Impaired cognition, sensorimotor gating, and hippocampal long-term depression in mice lacking the prostaglandin E2 EP2 receptor.

Cyclooxygenase-2 (COX-2) is a neuronal immediate early gene that is regulated by N-methyl d aspartate (NMDA) receptor activity. COX-2 enzymatic activity catalyzes the first committed step in prostaglandin synthesis. Recent studies demonstrate an emerging role for the downstream PGE(2) EP2 receptor in diverse models of activity-dependent synaptic plasticity and a significant function in models of neurological disease including cerebral ischemia, Familial Alzheimer's disease, and Familial amyotrophic lateral sclerosis. Little is known, however, about the normal function of the EP2 receptor in behavior and cognition. Here we report that deletion of the EP2 receptor leads to significant cognitive deficits in standard tests of fear and social memory. EP2-/- mice also demonstrated impaired prepulse inhibition (PPI) and heightened anxiety, but normal startle reactivity, exploratory behavior, and spatial reference memory. This complex behavioral phenotype of EP2-/- mice was associated with a deficit in long-term depression (LTD) in hippocampus. Our findings suggest that PGE(2) signaling via the EP2 receptors plays an important role in cognitive and emotional behaviors that recapitulate some aspects of human psychopathology related to schizophrenia.

EP2, a receptor for PGE2, regulates tumor angiogenesis through direct effects on endothelial cell motility and survival.

Prostaglandin E2 (PGE2), a major cyclooxygenase (COX) metabolite, plays important roles in tumor biology. We studied the role of EP2, a receptor for PGE2, in tumor angiogenesis using EP2 knockout mice. We found that deletion of the EP2 receptor impaired tumor angiogenesis and this finding was confirmed by an in vivo corneal angiogenesis model and an ex vivo aortic ring assay. To further characterize the cellular mechanisms of the EP2 receptor in angiogenesis, we isolated primary pulmonary endothelial cells (ECs) from wild-type (wt) and EP2-/- mice and observed that EP2-/- ECs exhibited defects in vascular branch formation when compared to wt ECs. In addition, EP2-/- ECs showed impaired cell motility on collagen-coated surface and they responded poorly to PGE2-induced cell migration compared to control cells. However, no difference in cell proliferation was observed between the EP2-/- and wt Ecs. In addition, EP2-/- ECs were more susceptible to apoptosis than wt cells under growth factor depletion conditions. Collectively, our data demonstrate that EP2 signaling in endothelium directly regulates tumor angiogenesis by contributing to cell survival and endothelial cell motility. Moreover, our finding suggests that EP2 is a major receptor in PGE2-mediated cell motility in ECs.

Effects of prostaglandin E2 on gene expression in primary osteoblastic cells from prostaglandin receptor knockout mice.

Recent studies have shown that stimulation of osteoclastogenesis in cocultures of osteoblasts and spleen cells in response to prostaglandin E2 (PGE2) is markedly decreased when the osteoblasts are derived from cells lacking either the EP2 or the EP4 receptor. Induction of osteoclast formation requires upregulation of receptor activator of nuclear factor-kappaB ligand (RANKL) on cells of the osteoblastic lineage, which then binds to the RANK receptor on cells of the osteoclast lineage. Osteoprotegerin (OPG) is a decoy receptor for RANKL that can block its interaction with RANK. In addition, macrophage-colony stimulating factor (M-CSF) is essential for osteoclast formation. Finally, PGE2 can increase interleukin-6 (IL-6), which may further enhance osteoclastogenesis. To study the relative influence of the EP2 and EP4 receptors on response of these factors to PGE2, we examined mRNA levels for RANKL, OPG, M-CSF, and IL-6 in primary osteoblastic cell cultures derived from two lines of EP2 knockout mice (EP2-/-) and one line of EP4 knockout mice (EP4-/-) and the relevant wild-type controls (EP2+/+ and EP4+/+). The responses of cells from wild-type animals of all three lines were similar. After PGE2 treatment, RANKL mRNA levels were increased at 2 h, and this was sustained over 72 h. Basal RANKL expression was moderately reduced in EP2-/- cells and markedly reduced in EP4-/- cells. PGE2 increased RANKL mRNA in EP2-/- cells and EP4-/- cells, but the levels were significantly reduced compared with wild-type cells. There were no consistent changes in expression of M-CSF or OPG in the different genotypes or with PGE2 treatment. IL-6 mRNA was variably increased by PGE2 in both wild-type and knockout cells, although the absolute levels were somewhat lower in both EP2-/- and EP4 -/- cultures. Parathyroid hormone (PTH) increased RANKL and IL-6 and decreased OPG mRNA levels similarly in both wild-type and EP2-/- or EP4-/- cells. The major defect in the response to PGE2 in animals lacking either EP2 or EP4 receptors is a reduction in basal and stimulated RANKL levels. Loss of EP4 receptor appears to have a greater effect on basal RANKL expression than EP2.

