Immunolocalization of the four prostaglandin E2 receptor proteins EP1, EP2, EP3, and EP4 in human kidney.

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.     

Role of EP4 prostaglandin E2 receptor in the ischemic liver

Prostaglandin E(2) (PGE(2)) mediates a variety of both innate and adaptive immunity responses through 4 distinct receptors, EP1-4. Recent studies have suggested the physiological and pathological role of EP4 in various inflammatory diseases. In this study, we investigated the importance of the EP4 receptor, and the efficacy of a selective EP4 agonist to alter hepatic ischemia/reperfusion (I/R) injury, an important cause of damage in liver resection and transplantation. We used an established murine I/R injury model, 70% partial hepatic ischemia for 90 minutes in male C57BL/6 mice. The local expression of EP4 messenger RNA (mRNA) in the naive and the ischemic liver at 2 hours after reperfusion was examined using RT-PCR analysis. Some mice received the EP4 selective agonist during I/R. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as markers of hepatic injury. EP4 expression in the liver was significantly up-regulated at 2 hours after reperfusion. Furthermore, treatment with EP4 agonist significantly inhibited hepatic injury at 6 hours after reperfusion. Our data suggest an inhibitory role of EP4 PGE(2) receptor in hepatic I/R injury and the therapeutic efficacy of a selective EP4 agonist for liver protection.     

A prostaglandin E2 receptor subtype EP1 receptor antagonist (ONO-8711) reduces hyperalgesia, allodynia, and c-fos gene expression in rats with chronic nerve constriction

Chronic constriction injury (CCI) of the sciatic nerve in rats induces persistent mechanical hyperalgesia and allodynia. CCI is widely known as a model of neuropathic pain, and many studies using this model have been reported. Recently, c-fos has been used as a neural marker of pain, and various studies have assessed the relationship between hyperalgesia and c-fos expression in the lumbar spinal cord. In this study, we examined the role of a prostaglandin E2 receptor subtype EP1 receptor antagonist (ONO-8711) in a rat CCI model. EP1 receptor antagonist (EP1-ra) oral administration from day 8 to day 14 significantly reduced hyperalgesia and allodynia in the three pain tests on day 15. EP1-ra treatment from day 8 to 14 also reduced c-fos-positive cells in laminae I-II, III-IV, and V-X compared with saline treatment. A single dose of EP1-ra treatment on day 8 significantly reduced hyperalgesia and allodynia at 1 h and 2 h after administration, but the efficacy was not observed at 24 h. We conclude that EP1-ra treatment may be useful for hyperalgesia and allodynia and that EP1 receptor mechanisms are involved in the maintenance of c-fos gene expression induced by nerve injury. IMPLICATIONS: We examined whether a prostaglandin E2 receptor subtype EP1 receptor antagonist abrogates neuropathic pain induced by chronic constriction injury model in rats. The EP1 receptor antagonist significantly reduced hyperalgesia, allodynia, and c-fos positive cells. These findings suggested that EP1 receptor antagonists may have a role in treatment of neuropathic pain.     

A selective EP4 receptor antagonist abrogates the stimulation of osteoblast recruitment from bone marrow stromal cells by prostaglandin E2 in vivo and in vitro

Recent evidence indicates that systemic administration of PGE2 increases bone formation and bone mass via activation of the EP4 receptor. Previously, we demonstrated that osteoblastic recruitment from rat bone marrow stromal cells (BMSC) is a major mechanism for the anabolic effect of PGE2. In this study, we used a selective EP4 antagonist to test if the stimulation of osteoblast differentiation from rat BMSC in vitro and in vivo involves the EP4 receptor. In vitro, PGE2 (100 nM) increased nodule formation and alkaline phosphatase (ALP) activity in cultures of rat BMSC 1.5- to 2-fold. These effects were abolished by the EP4 antagonist at 10(-6) M but not 10(-9) M. Furthermore, PGE2 increased the number of surviving adherent BMSC by approximately 225% and the EP4 antagonist prevented this effect as well. The antagonist had no effect on basal levels of nodule formation and adherent cell number. In vivo, daily systemic administration of PGE2 at 6 mg/kg for 2 weeks increased cancellous bone area (by approximately 50%) and increased nodule formation (measured as mineralized area) in ex vivo stromal cultures by approximately 50%. Pre-administration of the EP4 antagonist at 10 mg/kg abrogated both the increase in bone mass as well as the increase in nodule formation. These data indicate that PGE2 stimulates osteoblastic commitment of BMSC via activation of the EP4 receptor.     

