Expression Of Prostanoid Receptors Research Articles

Docosahexaenoic acid and eicosapentaenoic acid strongly inhibit prostanoid TP receptor-dependent contractions of guinea pig gastric fundus smooth muscle.

The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT‐qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D2 and U46619. The mRNA expression levels of the prostanoid receptors were TP ≈ EP3 >> FP > EP1. The inhibition by DHA, EPA, and LA was positively correlated with that by SQ 29,548 (a TP receptor antagonist) but not with that by L‐798,106 (an EP3 receptor antagonist). DHA and EPA suppressed high KCl‐induced contractions by 35% and 25%, respectively, and the contractions induced by the prostanoids and U46619 were suppressed by verapamil, a voltage‐dependent Ca2+ channel (VDCC) inhibitor, by 40%–85%. Although LA did not suppress high KCl‐induced contractions, it suppressed U46619‐induced contractions in the presence of verapamil. However, LA did not show significant inhibitory effects on U46619‐induced Ca2+ increases in TP receptor‐expressing cells. In contrast, LA inhibited U46619‐induced contractions in the presence of verapamil, which was also suppressed by SKF‐96365 (a store‐operated Ca2+ channel [SOCC] inhibitor). These findings suggest that the TP receptor and VDCC are targets of DHA and EPA to inhibit prostanoid‐induced contractions of guinea pig GFSM, and SOCCs play a significant role in LA‐induced inhibition of U46619‐induced contractions.

Bronchodilation induced by PGE2 is impaired in Group III pulmonary hypertension

In patients with pulmonary hypertension (PH) associated with lung disease and/or hypoxia (Group III), decreased pulmonary vascular tone and tissue hypoxia is therapeutically beneficial. PGE2 and PGI2 induce potent relaxation of human bronchi from non-PH (control) patients via EP4 and IP receptors, respectively. However, the effects of PGE2 /PGI2 and their mimetics on human bronchi from PH patients are unknown. Here, we have compared relaxant effects of several PGI2 -mimetics approved for treating PH Group I with several PGE2 -mimetics, in bronchial preparations derived from PH Group III and control patients. Relaxation of bronchial muscle was assessed in samples isolated from control and PH Group III patients. Expression of prostanoid receptors was analysed by western blot and real-time PCR, and endogenous PGE2 , PGI2 , and cAMP levels were determined by ELISA. Maximal relaxations induced by different EP4 receptor agonists (PGE2 , L-902688, and ONO-AE1-329) were decreased in human bronchi from PH patients, compared with controls. However, maximal relaxations produced by PGI2 -mimetics (iloprost, treprostinil, and beraprost) were similar for both groups of patients. Both EP4 and IP receptor protein and mRNA expressions were significantly lower in human bronchi from PH patients. cAMP levels significantly correlated with PGI2 but not with PGE2 levels. The PGI2 -mimetics retained maximal bronchodilation in PH Group III patients, whereas bronchodilation induced by EP4 receptor agonists was decreased. Restoration of EP4 receptor expression in airways of PH Group III patients with respiratory diseases could bring additional therapeutic benefit.

Prostacyclins have no direct inotropic effect on isolated atrial strips from the normal and pressure-overloaded human right heart.

Prostacyclins are vasodilatory agents used in the treatment of pulmonary arterial hypertension. The direct effects of prostacyclins on right heart function are still not clarified. The aim of this study was to investigate the possible direct inotropic properties of clinical available prostacyclin mimetics in the normal and the pressure-overloaded human right atrium. Trabeculae from the right atrium were collected during surgery from chronic thromboembolic pulmonary hypertension (CTEPH) patients with pressure-overloaded right hearts, undergoing pulmonary thromboendarterectomy (n = 10) and from patients with normal right hearts operated by valve replacement or coronary bypass surgery (n = 9). The trabeculae were placed in an organ bath, continuously paced at 1 Hz. They were subjected to increasing concentrations of iloprost, treprostinil, epoprostenol, or MRE-269, followed by isoprenaline to elicit a reference inotropic response. The force of contraction was measured continuously. The expression of prostanoid receptors was explored through quantitative polymerase chain reaction (qPCR).Iloprost, treprostinil, epoprostenol, or MRE-269 did not alter force of contraction in any of the trabeculae. Isoprenaline showed a direct inotropic response in both trabeculae from the pressure-overloaded right atrium and from the normal right atrium. Control experiments on ventricular trabeculae from the pig failed to show an inotropic response to the prostacyclin mimetics. qPCR demonstrated varying expression of the different prostanoid receptors in the human atrium. In conclusion, prostacyclin mimetics did not increase the force of contraction of human atrial trabeculae from the normal or the pressure-overloaded right heart. These data suggest that prostacyclin mimetics have no direct inotropic effects in the human right atrium.

