Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage.

OBJECTIVES: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. METHODOLOGY: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). RESULTS: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. CONCLUSIONS: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage

Abstract Objectives: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. Methodology: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). Results: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. Conclusions: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Evaluation of prostaglandin E2 and E-series prostaglandin receptor in patients with interstitial cystitis.

PURPOSE: We evaluated PGE2 and EP receptor in patients with interstitial cystitis. MATERIALS AND METHODS: Enrolled in the study were 20 female patients with interstitial cystitis (11 with and 9 without Hunner lesions), 9 female controls with another urological disease who needed a cystoscopic procedure and 10 normal volunteers. In all participants we determined O'Leary-Sant symptom and problem scores, and obtained voluntary urine specimens for PGE2 analysis. Using anesthesia the bladder was distended by saline in stepwise fashion from 100 ml to maximum capacity in patients with interstitial cystitis. Each time the infused saline was retrieved for PGE2 analysis. We also measured PGE2 and the expression of EP receptor mRNA in bladder biopsy tissue in patients with interstitial cystitis. RESULTS: Symptom and problem indexes in patients with interstitial cystitis and Hunner lesions were significantly higher than in patients with interstitial cystitis without Hunner lesions. Urinary PGE2 in patients with interstitial cystitis and Hunner lesions was significantly higher than in patients with interstitial cystitis without lesions, controls and normal volunteers. PGE2 in retrieved saline in patients with interstitial cystitis and Hunner lesions increased depending on infusion volume but not in patients with interstitial cystitis without lesions. PGE2 content in bladder biopsy tissue was significantly higher in patients with interstitial cystitis and Hunner lesions than in controls. In patients with interstitial cystitis and Hunner lesions the expression of EP1 and EP2 mRNA was significantly higher than in controls. CONCLUSIONS: Our study showed increased PGE2 production and mRNA expression of EP1 and EP2 receptors in the bladder in patients with interstitial cystitis and Hunner lesions. Further studies are warranted to explore the pathophysiological and therapeutic implications.

[Expression and function of E prostanoid receptors in urological cancer].

The biological activities of prostaglandin E2 are mediated through their specific receptors, E prostanoid receptors (EPRs). This family comprises 4 subtypes (EP1R-4R), and has been associated with cancer development and progression. In urological cancers, expression of EP2R and EP4R can be significant predictors of survival for renal cell carcinoma (RCC). On the other hand, EP1R, EP2R, and EP4R are known to be associated with carcinogenesis and malignant aggressiveness in prostate cancer. In addition, EP4R has been associated with tumor progression and prognosis in urothelial cancer of the upper urinary tract. There is a general agreement that non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of several malignancies including colorectal cancer. However, NSAIDs often cause gastrointestinal injury and nephropathy. On the other hand, cyclooxygenase (COX)-2-selective inhibitors can reduce the progression of cancer via the suppression of cell proliferation angiogenesis without decreasing adverse reactions. However, COX-2-selective inhibitors might increase the risk of cardiovascular disease, including myocardial infarction. More selective and detailed control of COX-2-mediated signals is thus needed to improve anti-tumor effects and to decrease adverse reactions. EPRs are expected to serve as new therapeutic targets in urological cancer, because they are more selective in malignant phenotypes. Finally, we speculate that some EPRs inhibitors may reduce adverse events and exert more intense effects on urological cancer.

Platelet receptors.

Well-understood functions for "traditional" platelet receptors are described, but "newer" receptors are equally discussed. Receptors are described biochemically (structure, ligand(s), protein partners, and function) and whenever possible, their clinical importance (mutations, polymorphisms, syndrome) are highlighted.

EP receptor expression in human intestinal epithelium and localization relative to the stem cell zone of the crypts.