Peroxisome proliferator-activated receptor-gamma activity is associated with renal microvasculature.

Peroxisome proliferator-activated receptor-gamma (PPAR gamma) is a nuclear transcription factor and the pharmacological target for antidiabetic thiazolidinediones (TZDs). TZDs ameliorate diabetic nephropathy and have direct effects on cultured mesangial cells (MCs); however, in situ hybridization failed to detect expression of PPAR gamma in glomeruli in vivo. The purpose of this study was to determine whether PPAR gamma is expressed in renal glomeruli. Two rabbit PPAR gamma isoforms were cloned. Nuclease protection assays demonstrate that both PPAR gamma isoforms are expressed in freshly isolated glomeruli. Treatment of rabbits with the TZD troglitazone selectively induced expression of an endogenous PPAR gamma target gene, adipocyte fatty acid-binding protein (A-FABP), in renal glomerular cells and renal medullary microvascular endothelial cells, demonstrated by both in situ hybridization and immunostain. Troglitazone also dramatically increased A-FABP expression in cultured MCs. Constitutive PPAR gamma expression was detected in cultured rabbit MCs. Endogenous MC PPAR gamma can also drive PPAR gamma reporter. Troglitazone and 15-deoxy-Delta 12,14 prostaglandin J(2) at low concentrations reduced mesangial cell [(3)H]thymidine incorporation without affecting viability. These data suggest that constitutive PPAR gamma activity exists in renal glomeruli in vivo and could provide a pharmacological target to directly modulate glomerular injury.

Urogenital distribution of a mouse membrane-associated prostaglandin E(2) synthase.

PGE(2) plays a critical role in regulating renal function and facilitating reproduction. One of the rate-limiting biosynthetic enzymes in PGE(2) synthesis is the terminal PGE(2) synthase (PGES). In the present studies, we report the functional expression of a membrane-associated murine PGES (mPGES) and its expression in urogenital tissues. Two independent cDNA clones sharing an identical open reading frame of 459 bp and encoding a peptide of 153 amino acids, but differing in the 3'-untranslated region, were identified. Assays for enzymatic activity, using microsomes prepared from cells transfected with mPGES cDNA, showed that these cDNA sequences encode a functional protein that catalyzes the conversion of PGH(2) to PGE(2). Constitutive expression of mPGES was highest in the mouse kidney, ovary, and urinary bladder but was also expressed at lower levels in uterus and testis. Renal mPGES expression was predominantly localized to epithelia of distal tubules and medullary collecting ducts. High expression was also seen in transitional epithelial cells of bladder and ureter and in the primary and secondary follicles in the ovary. In conclusion, mPGES is constitutively expressed along the urogenital tract, where it may have important roles in normal physiology and disease.

EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2.

Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2 from airway epithelium in other species, suggesting PGE2 as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2 receptor [EP2(-/-)], one of the PGE2 receptors, we tested the hypothesis that PGE2 is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(-/-) mice. There were no differences between EP2(-/-) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(-/-) and wild-type tracheas in basal or stimulated PGE2 production. Exogenous PGE2 produced significantly less relaxation in EP2(-/-) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2 receptors are of primary importance in airway relaxation to PGE2 and relaxation to SP or ATP is mediated through PGE2 acting on EP2 receptors.

Dysregulated cytokine production in human cystic fibrosis bronchial epithelial cells.

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) alpha and IL-1beta stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNFalpha stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNFalpha stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1beta. Finally, when CF cells were grown at 27 degrees C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNFalpha-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.

Diversification of cyclooxygenase-2-derived prostaglandins in ovulation and implantation.