Expression of prostanoid receptors (EP1, 2, 3, and 4) in normal and methimazole-treated mouse olfactory epithelium

Conclusion: Prostanoid receptors (EP1, EP2, EP3, and EP4) are expressed in the olfactory epithelium (OE), and the EP4 prostanoid receptor may play an important role in the OE. Objective: The purpose of the present study was to investigate the expression and localization of the four types of prostanoid receptors (EP1, EP2, EP3, and EP4) in the OE of normal and methimazole-treated mice to gain more complete knowledge about the functional significance of the prostanoid receptors in OE. Methods: CBA/J mice were used in this study. The localization of the prostanoid receptors (EP1, EP2, EP3, and EP4) in the OE was investigated by immunohistochemistry. The changes in expression of prostanoid receptors were studied in methimazole-treated mice. Furthermore, the effect of EP agonists on the methimazole-induced degeneration of OE was assessed by morphological analysis and by assessment of apoptosis. Results: All four types of EP receptors were recognized in mouse OE. Expression of EP4 in the OE was significantly reduced after methimazole treatment. In the methimazole-treated mice, an EP4 agonist reduced OE damage and apoptosis.

Localization of prostanoid receptors in the mouse inner ear

Conclusion: EP4, EP2, and IP prostanoid receptors exert an otoprotective function and FP may be important for fluid homeostasis in the inner ear. Objective: To investigate the expression of prostanoid receptors in the normal mouse inner ear. Methods: CBA/J mice were used in this study. The localization of prostanoid receptors, i.e. DP, EP1, EP2, EP3, EP4, FP, IP, and TP, in the inner ear, i.e. the cochlea, vestibular end organs, endolymphatic sac, was studied by immunohistochemical techniques. Results: The EP4, IP, and FP prostanoid receptors were found to be abundantly distributed in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, and the endolymphatic sac. EP2 and EP3 are also localized in the inner ear whereas DP, EP1, and TP are only weakly expressed.

Gi-coupled prostanoid receptors are the likely targets for COX-1-generated prostanoids in rat pheochromocytoma (PC12) cells

Cyclooxygenase-1 (COX-1) behaves as a delayed response gene in rat pheochromocytoma (PC12) cells exposed to nerve growth factor (NGF). To investigate the possible targets for COX-1 generated prostanoids in the early stages of neuronal differentiation, we have examined the expression of prostanoid receptors by PC12 cells using functional assays. Prostanoid receptor-specific agonists failed to activate adenylyl cyclase in undifferentiated and NGF-treated PC12 cells; neither did they stimulate phospholipase C activity. EP3 receptor agonists and PGF 2α were the only active ligands, able to inhibit forskolin-stimulated adenylyl cyclase activity. PC12 cells expressed EP3 and FP receptor mRNA, but only the responses to EP3 receptor agonists were inhibited by the EP3 receptor antagonist ONO-AE3-240. The functional role of NGF-stimulated COX-1 remains to be determined since we found no strong evidence of a role for EP3 receptors in the morphological changes induced by NGF during the early stages of differentiation of PC12 cells.

Characterization of prostanoid receptors present on adrenergic neurons innervating the porcine uterine longitudinal muscle

The cyclooxygenase–prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [ 3H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [ 3H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca 2+-free (EGTA, 1 mM) incubation medium and by tetrodotoxin or ω-conotoxin GVIA, suggesting that [ 3H]-noradrenaline was released from neural components. The EFS-evoked [ 3H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE 2 and PGF 2α, but not PGD 2, inhibited the EFS-evoked [ 3H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP 1/EP 3) decreased the EFS-evoked [ 3H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP 2) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP 1 receptor antagonist) did not change the inhibition by sulprostone or PGE 2. Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP 3 receptor and TP receptor antibodies suggested the localization of EP 3 or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP 3 and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.