There is substantial evidence for PGE2 affecting intestinal epithelial proliferation. PGE2 is also reported to be involved in the regulation of growth and differentiation in adult stem cells, both effects mediated by binding to EP-receptors. We have used the Lgr5 as a marker to scrutinize EP-receptor and COX expression in human intestinal epithelial cells with focus on the stem cell area of the crypts. Normal tissue from ileum and colon, but also duodenal biopsies from patients with untreated celiac disease, were investigated by immunohistochemistry and RT-PCR. The combination of fresh flash-frozen tissue and laser microdissection made it possible to isolate RNA from the epithelial cell layer, only. In the small intestine, Lgr5 labels cells are in the +4 position, while in the colon, Lgr5 positive cells are localized to the crypt bottoms. Epithelial crypt cells of normal small intestine expressed neither EP-receptor mRNA nor COX1/2. However, crypt cells in tissue from patients with untreated celiac disease expressed EP2/4 receptor and COX1 mRNA. In the colon, the situation was different. Epithelial crypt cells from normal colon were found to express EP2/4 receptor and COX1/2 transcripts. Thus, there are distinct differences between normal human small intestine and colon with regard to expression of EP2/4 receptors and COX1/2. In normal colon tissue, PGE2-mediated signaling through EP-receptors 2/4 could be involved in regulation of growth and differentiation of the epithelium, while the lack of EP-receptor expression in the small intestinal tissue exclude the possibility of a direct effect of PGE2 on the crypt epithelial cells.

Expression and regulation of prostaglandin receptors in the human placenta and fetal membranes at term and preterm.

Prostaglandins (PGs) play an important role in parturition in many species, including humans. The present study examined the distribution of PG receptor subtypes (EP1-4 and FP) in intrauterine tissues at term and preterm birth. Placentas and fetal membranes were collected from patients at term in labour (n = 12) or not in labour (n = 12). Preterm tissue was collected from three different groups of patients: (1) idiopathic preterm labour (PTL) without chorioamnionitis or betamethasone (BM) treatment (n = 9), (2) idiopathic PTL that received BM with no chorioamnionitis (PTL-BM; n = 9) and (3) pregnancies that were complicated with chorioamnionitis and had no BM (PTL-CHA; n = 6). EP1-4 and FP receptors were localised and levels of expression were determined by western blot analysis. All EP receptors and FP were localised to the amnion, placenta and choriodecidua. Moreover, isolated amnion mesenchymal, amnion epithelial, chorion trophoblast and syncytiotrophoblast cells in primary culture also expressed PG receptors. A significant increase was observed in EP1, EP3 and FP expression in placenta, chorion and amnion with labour. Maternal betamethasone treatment increased EP1, EP3 and FP receptor protein expression and chorioamnionitis decreased expression in all the receptor subtypes. These changes in PG receptors in the fetal membranes are consistent with the development of a feed-forwards cascade mediated through PG action that may contribute to the birth process.

Prostaglandin receptors EP2 and IP are detectable in atherosclerotic arteries and plaques.

AIM: Prostaglandin (PG) receptor agonists are frequently used for the pharmacological treatment of arteriosclerosis obliterans (ASO). In particular, the PG receptors EP2 and IP stimulate vasodilation and inhibit platelet aggregation, biological processes thought to be protective against ASO and important for physiological homeostasis. However it is uncertain whether EP2 and IP exist in diseased arteries, or what their distribution within the artery might be. In this study, we analyzed the distribution of these PG receptors in patients with severe ASO to determine the potential application of stimulation of these receptors as targets for pharmacological treatment. METHODS: We collected segments of atherosclerotic femoral arteries during femoropopliteal bypass surgery and determined the expression levels of EP2 and IP receptors by western blotting. Immunofluorescence was used to observe receptor localization. RESULTS: Findings of western blotting showed an increased Cox-2 expression in patients with ASO. The EP2 as well as IP receptors were each induced approximately 3-fold in comparison to normal samples. The expression of these receptors was increased in the intimal layer as well as the medial layer; their expression was also detectable within the atherosclerotic plaque. CONCLUSION: We observed induction of the PG receptors EP2 and IP in atherosclerotic femoral arteries in the arterial intima, medial layer, as well as the associated atherosclerotic plaque. These results suggest that receptor-selective PG agonists specifically target atherosclerotic arteries and therefore, may find potential application in the pharmacological management of patients with ASO.

Prostaglandin receptor EP1 and EP2 site in guinea pig bladder urothelium and lamina propria.

