system in cultured rat mesangial cells.

Inhibition of human glioma cell growth by a PHS-2 inhibitor, NS398, and a prostaglandin E receptor subtype EP1-selective antagonist, SC51089.

Prostaglandin H synthase (PHS) is a key enzyme in the synthesis of prostaglandins (PGs). Recently, enhanced expression of PHS-2 in brain tumors and the correlation between the PHS-2 level and the histopathological grade of glioma has been reported. Furthermore, in vitro inhibition of glioma cell growth by a specific PHS-2 inhibitor, NS398, has been demonstrated. It has also been shown that prostaglandin E2 (PGE2) contributes to colon carcinogenesis by binding to the prostaglandin E receptor subtype EP1. We therefore evaluated the effects of NS398 and two EP1 antagonists, SC51089 and AH6809, on glioma cell lines. To evaluate mechanisms of NS398's action, two glioma cell lines, a PHS-2-positive cell line (KMG4) and a PHS-2-deficient cell line (A 172), were used. NS398 inhibited both the anchorage-dependent and -independent growth of glioma cell lines regardless of PHS-2 expression, suggesting that some PHS-2-independent mechanisms underlie the antineoplastic effect of NS398. However, the antineoplastic effect was attenuated by the addition of PGE2, which is one of the main products of PHS, suggesting the predominant mechanism is PHS-dependent. The EP1 antagonists, SC51089 and AH6809, inhibited the growth of glioma cell lines in vitro. Furthermore, NS398 or SC51089 slowed tumor growth in vivo, which was assessed using KMG4 tumor xenografts on SCID mice. PHS-2 inhibitors and EP1 antagonists might be useful in the prevention and/or treatment of glioma.

Cooperation of Two Subtypes of PGE2 Receptor, Gi Coupled EP3 and Gs Coupled EP2 or EP4 Subtype

AbstractFor Abstract see ChemInform Abstract in Full Text.