Selectivity profiling of the novel EP2 receptor antagonist,PF-04418948, in functional bioassay systems: atypical affinity at the guinea pig EP2 receptor

BACKGROUND AND PURPOSE

Understanding the role of the EP₂ receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP₂ receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP₂ receptor over other EP receptors using a range of isolated tissue systems.

EXPERIMENTAL APPROACH

PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP₁); ONO-AE1-259 and PGE₂- induced relaxation of mouse and guinea pig trachea (EP₂); PGE₂-induced depolarization of guinea pig isolated vagus (EP₃); PGE₂-induced relaxation of human and rat trachea (EP₄). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays.

KEY RESULTS

In bioassay systems, where assessment of potency/selectivity is made against the ‘native’ receptor, PF-04418948 only acted as an antagonist of EP₂ receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE₂ respectively. However, the affinity of PF-04418948 was not equal in the two preparations.

CONCLUSIONS AND IMPLICATIONS

Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP₂-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP₂ receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE₂ and the role of EP₂ receptors in health and disease.     

Prostanoid EP1 receptor as the target of (?)-epigallocatechin-3-gallate in suppressing hepatocellular carcinoma cells in vitro

Aim:

To investigate the effects of (−)-epigallocatechin-3-gallate (EGCG), an active compound in green tea, on prostaglandin E₂ (PGE₂)-induced proliferation and migration, and the expression of prostanoid EP₁ receptors in hepatocellular carcinoma (HCC) cells.

Methods:

HCC cell line HepG2, human hepatoma cell lines MHCC-97L, MHCC-97H and human hepatocyte cell line L02 were used. Cell viability was analyzed using MTT assay. PGE₂ production was determined with immunoassay. Wound healing assay and transwell filter assay were employed to assess the extent of HCC cell migration. The expression of EP₁ receptor and Gq protein were examined using Western blot assay.

Results:

PGE₂ (4-40000 nmol/L) or the EP₁ receptor agonist ONO-DI-004 (400-4000 nmol/L) increased the viability and migration of HepG2 cells in concentration-dependent manners. EGCG (100 μg/mL) significantly inhibited the viability and migration of HepG2 cells induced by PGE₂ or ONO-DI-004. HepG2 cells secreted an abundant amount of PGE₂ into the medium, and EGCG (100 μg/mL) significantly inhibited the PGE₂production and EP₁ receptor expression in HepG2 cells. EGCG (100 μg/mL) also inhibited the viability of MHCC-97L cells, but not that of MHCC-97H cells. Both EGCG (100 μg/mL) and EP₁ receptor antagonist ONO-8711 inhibited PGE₂ 4 μmol/L and ONO-DI-004 400 nmol/L-induced growth and migration of HepG2 cells. Both EGCG (100 μg/mL) and ONO-8711 210 nmol/L inhibited PGE₂- and ONO-DI-004-induced EP₁ expression. EGCG and ONO-8711 had synergistic effects in inhibiting EP₁ receptor expression. PGE₂, ONO-DI-004, ONO-8711, and EGCG had no effects on Gq expression in HepG2 cells, respectively.

Conclusion:

These findings suggest that the anti-HCC effects of EGCG might be mediated, at least partially, through the suppressing EP₁ receptor expression and PGE₂ production.     

BGC20-1531, a novel,potent and selective prostanoid EP4 receptor antagonist: a putative new treatment for migraine headache

Background and purpose:

Prostanoid EP₄ receptor antagonists may have therapeutic utility in the treatment of migraine since EP₄ receptors have been shown to be involved in prostaglandin (PG)E₂-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP₄ receptor antagonist.

Experimental approach:

BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP₄ receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo.