PURPOSE: Urothelium has 2 main functions. It is a barrier to urine and has a sensory role. In response to stretch urothelium releases various substances that modulate afferent nerve activity. Recent data on the localization of cyclooxygenase type 1, the enzyme responsible for prostaglandin production, suggests that prostaglandin may have complex local action. MATERIALS AND METHODS: The bladders of 7 guinea pigs were stained for prostaglandin receptors type 1 and 2, and costained for vimentin and cyclooxygenase I. RESULTS: Prostaglandin receptor type 1 staining was seen in urothelial cells and in the suburothelium. Urothelial staining, which was often punctuate and weak, was detected in all urothelial cell layers, including suburothelial cells. In contrast, strong prostaglandin receptor type 2 staining was seen in the urothelium and in suburothelial cells. Cyclooxygenase I was absent in interstitial cells and umbrella cells with the highest concentration in the basal cell layer. CONCLUSIONS: Interstitial cells express prostaglandin receptor types 1 and 2, indicating that they can respond to prostaglandin. Umbrella cells do not express cyclooxygenase I. Cyclooxygenase I was present in basal urothelial cells, making them a possible site of prostaglandin synthesis. Thus, prostaglandin produced by urothelium may target prostaglandin receptor types 1 and 2 in the urothelium and suburothelium. Therefore prostaglandin is hypothesized to have a role in signal regulation in the bladder wall.

Quantitative detection of EP3-II, III and VI messenger RNA in gravid and non-gravid human myometrium using real-time RT-PCR.

OBJECTIVE: To examine mRNA expression of prostaglandin E2 receptor isoforms EP3-II, III and VI in lower uterine segment myometrium in the non-pregnant and pregnant state using quantitative real-time RT-PCR. METHODS: Myometrial samples were obtained at the time of cesarean delivery or hysterectomy. Pregnant subjects were categorised based on the presence or absence of labour. Labour was defined as regular uterine contractions resulting in cervical change. Quantification for EP3 isoforms II, III and VI mRNA was performed using real-time RT-PCR. RESULTS: There were no differences between non-pregnant and non-labouring pregnant subjects in mRNA expression of EP3-II, III and VI. However, when compared to pregnant subjects not in labour, labouring subjects had a 3.8-fold reduction in EP3-II expression (p < 0.001) and 5.3-fold reduction in EP3-VI expression (p = 0.022). CONCLUSIONS: In human parturition, there is decreased mRNA expression of lower-uterine segment EP3 receptor isoforms II and VI during labour. This may reflect differential relaxation of the lower segment of the uterus allowing dilatation and descent of the fetus.

Coiled-coil tag--probe system for quick labeling of membrane receptors in living cell.

The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeting the tag has been craved as an alternative to widely used larger fluorescent proteins. We describe a rapid method with a small tag (21 amino acids) for the fluorescence labeling of cell-surface receptors using a high affinity coiled-coil formation without metals or enzymes. The peptide probes K3 (KIAALKE)3 and K4 (KIAALKE)4 labeled with a fluorophore specifically stained the surface-exposed tag sequence E3 (EIAALEK)3 attached to the N-terminus of the mouse-derived prostaglandin EP3 receptor in living cells (Kd = 64 and 6 nM for K3 and K4, respectively). The labeling was quick (<1 min), nontoxic, and available even in culture medium without affecting receptor function. As an application of this tractable method, the agonist-induced internalization of the human-derived 2-adrenergic receptor and epidermal growth factor receptor was successfully visualized.

E series of prostaglandin receptor 2-mediated activation of extracellular signal-regulated kinase/activator protein-1 signaling is required for the mitogenic action of prostaglandin E2 in esophageal squamous-cell carcinoma.

