Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor.
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Prostaglandins (PGs) are bioactive lipids that modulate a broad spectrum of biologic processes including reproduction and circulatory homeostasis. Although reproductive functions of mammals are influenced by PGs at numerous levels, including ovulation, fertilization, implantation, and decidualization, it is not clear which PGs are involved and whether a single mechanism affects all reproductive functions. Using mice deficient in 1 of 4 prostaglandin E2 (PGE2) receptors -- specifically, the EP2 receptor -- we show that Ep2(-/-) females are infertile secondary to failure of the released ovum to become fertilized in vivo. Ep2(-/-) ova could be fertilized in vitro, suggesting that in addition to previously defined roles, PGs may contribute to the microenvironment in which fertilization takes place. In addition to its effects on reproduction, PGE2 regulates regional blood flow in various vascular beds. However, its role in systemic blood pressure homeostasis is not clear. Mice deficient in the EP2 PGE2 receptor displayed resting systolic blood pressure that was significantly lower than in wild-type controls. Blood pressure increased in these animals when they were placed on a high-salt diet, suggesting that the EP2 receptor may be involved in sodium handling by the kidney. These studies demonstrate that PGE2, acting through the EP2 receptor, exerts potent regulatory effects on two major physiologic processes: blood pressure homeostasis and in vivo fertilization of the ovum. (+info)
Agonist-induced phosphorylation by G protein-coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E(2) receptor hybrid.
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Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R. (+info)
Differential regulation of renal prostaglandin receptor mRNAs by dietary salt intake in the rat.
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BACKGROUND: In this study, we tested the hypothesis that prostaglandin (PG) receptor expression in the rat kidney is subject to physiological regulation by dietary salt intake. METHODS: Rats were fed diets with 0.02 or 4% NaCl for two weeks. PG receptor expression was assayed in kidney regions and cells by ribonuclease protection assay and reverse transcription-polymerase chain reaction analysis. Functional correlates were studied by measurement of PGE2-induced cAMP formation and renin secretion in juxtaglomerular (JG) cells isolated from animals on various salt intakes. RESULTS: EP1 and EP3 receptors were predominantly expressed, and the EP2 receptor was exclusively expressed in the rat kidney medulla. The EP4 receptor was strongly expressed in glomeruli and in renin-secreting JG granular cells. IP receptor transcripts were found mainly in cortex. Maintaining rats on a low- or high-NaCl diet did not affect the expression of EP1 or IP receptors, whereas EP4 transcripts in glomeruli were increased twofold by salt deprivation. Consistent with this, we found that PGE2-evoked cAMP production and renin secretion by JG cells from salt-deprived animals were significantly higher compared with cells obtained from salt-loaded animals. In the outer medulla, EP3 transcripts correlated directly with salt intake, and mRNA abundance was increased twofold by a high-NaCl diet. CONCLUSIONS: Our results suggest that subtype-specific, regional changes in PG receptor expression are involved in the renal adaptation to changes in salt intake. The results are in accord with the general concept that renocortical PGE2 stimulates renin secretion and maintains renal blood flow during low-salt states, whereas medullary PGE2 promotes salt excretion in response to a high salt intake. (+info)
Importance of the extracellular domain for prostaglandin EP(2) receptor function.