Key results:

BGC20-1531 exhibited high affinity at recombinant human EP₄ receptors expressed in cell lines (pK_B 7.6) and native EP₄ receptors in human cerebral and meningeal artery (pK_B 7.6–7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE₂-induced vasodilatation of human middle cerebral (pK_B 7.8) and meningeal (pK_B 7.6) arteries in vitro, but had no effect on responses induced by PGE₂ on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1–10 mg·kg⁻¹ i.v.) caused a dose-dependent antagonism of the PGE₂-induced increase in canine carotid blood flow in vivo.

Conclusions and implications:

BGC20-1531 is a potent and selective antagonist at EP₄ receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache.     

Interaction of prostanoid EP₃ and TP receptors in guinea-pig isolated aorta: contractile self-synergism of 11-deoxy-16,16-dimethyl PGE₂

BACKGROUND AND PURPOSE

Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E₂ (DX-DM PGE₂) on pig cerebral artery when used as a selective EP₃ receptor agonist. This study investigated the selectivity profile of DX-DM PGE₂, focusing on the interaction between its EP₃ and TP (thromboxane A₂-like) agonist activities.

EXPERIMENTAL APPROACH

Contraction of guinea-pig trachea (EP₁ system) and aorta (EP₃ and TP systems) was measured in conventional organ baths.

KEY RESULTS

Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE₂ (EP₃ agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE₂ induced strong contraction, which on the basis of treatment with (DG)-3ap (EP₃ antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP₃ and TP receptors. EP₃/TP self-synergism also accounted for contraction induced by PGF_2α and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE₂ also showed significant EP₁ agonism on guinea-pig trachea as defined by the EP₁ antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM.

CONCLUSIONS AND IMPLICATIONS

EP₃/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP₃ agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP₃ system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

Background and Purpose

Hyper-nociceptive PGE₂ EP₄ receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other G_s-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity.

Experimental Approach

Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures.

Key Results

Pharmacological analysis showed the presence of G_s-coupled β₂-adrenoceptors and CGRP receptors, but not β₁-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation.

Conclusions and Implications

G_s-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP₄ and IP receptors, but not β₂-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses.     

A novel antagonist of the prostaglandin E2 EP4 receptor inhibits Th1 differentiation and Th17 expansion and is orally active in arthritis models

Background and purpose:

Rheumatoid arthritis (RA) is an autoimmune disorder involving subsets of activated T cells, in particular T helper (Th) 1 and Th17 cells, which infiltrate and damage tissues and induce inflammation. Prostaglandin E₂ (PGE₂) enhances the Th17 response, exacerbates collagen-induced arthritis (CIA) and promotes inflammatory pain. The current study investigated whether selective antagonism of the PGE₂ EP₄ receptor would suppress Th1/Th17 cell development and inflammatory arthritis in animal models of RA.

Experimental approach:

Effects of PGE₂ and a novel EP₄ receptor antagonist ER-819762 on Th1 differentiation, interleukin-23 (IL-23) production by dendritic cells (DCs), and Th17 development were assessed in vitro. The effect of ER-819762 was evaluated in CIA and glucose-6-phosphate isomerase (GPI)-induced arthritis models. In addition, the effects of ER-819762 on pain were evaluated in a model of chronic inflammatory pain induced by complete Freund''s adjuvant (CFA) in the rat.

Key results:

Stimulation of the EP₄ receptor enhanced Th1 differentiation via phosphatidylinositol 3 kinase signalling, selectively promoted Th17 cell expansion, and induced IL-23 secretion by activated DCs, effects suppressed by ER-819762 or anti-PGE₂ antibody. Oral administration of ER-19762 suppressed Th1 and Th17 cytokine production, suppressed disease in collagen- and GPI-induced arthritis in mice, and suppressed CFA-induced inflammatory pain in rats.

Conclusion and implications:

PGE₂ stimulates EP₄ receptors to promote Th1 differentiation and Th17 expansion and is critically involved in development of arthritis in two animal models. Selective suppression of EP₄ receptor signalling may have therapeutic value in RA both by modifying inflammatory arthritis and by relieving pain.     