The use of nonsteroidal anti-inflammatory drugs is associated with a lower risk for esophageal squamous cell carcinoma, in which overexpression of cyclooxygenase-2 (COX-2) is frequently reported. Prostaglandin E(2) (PGE(2)), a COX-2-derived eicosanoid, is implicated in the promotion of cancer growth. However, the precise role of PGE(2) in the disease development of esophageal squamous cell carcinoma remains elusive. In this study, we investigated the effect of PGE(2) on the proliferation of cultured esophageal squamous cell carcinoma cells (HKESC-1). Results showed that HKESC-1 cells expressed all four series of prostaglandin (EP) receptors, namely, EP1 to EP4 receptors. In this regard, PGE(2) and the EP2 receptor agonist (+/-)-15-deoxy-16S-hydroxy-17-cyclobutyl PGE(1) methyl ester (butaprost) markedly increased HKESC-1 cell proliferation. Moreover, the mitogenic effect of PGE(2) was significantly attenuated by RNA interference-mediated knockdown of the EP2 receptor, indicating that this receptor mediated the mitogenic effect of PGE(2). In this connection, PGE(2) and butaprost induced phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), whose down-regulation by RNA interference significantly attenuated PGE(2)-induced cell proliferation. In addition, PGE(2) and butaprost increased c-Fos expression and activator protein 1 (AP-1) transcriptional activity, which were abolished by the mitogen-activated protein kinase/Erk kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)-butadiene ethanolate (U0126). AP-1-binding inhibitor curcumin also partially reversed the mitogenic effect of PGE(2). Taken together, these data demonstrate for the first time that the EP2 receptor mediates the mitogenic effect of PGE(2) in esophageal squamous cell carcinoma via activation of the Erk/AP-1 pathway. This study supports the growth-promoting action of PGE(2) in esophageal squamous cell carcinoma and the potential application of EP2 receptor antagonists in the treatment of this disease.

Prostanoid receptor EP1 expression in breast cancer.

Cyclooxygenase enzymes play an important role in carcinogenesis, and increased expression of cyclooxygenase enzymes has been reported in cancers arising at a number of different sites. Most, if not all of these actions are thought to be mediated by prostaglandin E2 (PGE2). The actions of PGE2 are mediated via four main prostanoid receptors, designated EP1, EP2, EP3 and EP4, based on their different pharmacological properties and secondary messenger pathways. Recently, expression of EP1 has been reported in rat mammary gland and the inhibition of this receptor has been documented to have chemopreventive effect in this animal model. EP1 has also been shown to decrease the incidence of colon cancer in mouse models. In this study, we analysed the expression of EP1 in normal and malignant breast tissues. Expression of EP1 was analysed in breast (benign and cancer) cell lines by reverse-transcriptase polymerase chain reaction and by western blot analyses. Expression was also analysed by immunohistochemistry in normal breast tissues and in 89 cases of breast cancer. Semiquantitative analysis of the staining was performed. The data were compared with and correlated with other prognostic factors like tumour size, tumour grade, lymph node status, oestrogen receptor, progesterone receptor (PR), HER2/neu and cyclooxygenase-2. EP1 expression was demonstrated in human breast cancer by immunohistochemistry. Expression of EP1 was seen both in the cytoplasm and/or in the nuclear membrane in majority of cases. Nuclear EP1 expression correlated with PR (P=0.032) and inversely with node positivity (P=0.025). However, EP1 expression did not correlate with expression of cyclooxygenase-2 (P=0.059). Expression of EP1 is frequently seen in human breast cancers. Nuclear expression of EP1 correlates with good prognosis markers like node negative status and PR expression.

Prostaglandin E2 acts on EP1 receptor and amplifies both dopamine D1 and D2 receptor signaling in the striatum.