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The ligand binding pocket of biogenic amine G protein-coupled receptors is embedded in the membrane-spanning regions of these receptors, whereas the extracellular domains of the peptidergic receptors play a key role in the structure and function of this class of receptors. To examine the role of the extracellular sequences in prostaglandin receptor-ligand interaction, chimeras were constructed with the two G(s)-coupled E-prostanoid (EP) receptors, replacing each of the extracellular sequences of the human EP(2) receptor with the corresponding human EP(4) receptor residues. Replacement of the third extracellular loop (ECIII) yielded a receptor that binds [(3)H]prostaglandin E(2) (PGE(2); K(d) = 6.3 nM) with similar affinity as the EP(2) wild-type receptor (K(d) = 12.9 nM). Similarly, replacement of the nonconserved carboxyl-terminal portion of ECII resulted in a receptor that maintains [(3)H]PGE(2) binding (K(d) = 8.8 nM). In contrast, replacement of the amino terminus, ECI, the entire ECII region, or the residues within the highly conserved motif of the amino-terminal half of ECII yielded chimeras that displayed neither detectable [(3)H]PGE(2) binding nor receptor-evoked cAMP generation. Immunoprecipitation demonstrated that each chimera is expressed at levels near that of wild-type receptors; however, enzyme-linked immunosorbent assay revealed that inactive chimeras have reduced cell surface expression. Similarly, chimeras that exchange the multiple extracellular loop sequences N/ECI, ECII/ECIII, or all four sequences lacked detectable binding and signal transduction, and although expressed, were not detected on the cell surface. These data suggest that the extracellular sequences of the EP(2) receptor are critical determinants of receptor structure and/or function, unlike other G protein-coupled receptors that bind small molecules. (+info)
Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2).
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Female mice lacking the gene encoding the prostaglandin (PG) E(2) receptor subtype EP(2) (EP(2)(-/-)) become pregnant and deliver their pups at term, but with a much reduced litter size. A decrease in ovulation number and a much reduced fertilization rate were observed in EP(2)(-/-) females without difference of the uterus to support implantation of wild-type embryos. Treatment with gonadotropins induced EP(2) mRNA expression in the cumulus cells of ovarian follicles of wild-type mice. The immature cumuli oophori from wild-type mice expanded in vitro in response to both follicle-stimulating hormone and PGE(2), but the response to PGE(2) was absent in those from EP(2)(-/-) mice. Cumulus expansion proceeded normally in preovulatory follicles but became abortive in a number of ovulated complexes in EP(2)(-/-) mice, indicating that EP(2) is involved in cumulus expansion in the oviduct in vivo. No difference in the fertilization rate between wild-type and EP(2)(-/-) mice was found in in vitro studies using cumulus-free oocytes. These results indicate that PGE(2) cooperates with gonadotropin to complete cumulus expansion for successful fertilization. (+info)
Immunolocalization of the four prostaglandin E2 receptor proteins EP1, EP2, EP3, and EP4 in human kidney.
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Four prostaglandin E2 receptor subtypes designated EP1, EP2, EP3, and EP4 have been shown to mediate a variety of effects of prostaglandin E2 (PGE2) on glomerular hemodynamics, tubular salt and water reabsorption, and on blood vessels in the human kidney. Despite the important role of renal PGE2, the localization of PGE2 receptor proteins in the human kidney is unknown. The present study used antipeptide antibodies to the EP1 to EP4 receptor proteins for immunolocalization in human kidney tissue. Immunoblot studies using these antibodies demonstrated distinct bands in membrane fraction from human kidney. By means of immunohistochemistry, expression of the human EP1 receptor subtype protein in renal tissue was detected mainly in connecting segments, cortical and medullary collecting ducts, and in the media of arteries and afferent and efferent arterioles. The human EP2 receptor subtype protein was detectable only in the media of arteries and arterioles. The human EP3 receptor subtype protein was strongly expressed in glomeruli, Tamm-Horsfall negative late distal convoluted tubules, connecting segments, cortical and medullary collecting ducts, as well as in the media and the endothelial cells of arteries and arterioles. Staining of the human EP4 receptor subtype protein was observed in glomeruli and in the media of arteries. However, no signal of either receptor subtype was detected in the thick ascending limb, the macula densa, or in adjacent juxtaglomerular cells. These results support the concept that PGE2 modulates specific functions in different anatomical structures of the human kidney. (+info)
Flow after prostaglandin E1 is mediated by receptor-coupled adenylyl cyclase in human anterior segments.