The constitutive activity of ghrelin receptors is decreased by co-expression with vasoactive prostanoid receptors when over-expressed in human embryonic kidney 293 cells

The functional activity of G protein-coupled receptors can be modified by their ability to form oligomeric complexes with G protein-coupled receptors from other receptor families. Emerging evidence suggests that the appetite-regulating hormone ghrelin is a directly acting vasodilator peptide with anti-inflammatory activity, therefore, we have examined the ability of ghrelin receptors to oligomerize with members of the prostanoid receptor family which are also involved in modulating vascular activity and inflammatory responses. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the ability of ghrelin receptors to hetero-oligomerize with prostaglandin E 2 receptor subtype EP 3-I, prostacyclin receptors, and thromboxane A 2 (TPα) receptors, when transiently over-expressed in human embryonic kidney 293 cells. These results suggest that hetero-oligomeric interactions between ghrelin receptors and prostanoid receptors are likely to be of biological relevance. Co-transfection of cells with ghrelin receptor and prostanoid receptors significantly decreased ghrelin receptor expression and attenuated its constitutive activation of phospholipase C without changing its affinity for ghrelin. We also observed an increase in the proportion of ghrelin receptors localized intracellularly in the presence of prostanoid receptors. Taken together, these results suggest that the increased expression of prostanoid receptors in conditions of vascular inflammation, such as in atherosclerotic plaques, could influence those cellular responses dependent on the constitutive activation of ghrelin receptors.

Prostanoid receptor expression in colorectal cancer related to tumor stage, differentiation and progression

Introduction: Alterations in eicosanoid metabolism is well established in a variety of malignant tumors, particularly colorectal carcinoma. Recent studies in our laboratory have emphasized a role for EP subtype receptors in progression of colorectal cancer and disease specific mortality. Therefore, the aim of the present study was to extend our knowledge to include additional receptor expression (DP1, DP2, FP, IP, TP) for prostanoids (PGD2, TXA2, PGF2α, PGI2) in relationship to tumor stage, differentiation and progression of colorectal cancer. Material and methods: Total RNA from 62 tumors and adjacent normal colon tissue (n = 48) was extracted. Quantification of receptor expression was performed by realtime PCR and related to the expression of an appropriate housekeeping gene (GAPDH). Tumors were assessed according to Dukes A-D (stage I-IV). Results: DP1, DP2, FP and IP receptor subtypes displayed significantly reduced overall expression in tumor tissue compared to normal colon tissue, while the TP receptor subtype showed significantly higher expression in tumor tissue. Overall expression of the prostanoid receptors in tumor tissue was not related to clinical indexes as tumor stage and tumor cell differentiation evaluated by multivariate analyses. Cultured colorectal cancer cell lines with low (HT-29) and high (HCA-7) intrinsic PGE2 production at confluent state did not express DP1 and IP receptor subtypes, but displayed low expression of DP2, FP and TP receptor subtypes. Conclusion: The results in the present study indicate imbalanced expression of prostanoid receptors in colorectal cancer compared to normal colon tissue without clear cut relationship to disease progression. Therefore, future studies should be performed on defined cells within the tumor tissue compartment determining whether any prostanoid receptor(s) is useful as a molecular target in treatment or prevention of colorectal cancer.

Prostanoid receptors in human uterine myocytes: the effect of reproductive state and stretch

In the human, prostanoids are known to be important mediators of uterine relaxation and contraction during pregnancy and parturition. We have previously shown that stretch of uterine smooth muscle cells increased prostaglandin H synthase 2 (PGHS-2) mRNA expression, PGHS-2 peptide synthesis and activity, however, the net effect on uterine contractility of this increase in prostaglandin synthesis would be determined by the expression of the different prostanoid receptors. Therefore, the aims of this study were to establish the expression of prostanoid receptor mRNA in uterine myocytes obtained from women in different reproductive states and to test the hypothesis that stretch of uterine myocytes alters prostanoid receptor mRNA expression to promote uterine contractility. Myocytes were isolated from women undergoing hysterectomy (NP) and pregnant women undergoing LSCS either before (NL) or after the onset of labour (L) and were subjected to 11% stretch for 1 h (n = 6 in all cases). Copy numbers of the individual receptors varied widely with reproductive state but followed the pattern: FP > IP = DP = EP-4 > TP = EP-3 = EP-2 > EP-1. FP mRNA expression was significantly lower in the NL group compared to the NP group and EP-3, EP-4 and TP mRNA expression was significantly lower in both NL and L groups compared to NP group levels. The level of mRNA expression of EP-1, EP-2, DP and IP did not differ between NP, NL and L groups. Stretch of cells derived from the NP group resulted in a significant decrease in EP-4 mRNA expression alone and of the NL group a significant decrease in EP-2 mRNA expression alone. Stretch had no effect on cells derived from the L group. These data show that pregnancy is associated with a significant reduction in 3 of 4 pro-contraction associated prostanoid receptor mRNA expression and 1 of 4 pro-relaxant. Stretch elicited no consistent change in prostanoid receptor mRNA expression.