Dopamine is involved in multiple neural functions including motor control, reward and motivational processing, learning and reinforcement, and cognitive attention. Dopamine binds to two distinct classes of receptors, namely D1 and D2, to exert these functions. Various endogenous substances regulate dopamine signaling, although their physiological functions are not fully understood. Here, we examined the role of prostaglandin E2 (PGE2) and one of its receptors, EP1, in dopaminergic function in the striatum. EP1 was expressed in both preprodynorphin-containing D1 and preproenkephalin-containing D2 neurons, and PGE2 was produced in striatal slices in response to both D1 and D2 dopamine receptor stimulation. EP1-deficient mice exhibited significant suppression of hyperlocomotion induced by cocaine or SKF81297 (6-chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide), a D1 agonist, and significant attenuation of catalepsy induced by raclopride, a D2 antagonist. Despite these behavioral defects, the extracellular concentration of dopamine was not suppressed in the striatum of EP1-deficient mice, and the densities of D1 and D2 receptors in the striatum were not different between the two genotypes. Stimulation of the D1 receptor induced phosphorylation of dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at Thr34 in striatal slices, and the addition of indomethacin, a PG synthesis inhibitor, attenuated the D1 agonist-induced increase in DARPP-32-Thr34 phosphorylation. The further addition of an EP1 agonist restored the indomethacin-attenuated phosphorylation. Furthermore, both D1- and D2-mediated changes in the DARPP-32-Thr34 phosphorylation were attenuated in EP1-/- slices. These results suggest that PGE2 is formed in response to dopamine receptor stimulation in the striatum and amplifies both D1 and D2 receptor signaling via EP1.

Spinal prostaglandins facilitate exaggerated A- and C-fiber-mediated reflex responses and are critical to the development of allodynia early after L5-L6 spinal nerve ligation.

BACKGROUND: Spinal prostaglandins are important in the early pathogenesis of spinal nerve ligation (SNL)-induced allodynia. This study examined the effect of SNL on the expression of cyclooxygenase (COX)-1, COX-2, and prostaglandin E2 receptors in the rat lumbar spinal cord, and the temporal and pharmacologic relation of these changes to the exaggerated A- and C-fiber-mediated reflex responses and allodynia, 24 h after injury. METHODS: Male Sprague-Dawley rats, fitted with intrathecal catheters, underwent SNL or sham surgery. Paw withdrawal threshold, electromyographic analysis of the biceps femoris flexor reflex, and immunoblotting of the spinal cord were used. RESULTS: Both allodynia (paw withdrawal threshold of < or = 4 g) and exaggerated A- and C-fiber-mediated reflex responses (i.e., decrease in activation threshold, increase in evoked activity, including windup; P < 0.05) were evident 24 h after SNL but not sham surgery. Allodynic animals exhibited significant increases in prostaglandin E2 receptor (subtypes 1-3) and COX-1 (but not COX-2) expression in the ipsilateral lumbar dorsal horn. The corresponding ventral horns and contralateral dorsal horn were unchanged from sham controls. Exaggerated A- and C-fiber-mediated reflex responses were significantly attenuated by intrathecal SC-560 or SC-51322, but not SC-236, given 24 h after SNL. CONCLUSION: These results provide further evidence that spinal prostaglandins, derived primarily from COX-1, are critical in the exaggeration of A- and C-fiber input and allodynia, 24 h after SNL.

Effects of UVB on E prostanoid receptor expression in murine skin.

Prostaglandin E2 (PGE2) upregulation in response to UV light exposure is a significant factor in the development of non-melanoma skin cancer. It is known that PGE2 signals via the E prostanoid receptors, EP1-4, but the role that each receptor plays in skin carcinogenesis is unclear. Immunohistochemical analysis of EP receptor staining in unirradiated and UVB-exposed SKH-1 mouse skin demonstrated the localization of EP1 and EP2 to the plasma membrane of differentiated epidermal keratinocytes. In contrast, the EP3 receptor localized to the basal layer of the epidermis in unirradiated skin and throughout the epidermis in UVB-exposed skin. In unirradiated skin, cytoplasmic EP4 staining was seen throughout the epidermis, in dermal leukocytes, and in vascular endothelium. However, UVB exposure resulted in relocalization of the EP4 receptor to the plasma membrane of keratinocytes, with no change in the dermal staining pattern. In tumors isolated from UVB-exposed mice, EP1 and EP2 staining was detected in the more differentiated cells surrounding keratin pearls, whereas EP3 and EP4 were detectable throughout the tumors. Differential expression of the EP receptors suggests that each receptor may play a distinct role in skin tumor development.

Direct vasoconstrictor effect of prostaglandin E2 on renal interlobular arteries: role of the EP3 receptor.