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PURPOSE: To assess the effect of prostaglandin (PG) F2alpha and PGE1 on flow through the trabecular meshwork in organ preserved human anterior segments. METHODS: Isolated human anterior segments were perfused under standard conditions at a constant pressure of 10 mm Hg, while flow was continuously monitored. After a stabilization period, 6 consecutive concentrations of PGs were administered. cAMP levels were determined in the perfusate at baseline conditions and at 10(-6) M PG. RESULTS: Perfusion with concentrations ranging from 10(-10) to 10(-5) M PGE1 resulted in a dose-dependent increase in flow (P < 0.0001), reaching a plateau of a 26% increase at 10(-7) M. Perfusion with PGF2alpha or placebo (Eagle's minimum essential medium) did not influence baseline flow. cAMP produced by human anterior segments increased from 4.8+/-0.6 pmol x 30 min(-1) per anterior segment at baseline to 19.2+/-4.8 pmol x 30 min(-1) per anterior segment after perfusion with 10(-6) M PGE1 (P < 0.005). Perfusion with 10(-6) M PGF2alpha did not influence baseline cAMP production. Perfusion with 10(-5) M GDP-beta-S, an inhibitor of G protein, before and in combination with 10(-6) M PGE1 completely inhibited the increase in flow and cAMP production as observed after PGE1 alone. Perfusion with 10(-5) M GDP-beta-S alone did not affect baseline cAMP production. CONCLUSIONS: In organ preserved perfused human anterior segments, flow and cAMP production in the perfusate are not mediated by receptor-coupled adenylyl cyclase activity at baseline conditions. Perfusion with PGE1 is suggested to increase flow through the trabecular meshwork by stimulation of prostanoid EP2 receptor subtype, EP4 receptor subtype, or both, coupled to G(s) protein, inducing activation of the adenylyl cyclase catalytic unit. The results may indicate a physiological role for EP2 receptor subtype, EP4 receptor subtype, or both in the modulation of flow through the trabecular meshwork after stimulation. (+info)
Pregnancy and exogenous steroid treatments modulate the expression of relaxant EP(2) and contractile FP receptors in the rat uterus.
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Prostaglandins (PGs) interact with specific receptors on plasma membranes to regulate myometrial activity in many species. The present study examined whether the expression of relaxant prostaglandin E receptor subtype two (EP(2)) and contractile prostaglandin F receptor (FP) mRNA in the rat uterus is changed during various states of pregnancy and regulated by steroid hormones. Expression of mRNA for EP(2) and FP receptors in the full-thickness uteri was analyzed by reverse transcription-polymerase chain reaction using specific primers. Abundance of receptor mRNA was expressed relative to beta-actin mRNA. Results showed that 1) mRNA for EP(2) receptors in the rat uterus was substantially increased during pregnancy (320%) compared with the nonpregnant state (100%, P < 0.01), and declined during labor at term (36% vs. 100% in control, P < 0.01); 2) mRNA expression for FP receptors in rat uterus was increased during pregnancy (333% vs. 100% in nonpregnant rats, P < 0. 01) and reached maximal levels during labor (515% vs. 100% in control, P < 0.01); 3) upon RU-486 treatment on Day 19 of pregnancy, uterine EP(2) receptor mRNA levels were decreased (18% vs. 100% in control, P < 0.01), and FP mRNA levels were increased (357% vs. 100% in control, P < 0.01); 4) with ICI 164384 (an antiestrogen) treatment on Day 19 of gestation, uterine FP receptor mRNA levels were decreased without effects on EP(2) receptors; 5) in ovariectomized (ovx) rats, progesterone increased EP(2) (163% vs. 100% in control, P < 0.01) and had no effects on FP receptor mRNA expression in the rat uterus; 6) estradiol increased FP receptor mRNA levels (358% vs. 100% in control, P < 0.01) and had no effects on EP(2) mRNA in the ovx rat uterus. Therefore, we conclude that steroid hormones modulate the mRNA for relaxant EP(2) and contractile FP receptors for PGs in the uterus and thus regulate uterine activity during pregnancy and labor. (+info)