Lubiprostone targets prostanoid EP₄ receptors in ovine airways

BACKGROUND AND PURPOSE

Lubiprostone, a prostaglandin E₁ derivative, is reported to activate ClC-2 chloride channels located in the apical membranes of a number of transporting epithelia. Lack of functioning CFTR chloride channels in epithelia is responsible for the genetic disease cystic fibrosis, therefore, surrogate channels that can operate independently of CFTR are of interest. This study explores the target receptor(s) for lubiprostone in airway epithelium.

EXPERIMENTAL APPROACH

All experiments were performed on the ventral tracheal epithelium of sheep. Epithelia were used to measure anion secretion from the apical surface as short circuit current or as fluid secretion from individual airway submucosal glands, using an optical method.

KEY RESULTS

The EP₄ antagonists L-161982 and {"type":"entrez-nucleotide","attrs":{"text":"GW627368","term_id":"290498219","term_text":"GW627368"}}GW627368 inhibited short circuit current responses to lubiprostone, while EP_1,2&3 receptor antagonists were without effect. Similarly, lubiprostone induced secretion in airway submucosal glands was inhibited by L-161982. L-161982 effectively competed with lubiprostone with a K_d value of 0.058 µM, close to its value for binding to human EP₄ receptors (0.024 µM). The selective EP₄ agonist L-902688 and lubiprostone behaved similarly with respect to EP₄ receptor antagonists. Results of experiments with H89, a protein kinase A inhibitor, were consistent with lubiprostone acting through a G_s-protein coupled EP₄ receptor/cAMP cascade.

CONCLUSIONS AND IMPLICATIONS

Lubiprostone-induced short-circuit currents and submucosal gland secretions were inhibited by selective EP₄ receptor antagonists. The results suggest EP₄ receptor activation by lubiprostone triggers cAMP production necessary for CFTR activation and the secretory responses, a possibility precluded in CF tissues.     

PGE(2) -EP(2) signalling in endothelium is activated by haemodynamic stress and induces cerebral aneurysm through an amplifying loop via NF-κB

BACKGROUND AND PURPOSE

Cerebral aneurysm is a frequent cerebrovascular event and a major cause of fatal subarachnoid haemorrhage, but there is no medical treatment for this condition. Haemodynamic stress and, recently, chronic inflammation have been proposed as major causes of cerebral aneurysm. Nevertheless, links between haemodynamic stress and chronic inflammation remain ill-defined, and to clarify such links, we evaluated the effects of prostaglandin E₂ (PGE₂), a mediator of inflammation, on the formation of cerebral aneurysms.

EXPERIMENTAL APPROACH

Expression of COX and prostaglandin E synthase (PGES) and PGE receptors were examined in human and rodent cerebral aneurysm. The incidence, size and inflammation of cerebral aneurysms were evaluated in rats treated with COX-2 inhibitors and mice lacking each prostaglandin receptor. Effects of shear stress and PGE receptor signalling on expression of pro-inflammatory molecules were studied in primary cultures of human endothelial cells (ECs).

KEY RESULTS

COX-2, microsomal PGES-1 and prostaglandin E receptor 2 (EP₂) were induced in ECs in the walls of cerebral aneurysms. Shear stress applied to primary ECs induced COX-2 and EP₂. Inhibition or loss of COX-2 or EP₂in vivo attenuated each other''s expression, suppressed nuclear factor κB (NF-κB)-mediated chronic inflammation and reduced incidence of cerebral aneurysm. EP₂ stimulation in primary ECs induced NF-κB activation and expression of the chemokine (C-C motif) ligand 2, essential for cerebral aneurysm.

CONCLUSIONS AND IMPLICATIONS

These results suggest that shear stress activated PGE₂-EP₂ pathway in ECs and amplified chronic inflammation via NF-κB. We propose EP₂ as a therapeutic target in cerebral aneurysm.     