Previous observations of ovulation and fertilization defects in cyclooxygenase-2 (COX-2)-deficient mice suggested that COX-2-derived ovarian prostaglandins (PGs) participate in these events. However, the specific PG and its mode of action were unknown. Subsequent studies revealed that mice deficient in EP(2), a PGE(2)-receptor subtype, have reduced litter size, apparently resulting from poor ovulation but more dramatically from impaired fertilization. Using a superovulation regimen and in vitro culture system, we demonstrate herein that the ovulatory process, not follicular growth, oocyte maturation, or fertilization, is primarily affected in adult COX-2- or EP(2)-deficient mice. Furthermore, our results show that in vitro-matured and -fertilized eggs are capable of subsequent preimplantation development. However, severely compromised ovulation in adult COX-2- or EP(2)-deficient mice is not manifested in immature (3-wk-old) COX-2- or EP(2)-deficient mice, suggesting that the process of ovulation is more dependent on PGs in adult mice. Although the processes of implantation and decidualization are defective in COX-2(-/-) mice, our present results demonstrate that these events are normal in EP(2)-deficient mice, as determined by embryo transfer and experimentally induced decidualization. Collectively, previous and present results suggest that whereas COX-2-derived PGE(2) is essential for ovulation via activation of EP(2), COX-2-derived prostacyclin is involved in implantation and decidualization via activation of peroxisome proliferator-activated receptor delta.

Prostanoid receptors: subtypes and signaling.

Cyclooxygenases metabolize arachidonate to five primary prostanoids: PGE(2), PGF(2 alpha), PGI(2), TxA(2), and PGD(2). These autacrine lipid mediators interact with specific members of a family of distinct G-protein-coupled prostanoid receptors, designated EP, FP, IP, TP, and DP, respectively. Each of these receptors has been cloned, expressed, and characterized. This family of eight prostanoid receptor complementary DNAs encodes seven transmembrane proteins which are typical of G-protein-coupled receptors and these receptors are distinguished by their ligand-binding profiles and the signal transduction pathways activated on ligand binding. Ligand-binding selectivity of these receptors is determined by both the transmembrane sequences and amino acid residues in the putative extracellular-loop regions. The selectivity of interaction between the receptors and G proteins appears to be mediated at least in part by the C-terminal tail region. Each of the EP(1), EP(3), FP, and TP receptors has alternative splice variants described that alter the coding sequence in the C-terminal intracellular tail region. The C-terminal variants modulate signal transduction, phosphorylation, and desensitization of these receptors, as well as altering agonist-independent constitutive activity.

Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes.

The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.

G protein-coupled prostanoid receptors and the kidney.

Renal cyclooxygenase 1 and 2 activity produces five primary prostanoids: prostaglandin E2, prostaglandin F2alpha, prostaglandin I2, thromboxane A2, and prostaglandin D2. These lipid mediators interact with a family of distinct G protein-coupled prostanoid receptors designated EP, FP, IP, TP, and DP, respectively, which exert important regulatory effects on renal function. The intrarenal distribution of these prostanoid receptors has been mapped, and the consequences of their activation have been partially characterized. FP, TP, and EP1 receptors preferentially couple to an increase in cell calcium. EP2, EP4, DP, and IP receptors stimulate cyclic AMP, whereas the EP3 receptor preferentially couples to Gi, inhibiting cyclic AMP generation. EP1 and EP3 mRNA expression predominates in the collecting duct and thick limb, respectively, where their stimulation reduces NaCl and water absorption, promoting natriuresis and diuresis. The FP receptor is highly expressed in the distal convoluted tubule, where it may have a distinct effect on renal salt transport. Although only low levels of EP2 receptor mRNA are detected in the kidney and its precise intrarenal localization is uncertain, mice with targeted disruption of the EP2 receptor exhibit salt-sensitive hypertension, suggesting that this receptor may also play an important role in salt excretion. In contrast, EP4 receptor mRNA is predominantly expressed in the glomerulus, where it may contribute to the regulation of glomerular hemodynamics and renin release. The IP receptor mRNA is highly expressed near the glomerulus, in the afferent arteriole, where it may also dilate renal arterioles and stimulate renin release. Conversely, TP receptors in the glomerulus may counteract the effects of these dilator prostanoids and increase glomerular resistance. At present there is little evidence for DP receptor expression in the kidney. These receptors act in a concerted fashion as physiological buffers, protecting the kidney from excessive functional changes during periods of physiological stress. Nonsteroidal anti-inflammatory drug (NSAID)-mediated cyclooxygenase inhibition results in the loss of these combined effects, which contributes to their renal effects. Selective prostanoid receptor antagonists may provide new therapeutic approaches for specific disease states.

Prostaglandin E receptors and the kidney.