Evidence indicates that prostaglandin E(2) (PGE(2)) preferentially affects preglomerular renal vessels. However, whether this is limited to small-caliber arterioles or whether larger vessels farther upstream also respond to PGE(2) is currently unclear. In the present study, we first investigated the effects of PGE(2) along the preglomerular vascular tree and subsequently focused on proximal interlobular arteries (ILAs). Proximal ILAs in hydronephrotic rat kidneys as well as isolated vessels from normal kidneys constricted in response to PGE(2), both under basal conditions and after the induction of vascular tone. By contrast, smaller vessels, i.e., distal ILAs and afferent arterioles, exhibited PGE(2)-induced vasodilation. Endothelium removal and pretreatment of single, isolated proximal ILAs with an EP1 receptor blocker (SC51322, 1 micromol/l) or a thromboxane A(2) receptor blocker (SQ29548, 1 micromol/l) did not prevent vasoconstriction to PGE(2). Furthermore, in the presence of SC51322, responses of these vessels to PGE(2) and the EP1/EP3 agonist sulprostone were superimposable, indicating that PGE(2)-induced vasoconstriction is mediated by EP3 receptors on smooth muscle cells. Immunohistochemical staining of proximal ILAs confirmed the presence of EP3 receptor protein on these cells and the endothelium. Adding PGE(2) to normal isolated kidneys induced a biphasic flow response, i.e., an initial flow increase at PGE(2) concentrations

Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM.

The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.

Localization and steroid regulation of prostaglandin E2 receptor protein expression in ovine cervix.

Although prostaglandin E2 (PGE2) has been identified as a central mediator of the cervical ripening process, the mechanisms responsible for PGE2 ripening are still poorly understood, partly because of the lack of information concerning the precise cellular localization and regulation of PGE2 (EP) receptors in the cervix. To provide new insights into the mechanisms of cervical ripening, we used indirect immunofluorescence to localize cervical EP receptor protein expression in ovariectomized ewes and examined the effect of administration of progesterone or estradiol. EP receptors were widely distributed in cervical blood vessels, epithelium of the cervical canal, circular and longitudinal muscles, and stroma. Estradiol replacement decreased EP1 and EP3 receptor protein in blood vessel media (by 23 and 31% respectively, P < 0.05) and decreased EP1 receptor protein expression in the longitudinal muscle layer (by 27%, P < 0.05). Stromal EP1 and EP3 receptor protein expression was also reduced by estradiol (by 29 and 20% respectively, P < 0.05). Progesterone replacement had no significant effect on EP receptor protein expression. The arterial changes would favor PGE2-induced vasodilatation, subsequent edema and leukocyte infiltration during the cervical ripening process whereas the muscular alterations would facilitate smooth muscle relaxation and cervical dilatation. Furthermore, estradiol provoked perinuclear localization of EP3 receptor protein in the longitudinal muscle layer. This latter result suggests that cellular EP receptor localization is regulated by estradiol and that PGE2 may also control smooth muscle contraction and regulate ovine cervical dilatation in an intracrine manner via EP3 receptors.

Decreased prostaglandin E2 production by inflammatory cytokine and lower expression of EP2 receptor result in increased collagen synthesis in keloid fibroblasts.

We investigated the metabolism of arachidonic acid in normal skin-derived fibroblasts (NF) as well as in keloid-derived fibroblasts (KF) in response to macrophage migration inhibitory factor (MIF), a pluripotent cytokine. We found that MIF enhanced cyclooxygenase-2 activity in NF more than in KF. Consistent with this finding, prostaglandin E(2) (PGE(2)), an antifibrogenic molecule, was more significantly increased in NF than in KF by MIF treatment. As regarding E prostanoid receptor 2, the level of expression was significantly lower in KF than in NF. On the other hand, Forskolin, a direct activator of adenylcyclase, decreased collagen synthesis in both NF and KF, which indicates that cAMP plays an important role in regulating collagen synthesis. As PGE(2) induces cAMP production, it is conceivable that increased collagen synthesis in KF might be owing to decreased PGE(2) and cAMP production. These findings may aid in the development of a therapeutic strategy for the regulation of collagen synthesis in keloid fibroblasts.