Characterization of the prostanoid receptor(s) on human blood monocytes at which prostaglandin E2 inhibits lipopolysaccharide-induced tumour necrosis factor-α generation

1. The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-α (TNFα) generation from human peripheral blood monocytes was classified by use of naturally occurring and synthetic prostanoid agonists and antagonists.
2. In human monocytes that were adherent to plastic, neither prostaglandin D₂ (PGD₂), prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNFα-like immunoreactivity, as assessed with a specific ELISA, indicating the absence of excitatory prostanoid receptors on these cells.
3. Exposure of human monocytes to LPS (3 ng ml⁻¹, ∼ EC₈₄) resulted in a time-dependent elaboration of TNFα which was suppressed in cells pretreated with prostaglandin E₁ (PGE₁), PGE₂ and cicaprost. This effect was concentration-dependent with mean pIC₅₀ values of 7.14, 7.34 and 8.00 for PGE₁, PGE₂ and cicaprost, respectively. PGD₂, PGF_2α and U-46619 failed to inhibit the generation of TNFα at concentrations up to 10 μM.
4. With respect to PGE₂, the EP-receptor agonists, 16,16-dimethyl PGE₂ (non-selective), misoprostol (EP₂/EP₃-selective), 11-deoxy PGE₁ (EP₂-selective) and butaprost (EP₂-selective) were essentially full agonists as inhibitors of LPS-induced TNFα generation with mean pIC₅₀ values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE₁, another EP₂-selective agonist, AH 13205, inhibited TNFα generation by only 21% at the highest concentration (10 μM) examined. EP-receptor agonists which have selectivity for the EP₁- (17-phenyl-ω-trinor PGE₂) and EP₃-receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNFα generation.
5. Pretreatment of human monocytes with the TP/EP₄-receptor antagonist, AH 23848B, at 10, 30 and 100 μM suppressed LPS-induced TNFα generation by 10%, 28% and 77%, respectively, but failed to shift significantly the location of the PGE₂ concentration-response curves.
6. Given that AH 13205 was a poor inhibitor of TNFα generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory effect of PGE₂. Pretreatment of human monocytes with 10 and 30 μM AH 13205 inhibited the generation of TNFα by 31% and 53%, respectively, but failed to shift significantly the location of the PGE₂ concentration-response curves at either concentration examined.
7. Since PGD₂ and 17-phenyl-ω-trinor PGE₂ (EP₁-agonist) did not suppress TNFα generation, the EP₁/EP₂/DP-receptor antagonist, AH 6809, was employed to assess if EP₂-receptors mediated the inhibitory effect of PGE₂. Pretreatment of human monocytes with 10 μM AH 6809 did not affect LPS-induced TNFα generation but produced a parallel 3.5 fold rightwards shift of the PGE₂ concentration-response curve.
8. Collectively, these data suggest that human peripheral blood monocytes express at least two distinct populations of inhibitory prostanoid receptors that mediate inhibition of LPS-induced TNFα generation. One of these probably represents IP receptors based upon the selectivity of cicaprost for this subtype. The other population has the pharmacology of EP-receptors, but the rank order of potency for a range of synthetic EP-receptor agonists was inconsistent with an interaction with any of the currently defined subtypes. Given the pharmacological behaviour of butaprost, AH 6809 and AH 23848B in these cells, we propose that multiple (EP₂- and/or EP₄- and/or IP) or novel EP-receptors mediate the inhibitory effect of PGE₂ on TNFα generation.

     

Intermolecular cross-talk between the prostaglandin E2 receptor (EP)3 subtype and thromboxane A2 receptor signalling in human erythroleukaemic cells

Background and purpose:

In previous studies investigating cross-talk of signalling between prostaglandin (PG)E₂ receptor (EP) and the TPα and TPβ isoforms of the human thromboxane (TX)A₂ receptor (TP), 17-phenyl trinor PGE₂-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP₁ subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells.