Prostaglandin E(2) is a major renal cyclooxygenase metabolite of arachidonate and interacts with four G protein-coupled E-prostanoid receptors designated EP(1), EP(2), EP(3), and EP(4). Through these receptors, PGE(2) modulates renal hemodynamics and salt and water excretion. The intrarenal distribution and function of EP receptors have been partially characterized, and each receptor has a distinct role. EP(1) expression predominates in the collecting duct where it inhibits Na(+) absorption, contributing to natriuresis. The EP(2) receptor regulates vascular reactivity, and EP(2) receptor-knockout mice have salt-sensitive hypertension. The EP(3) receptor is also expressed in vessels as well as in the thick ascending limb and collecting duct, where it antagonizes vasopressin-stimulated salt and water transport. EP(4) mRNA is expressed in the glomerulus and collecting duct and may regulate glomerular tone and renal renin release. The capacity of PGE(2) to bidirectionally modulate vascular tone and epithelial transport via constrictor EP(1) and EP(3) receptors vs. dilator EP(2) and EP(4) receptors allows PGE(2) to serve as a buffer, preventing excessive responses to physiological perturbations.

Knockout of the murine prostaglandin EP2 receptor impairs osteoclastogenesis in vitro.

Prostaglandin E2 (PGE2) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE2 receptors, designated EP2 and EP4. We used cells from mice in which the EP2 receptor had been disrupted to test its role in the formation of TRAP + MNC. EP2 heterozygous (+/-) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP2 -/-) and wild-type (EP2 +/+) mice. PGE2, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP2 +/+ mice. In cultures from EP2 -/- animals, responses to PGE2, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP4 receptor antagonist (EP4RA) further inhibited TRAP+ MNC formation in both EP2 +/+ and EP2 -/- cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE2 or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP2 -/- mice, by 88% or 68% when only osteoblastic cells were from EP2 -/- mice and by 58% or 35% when only spleen cells were from EP2 -/- mice. PGE2 increased receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP2 +/+ mice 2-fold but had little effect on cells from EP2 -/- mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE2 increased the number of TRAP+ MNC in spleen cell cultures from EP2 +/+ mice but not in cultures from EP2 -/- mice. EP4RA had no effect on the PGE2 response in spleen cell cultures. PGE2 decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP2 +/+ mice but had little effect on cells from EP2 -/- mice. These data demonstrate that the prostaglandin EP2 receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP2 -/- mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE2 in EP2 -/- mice.

Characterization of murine vasopressor and vasodepressor prostaglandin E(2) receptors.

Four E-prostanoid (EP) receptors, designated EP(1), EP(2), EP(3), and EP(4), mediate the cellular effects of prostaglandin E(2) (PGE(2)). The present studies pharmacologically characterize the vasopressor and vasodepressor EP receptors in wild-type mice (EP(2)(+/+) mice) and mice with targeted disruption of the EP(2) receptor (EP(2)(-/-) mice). Mean arterial pressure (MAP) was measured via a carotid artery catheter in anesthetized male mice. Intravenous infusion of PGE(2) decreased MAP in EP(2)(+/+) mice but increased MAP in EP(2)(-/-) mice. Infusion of EP(3)-selective agonists, including MB28767, SC46275, and sulprostone, increased MAP in both EP(2)(+/+) and EP(2)(-/-) mice. Pretreatment with SC46275 desensitized mice to the subsequent pressor effect of sulprostone, but the vasodepressor effect of PGE(2) in EP(2)(+/+) mice remained intact. Although PGE(2) alone increased MAP in EP(2)(-/-) mice, prior desensitization of the pressor effect with SC46275 allowed a residual vasodepressor effect of PGE(2) to be seen in the EP(2)(-/-) mice. An EP(4)-selective agonist (prostaglandin E(1)-OH) functioned also as a vasodepressor in both EP(2)(-/-) and EP(2)(+/+) mice. High levels of EP(3) receptor mRNA were detected in mouse aortas and rabbit preglomerular arterioles by nuclease protection, with lower expressions of EP(1), EP(2), and EP(4) mRNA. The findings suggest that combined vasodepressor effects of EP(2) and EP(4) receptors normally dominate, accounting for the depressor effects of PGE(2). In contrast, in EP(2)(-/-) mice, EP(4) receptor activity alone is insufficient to overcome the EP(3) vasopressor effect. These findings suggest that a balance between pressor and depressor PGE(2) receptors determines its net effect on arterial pressure and that these receptors may be important therapeutic targets.