The effects of non-selective, preferential-selective and selective COX-inhibitors on the growth of experimental and human tumors in mice related to prostanoid receptors

Earlier observations on cyclo-oxygenase inhibitors (NSAIDs) restricting tumor growth were re-evaluated by comparing the effects of non-selective, preferential selective and selective derivatives of COX-inhibitors on tumor growth in mouse models with either prostaglandin-sensitive (MCG-101, human tumors) and -insensitive transplants (K1735-M2). Tumor growth, with and without provision of a classical cyclo-oxygenase inhibitor (indomethacin), was related to tumor content of COX-1/COX-2 protein as well as to EP1-EP4 and prostacyclin receptor expression. Mouse serum amyloid protein (SAP) was measured as an indicator of systemic inflammation, which relates to pro-inflammatory cytokines. Indomethacin inhibited tumor growth and prolonged the survival of mice bearing MCG-101 tumors, which display a high production of PGE2, while K1735-M2 tumors with insignificant amounts of PGE2 did not respond to indomethacin at all. However, the effects of various NSAIDs on tumor growth were highly variable in combination with the fact that most preferential selective and selective COX-2 inhibitors attenuated poorly systemic inflammation evaluated by plasma concentrations of mouse SAP. The ability of NSAIDs to attenuate tumor growth was not related to the tumor content of COX-2 protein as expected. Multivariate analysis suggests that significant COX-inhibition of tumor growth may be related to tumor expression of subtype EP2, EP3 (p<0.005) and perhaps EP4 (p<0.09) in complex interplay. The extent of tumor growth inhibition by COX-inhibitors is not simply related to drug specificity on COX-1 or COX-2 pathways. Such effects may instead be related to tumor expression of prostanoid receptors in tumor tissue.

Expression of prostanoid receptors by myometrium and cervix in response to cyclooxygenase (COX-2) inhibition in the rabbit model of RU486-induced preterm delivery

Fetal membranes express elevated levels of COX-2 at the onset of labor. Selective COX-2 inhibitors are being considered for the management of preterm labor. Prostanoid receptors are linked to various second messengers and mediate vessel constriction or dilation. EP3 prostanoid receptor plays an important regulatory role in myometrial contractility. We evaluated the expression of prostaniod receptors in myometrium and cervix in response to COX-2 inhibition in a rabbit model of RU486-induced preterm delivery.

Expression of prostanoid receptors in human lower segment pregnant myometrium

Prostanoids, especially prostaglandin (PG) E 2, are important mediators of uterine relaxation and contractions during gestation and parturition. Inhibitors of PG formation as well as PG analogues are used to modulate uterine tonus. So far, only limited data are available regarding the expression of prostanoid receptors in human pregnant myometrium. In the present study, the expression of the receptors for PGE 2 (EP1, EP2, EP3, EP4), PGF 2 α (FP), prostacyclin (IP), and thromboxane A 2 (TP) in human pregnant myometrium was studied by RT-PCR, in situ hybridization and immunohistochemistry. Myometrial tissue was obtained from five women at term and not in labour and from two women who delivered preterm. Tissue specimens were excised from the upper edge of the transverse lower uterine segment incision. In all tissues analysed, EP1, EP2, EP3, EP4, FP, TP and IP receptor mRNA and protein was detected. mRNA expression for PGD 2 (DP) receptor was not detected in the majority of tissue specimens. EP1, EP2, EP4, IP, TP and FP receptor protein was detected on myometrial smooth muscle cells, whereas EP3 receptor protein was only expressed by stromal and endothelial cells. In situ hybridization experiments yielded similar results. The expression of the EP2 receptor mRNA was inversely related to gestational age. We suggest that the contractile effect of PGE 2 at term is probably mediated directly by the EP1 receptor expressed in myometrial smooth muscle cells and indirectly by the EP3 receptor expressed in stromal cells and a decrease in EP2 receptor expression.