Experimental approach:

Specificity of 17-phenyl trinor PGE₂ signalling and its possible cross-talk with signalling by TPα/TPβ receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)₃ generation and intracellular calcium ([Ca²⁺]_i) mobilization.

Key results:

While 17-Phenyl trinor PGE₂ led to activation of phospholipase (PL)Cβ to yield increases in IP₃ generation and [Ca²⁺]_i, it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA₂ mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE₂ was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G_i/G_βγ-dependent activation of PLCβ. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE₂-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP₃, rather than the EP₁, EP₂ or EP₄ receptor subtype in HEL cells.

Conclusions and Implications:

The EP₁ and EP₃ subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP₁ receptors impaired or desensitized, while that of EP₃ receptors augmented signalling through TPα/TPβ receptors, in a cell type-specific manner.     

AKAP-dependent sensitization of Cav3.2 channels via the EP4 receptor/cAMP pathway mediates PGE2-induced mechanical hyperalgesia

Background and Purpose

The Ca_v3.2 isoform of T-type Ca²⁺ channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE₂-induced hyperalgesia. Here we examined and analysed Ca_v3.2 sensitization via the PGE₂/cAMP pathway in NG108-15 cells that express Ca_v3.2 and produce cAMP in response to PGE₂, and its impact on mechanical nociceptive processing in rats.

Experimental Approach

In NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Ca_v3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats.

Key Results

In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE₂ increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE₂ was abolished by RQ-00015986-00, an EP₄ receptor antagonist. AKAP150 was co-immunoprecipitated with Ca_v3.2, regardless of stimulation with db-cAMP, and Ca_v3.2 was phosphorylated by db-cAMP or PGE₂. In rats, intraplantar (i.pl.) administration of db-cAMP or PGE₂ caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl₂, known to inhibit Ca_v3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE₂-induced mechanical hyperalgesia.

Conclusion and Implications

Our findings suggest that PGE₂ causes AKAP-dependent phosphorylation and sensitization of Ca_v3.2 through the EP₄ receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.     

Prostaglandin E2 couples through EP4 prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines

Background and purpose:

Prostaglandin (PG) E₂ and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE₂ signals principally through prostanoid receptors of the EP₂ and/or EP₄ subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP₂ and EP₄ receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE₂-induced IL-8 production.

Experimental approach:

To identify the causative receptor, we knocked-down and over-expressed EP₂ and EP₄ receptor subtypes in colonic epithelial cells and studied the effect of several selective EP₂/EP₄ receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE₂ and Bmax values for the EP₂ and EP₄ receptor on colonic epithelial cells were determined by radioligand-binding assays with [³H]PGE₂.

Key results:

PGE₂ had the highest affinity for the EP₄ receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP₄ receptor agonist, ONO-AE1-329, and abolished by silencing the EP₄ receptor gene by using siRNA techniques, a selective EP₄ receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase.

Conclusions and implications:

These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE₂ acting via the EP₄ receptor subtype.     

Myrtucommulone,a natural acylphloroglucinol,inhibits microsomal prostaglandin E2 synthase-1

Background and purpose:

The selective inhibition of prostaglandin (PG)E₂ formation via interference with microsomal PGE₂ synthase (mPGES)-1 could have advantages in the treatment of PGE₂-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.

Experimental approach:

The effect of MC on PGE₂ formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF_1α formation.

Key results:

MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH₂ to PGE₂ (IC₅₀ = 1 µmol·L⁻¹). PGE₂ formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 µmol·L⁻¹, and only moderate inhibition of cellular or cell-free COX-1 was evident (IC₅₀ > 15 µmol·L⁻¹).

Conclusions and implications:

MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE₂ formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.     

IL-1β reduces tonic contraction of mesenteric lymphatic muscle cells,with the involvement of cycloxygenase-2 and prostaglandin E2

Background and Purpose

The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, ‘lymphangions’. Here we have studied the effects of the inflammatory cytokine IL-1β on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC).