Structure-function analyses of eicosanoid receptors. Physiologic and therapeutic implications.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase (COX) metabolism of arachidonic acid that exert a broad range of physiologic activities including modulation of inflammation, ovulation, and arterial blood pressure. The physiologic actions of PGs are mediated in part by their interaction with specific G-protein-coupled PG receptors. Eight PG receptors have been cloned, including four for the major COX metabolite, PGE2. The physiologic roles of the PGE2 receptors have been investigated utilizing subtype-selective agonists, localization of receptor mRNA expression, and creation of mice with targeted disruption of PG receptor genes. These analyses have delineated discrete roles for the various PG receptor subtypes. Recent studies on mice lacking the PGE2 EP2 receptor have implicated the PGE2 EP2 receptor subtype in arterial dilatation and salt-sensitive hypertension, and also indicate that this receptor plays a key role in female fertility. The EP2 receptor may thus prove to be a productive target for pharmacological intervention in the treatment of hypertension and infertility.

Prostaglandin receptors: their role in regulating renal function.

Renal cyclooxygenase-1 and cyclooxygenase-2 actively metabolize arachidonate to metabolism five primary prostanoids: prostaglandin E2, prostaglandin F2a, prostaglandin I2, thromboxane A2, and prostaglandin D2. These lipid mediators interact with a family of distinct G-protein-coupled prostanoid receptors designated EP, FP, IP, TP, and DP, respectively, which exert important regulatory effects on renal function. The intrarenal distribution of these prostanoid receptors has been mapped and the consequences their activation are being characterized. The FP, TP, and EP1 receptors preferentially couple to increased cell Ca2+. EP2, EP4, DP, and IP receptors stimulate cyclic adenosine monophosphate, whereas the EP3 receptor preferentially couples to Gi, inhibiting cyclic adenosine monophosphate generation. EP1 and EP3 messenger RNA expression predominate in the collecting duct and thick limb, respectively, where their stimulation reduces sodium chloride and water absorption, promoting natriuresis and diuresis. Interestingly, only a mild change in renal water handling is seen in the EP3 receptor knockout mouse. Although only low levels EP2 receptor messenger RNA are detected in kidney and its precise intrarenal localization is uncertain, mice with targeted disruption of the EP2 receptor display salt-sensitive hypertension, suggesting it also plays an important role in salt excretion. In contrast, EP4 messenger RNA is readily detected in the glomerulus where it may contribute to the regulation of renin release and decrease glomerular resistance. TP receptors are also highly expressed in the glomerulus, where they may increase glomerular vascular resistance. The IP receptor messenger RNA is most highly expressed in the afferent arteriole and it may also modulate renal arterial resistance and renin release. At present there is little evidence for DP receptor expression in the kidney. Together these receptors act as physiologic buffers that protect the kidney from excessive functional changes during periods of physiologic stress. Loss of the combined effects of these receptors contributes to the side effects seen in the setting of nonsteroidal anti-inflammatory drug administration, whereas selective antagonists for these receptors may provide new therapeutic approaches in disease.

Importance of the extracellular domain for prostaglandin EP(2) receptor function.

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.

Prostaglandin E-prostanoid-3 receptor activation of cyclic AMP response element-mediated gene transcription.

The prostaglandin E-prostanoid (EP)3 receptor signals primarily through the inhibitory G protein Gi, thereby decreasing intracellular cAMP levels. To study the signal transduction properties of the rabbit EP3 receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A and the novel splice variant NT, which lacks the C-terminal sequence. The ability of the EP3 receptor splice variants to modulate expression of a beta-galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprostone-mediated increase in beta-galactosidase enzymatic activity with EC50 ranging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice variant. Substitution of either Asp338 with Ala, or Arg329 with Ala or Glu in the 77A splice variant resulted in a loss of receptor-evoked increases in beta-galactosidase activity, whereas substitution of Lys300 with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transduction was insensitive to pretreatment of cells with pertussis toxin, suggesting that a nonGi/Go pathway is activated by the EP3 receptor. Direct measurement of second messenger levels confirmed that there was no increase in cAMP levels mediated by the 77A splice variant, however, there was a modest increase in intracellular Ca2+. Partial blockade of the reporter activity with kinase inhibitors demonstrates that CRE activation is mediated in part by a Ca2+-dependent kinase pathway. These data suggest that the EP3 receptor signals through a novel cAMP response element binding protein/CRE pathway.

Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein-coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2-/-). EP2-/- mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2-/- mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2-/- mice developed profound systolic hypertension, whereas wild-type mice showed no change in systolic blood pressure. Analysis of wild-type and EP2-/- mice on day 5 of pregnancy indicated that the reduced litter size of EP2-/- mice is due to a pre-implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt-sensitive hypertension, and also plays an essential part in female fertility.