Expression of cyclooxygenase-2 and prostanoid receptors by human myometrium.

Prostanoids play an important role in the regulation of parturition. All reproductive tissues, including fetal membranes, decidua, and myometrium, have the capacity to synthesize prostanoids, and fetal membranes have been shown to express elevated levels of cyclooxygenase-2 (Cox-2) at the onset of labor. We have now investigated the expression of Cox-2 in human myometrium. Myometrial samples collected from women in labor during lower segment cesarean section expressed 15-fold higher levels of Cox-2 messenger ribonucleic acid (mRNA) compared to myometrial specimens collected from women not in labor, as detected by Northern blot analysis. Immunohistochemical detection of Cox-2 protein showed cytoplasmic staining in the smooth muscle cells of the myometrium. Cultured myometrial cells expressed low levels of Cox-2 mRNA under baseline conditions, but interleukin-1beta (IL-1beta) caused a 17-fold induction of expression of the Cox-2 transcript after incubation for 6 h. IL-1beta also induced expression of biologically active Cox-2 protein, as detected by immunofluorescence, Western blot analysis, and measuring the conversion of arachidonic acid to prostanoids in the presence and absence of a Cox-2-selective inhibitor, NS-398. PGE2 receptor subtype EP2 mRNA was expressed in cultured myometrial smooth muscle cells, whereas transcripts for EP1, EP3, EP4, FP, and IP were low or below the detection limit as measured by Northern blot analysis. However, IL-1beta stimulated expression of EP4 receptor mRNA. Our data suggest that expression of Cox-2 transcript is elevated at the onset of labor in myometrial smooth muscle cells, which may depend on induction by cytokines. As, in addition to Cox-2, the expression of prostanoid receptors is regulated, not only the production of prostanoids, but also responsiveness to them, may be modulated.

Differential expression of prostanoid receptors in hepatocytes, Kupffer cells, sinusoidal endothelial cells and stellate cells of rat liver

Background/Aims: Prostanoids produced by nonparenchymal cells modulate the function of parenchymal and nonparenchymal liver cells during homeostasis and inflammation via eight classes of prostanoid receptors coupled to different G-proteins. Prostanoid receptor expression in parenchymal and nonparenchymal cells was studied in order to get a better insight into the complex prostanoid-mediated intrahepatic signaling network. Methods: RNA was isolated from freshly purified parenchymal and nonparenchymal rat liver cells and the mRNA level of all eight prostanoid receptor classes was determined by newly developed semiquantitative reverse transcription-polymerase chain reaction protocols. Results: The mRNAs for the prostanoid receptors were differentially expressed. Hepatocytes were the only cell type which contained the mRNA of the G q-linked prostaglandin F 2α receptor; they were devoid of any mRNA for the G s-linked prostanoid receptors. Kupffer cells possessed the largest amount of mRNA for the G s-linked prostaglandin E 2 receptor subtype 2. Endothelial cells expressed high levels of mRNA for the G q-linked thromboxane receptor and medium levels of mRNA for the G s-linked prostacyclin receptor, while stellate cells had the highest levels of mRNA for the prostacyclin receptor. The mRNAs for the G q-linked prostaglandin E 2 receptor subtype 1 and the G i-linked prostaglandin E 2 receptor subtype 3 were expressed in hepatocytes and all nonparenchymal cell types atsimilar high levels, whereas the mRNA of the G s-linked prostaglandin D 2 receptor was expressed in all nonparenchymal cells at very low levels. Conclusions: In hepatocytes the prostaglandin F 2α receptor can mediate an increase in glucose output via an increase of intracellular InsP 3 while cAMP-dependent glucose output can be inhibited via the subtype 3 prostaglandin E 2 receptor. The subtype 2 prostaglandin E 2 receptor can restrain the inflammatory response of Kupffer cells via an increase in intracellular cAMP. The thromboxane receptor and the prostacyclin receptor in sinusoidal endothelial and the prostacyclin receptor in stellate cells may be involved in the regulation of sinusoidal blood flow and filtration.