Experimental Approach

We measured IL-1β in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1β or h-TNF-α (5, 10, 20 ng·mL⁻¹), with or without COX inhibitors [TFAP (10⁻⁵ M), diclofenac (0.2 × 10⁻⁵ M)], PGE₂ (10⁻⁵ M)], IL-1-receptor antagonist, Anakinra (5 μg·mL⁻¹), or a selective prostanoid EP₄ receptor antagonist, GW627368X (10⁻⁶ and 10⁻⁷ M).

Key Results

Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1β or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1β concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1β-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1β increased COX-2 protein and PGE₂ production in RMLMC.. PGE₂ induced relaxations in RMLMC, comparable to h-IL-1β. Conversely, COX-2 and EP₄ receptor inhibition reversed relaxation induced by IL-1β.

Conclusions and Implications

The IL-1β-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE₂. In experimental colitis, IL-1β and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1β restored tonic contraction.     

Stimulated release of a hyperpolarizing factor (ADHF) from mesenteric artery perivascular adipose tissue: involvement of myocyte BKCa channels and adiponectin

Background and Purpose

Perivascular adipose tissue (PVAT) releases adipocyte-derived hyperpolarizing factors (ADHFs) that may partly act by opening myocyte K⁺ channels. The present study in rat and mouse mesenteric arteries aimed to identify the myocyte K⁺ channel activated by PVAT and to determine whether adiponectin contributed to the hyperpolarizing effects of PVAT.

Experimental Approach

Myocyte membrane potential was recorded from de-endothelialized, non-contracted rat and mouse mesenteric arteries in the presence and absence of PVAT.

Key Results

The β₃-adrenoceptor agonist, CL-316,243 (10 μM), generated PVAT-dependent, iberiotoxin-sensitive myocyte hyperpolarizations resulting from BK_Ca channel opening and which were partially blocked by L-NMMA (100 μM). Adiponectin (5 μg·mL⁻¹) also produced iberiotoxin-sensitive hyperpolarizations in PVAT-denuded arterioles. Activation of myocyte AMP-activated protein kinase (AMPK) using 5 μM A-769662 also induced BK_Ca-mediated hyperpolarizations. Dorsomorphin abolished hyperpolarizations to CL-316,243, adiponectin and A-769662. In vessels from Adipo^−/− mice, hyperpolarizations to CL-316,243 were absent whereas those to A-769662 and adiponectin were normal. In rat vessels, adipocyte-dependent hyperpolarizations were blocked by glibenclamide and clotrimazole but those to NS1619 (33 μM) were unaltered.

Conclusions and Implications

Under basal, non-contracted conditions, β₃-adrenoceptor stimulation of PVAT releases an ADHF, which is probably adiponectin. This activates AMPK to open myocyte BK_Ca channels indirectly and additionally liberates NO, which also contributes to the observed PVAT-dependent myocyte hyperpolarizations. Clotrimazole and glibenclamide each reversed hyperpolarizations to adiponectin and A-769662, suggesting the involvement of myocyte TRPM4 channels in the ADHF-induced myocyte electrical changes mediated via the opening of BK_Ca channels.     

Stimulation of angiotensin AT2 receptors by the non-peptide agonist,Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:

Angiotensin type 2 receptor (AT₂ receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT₁ receptors). Recently, a novel non-peptide AT₂ receptor agonist, Compound 21, was described, which exhibited high AT₂ receptor selectivity.

Experimental approach:

Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR).

Key results:

Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT₂ receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng·kg⁻¹·min⁻¹ over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT₁ receptor antagonist, candesartan, Compound 21 (300 ng·kg⁻¹·min⁻¹) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng·kg⁻¹·min⁻¹) still evoked a significant depressor response in adult SHR (∼30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg·kg⁻¹). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 µg·kg⁻¹·min⁻¹ for 2 h) with the AT₂ receptor antagonist PD123319.

Conclusion and implications:

Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT₂ receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT₂ receptor function in cardiovascular disease.