Effects of selective prostaglandin EP4 receptor antagonist on osteoclast formation and bone resorption in vitro.

Prostaglandin estradiol (PGE(2)) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE(2) binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 micromol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNC) by 46%-48% in cultures treated with 0.1-1.0 micromol/L PGE(2) and by 96% in cultures treated with 0.01 micromol/L PGE(2). EP4RA also decreased TRAP(+) MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP(+) MNC formation. EP4RA decreased cAMP production in PGE(2)-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-kappaB ligand (RANKL) mRNA levels produced by PGE(2). In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%-25% and from PGE(2)-stimulated cultures by 22%-37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE(2) but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE(2)-stimulated osteoclastogenesis at 1 micromol/L, but almost complete inhibition at 0.01 micromol/L PGE(2). This could be due to the limited efficacy of the antagonist at high concentrations of PGE(2), or an alternative pathway, such as activation of the EP2 receptor.     

A nonprostanoid EP4 receptor selective prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats.

CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia. INTRODUCTION: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia. MATERIALS AND METHODS: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined. RESULTS: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls. CONCLUSIONS: CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.     

Biphasic effect of prostaglandin E2 on osteoclast formation in spleen cell cultures: role of the EP2 receptor.

We examined the effect of PGE2 on OC formation from spleen cells treated with M-CSF and RANKL. PGE2 decreased OC number at 5-6 days of culture and increased OC number, size, and resorptive activity at 7-8 days. A selective EP2 receptor agonist mimicked these effects. Deletion of the EP2 receptor or depletion of T-cells abrogated the increase in OC number. INTRODUCTION: Prostaglandin E2 (PGE2) has been reported to increase osteoclast (OC) number in spleen cells cultured with RANKL and macrophage-colony-stimulating factor (M-CSF). In this study, we examined the time course of PGE2 effects on spleen cells cultured with RANKL and M-CSF. We then investigated which PGE receptors and cell types were involved in these effects. MATERIALS AND METHODS: Spleen cells were cultured from wildtype C57BL/6 mice and EP2 or EP4 receptor-deficient (-/-) and wildtype (+/+) mice on a mixed genetic background. Spleen cells were cultured with M-CSF and RANKL for 5-9 days with or without PGE2 or selective agonists for the four PGE2 receptors (EP1A, EP2A, EP3A, or EP4A). Some cultures were performed using T-cell-depleted spleen cells. OC number and size were quantitated. OC apoptosis and pit formation were measured at 7 or 8 days. RESULTS: PGE2 decreased the number of OCs formed in the presence of RANKL and M-CSF at 5-6 days of culture and increased OC number at 8-9 days compared with cultures without PGE2. PGE2 also increased OC size at 7 and 8 days, decreased apoptosis of OC at 7 days, and increased pit formation at 8 days. EP1A or EP4A had no effect on OC. EP3A decreased OC number. EP2A mimicked effect of PGE2. EP2(-/-) spleen cells showed no increase in OC number in response to PGE2, whereas deletion of EP4 had no effect. Depletion of T-cells abrogated the late increase of OC number. CONCLUSIONS: We conclude that PGE2 has an initial inhibitory effect on OC formation in spleen cell cultures, possibly mediated by both EP2 and EP3 receptors, and a later stimulatory effect, mediated by the EP2 receptor, possibly acting on T-cells.     

Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

Previously, we discovered that periodontal ligament (PDL) cells not only support osteoclastogenesis through cell-to-cell contact, but also inhibit the formation of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells by a producing soluble factor(s). Furthermore, PDL cells express both receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) messenger RNA (mRNA). Clinically, "ankylosed teeth," which lack periodontal ligament, cannot be moved with orthodontic tooth treatment. From this, we hypothesized that PDL cells under mechanical stress should play a pivotal role in osteoclast formation during orthodontic tooth movement. This study examined how mechanical stress affects the osteoclastogenesis-supporting activity of PDL cells. PDL cells were compressed continuously and then cocultured with peripheral blood mononuclear cells (PBMCs) for 4 weeks. PDL cells under mechanical stress up-regulated osteoclastogenesis from PBMCs. Furthermore, the expression of RANKL mRNA and protein in PDL cells increased with compressive force in parallel with the change in the number of osteoclasts. In addition, cyclo-oxygenase 2 (COX-2) mRNA expression was induced by compressive force, and indomethacin inhibited the RANKL up-regulation resulting from compressive force. PDL cells under compressive force exhibited significantly increased prostaglandin E2 (PGE2) production in comparison with control PDL cells. Exogenous PGE2 treatment increased RANKL mRNA expression in PDL cells. Interestingly, OPG expression remained constant throughout compressive force or PGE2 treatment. In conclusion, compressive force up-regulated RANKL expression in PDL cells. Furthermore, RANKL up-regulation in mechanically stressed PDL cells was dependent on PGE2.     

Protective effect of prostaglandin E2 receptors EP2 and EP4 in alloimmune response in vivo

Prostaglandin E2 (PGE2) is produced during inflammatory responses mediating a variety of both innate and adaptive immune responses through 4 distinct receptors: EP1 to EP4. The use of gene-targeted mice and selective agonists/antagonists responsible for each receptor has gradually revealed that each receptor plays a unique and important role in various disease conditions. In addition, PGE2 is known to have some immunosuppressive properties. In this study, we investigated the role of PGE2 receptors by examining the therapeutic efficacy of highly selective receptor agonists on the alloimmune response in vivo. We used a fully major histocompatibility complex (MHC)-mismatched murine cardiac transplantation model. C57BL/6 cardiac allografts were heterotopically transplanted into BALB/c recipients. We treated mice with a highly selective agonist for each EP receptor. EP2 and EP4 agonists significantly prolonged allograft survival compared with controls. In particular, the EP4 agonist was more effective than the EP2 agonist in the inhibition of acute allograft rejection. In conclusion, PGE2 receptors merit further study as novel therapeutics for clinical transplantation.     

Bone biomechanical properties in prostaglandin EP1 and EP2 knockout mice

Prostaglandins play an important role in regulating the bone adaptation response to mechanical stimuli. Prostaglandin E2 (PGE2) is an effective modulator of bone metabolism. Administration of PGE2 to rodents results in increased cancellous and cortical bone mass translating into enhanced mechanical strength. The PGE2 influence on bone is mediated through four well-characterized receptors (EP1, EP2, EP3, and EP4). Although the PGE2 pathways and mechanisms of action on cells involved in bone adaptation are still under investigation, it is now known that each receptor plays a unique role in regulating PGE2-related bone cell function. The EP1 subtype is coupled with Ca2+ mobilization. The EP2 subtype stimulates cyclic adenosine monophosphate (cAMP) formation. cAMP in turn is responsible for the early cellular signal that stimulates bone formation. This study compared physical and biomechanical properties of bone in EP1 and EP2 knockout mice to their corresponding wild-type controls. Ash weight was measured in the ulnae, and femurs and vertebral bodies were tested in three-point bending and compression, respectively. The results suggest: (a) EP1 receptors have a minimal influence on skeletal strength or size in mice; and (b) EP2 receptors have a major influence on the biomechanical properties of bone in mice. The absence of EP2 receptors resulted in weak bone biomechanical strength properties in the EP2 knockout model as compared with the corresponding wild-type control mice.     

The effects of peripheral administration of a novel selective antagonist for prostaglandin E receptor subtype EP(1), ONO-8711, in a rat model of postoperative pain

Mechanically evoked pain, also known as incident pain, induced by coughing or deep breathing after surgery leads to potentially devastating consequences. It is generally thought that the prostaglandin receptor- (especially, the receptor for prostaglandin E(2), EP receptor) mediated sensitization of sensory nerve fibers is a key contributor to the generation of hyperalgesia. We examined whether a peripherally administered novel selective EP(1) antagonist, ONO-8711, would be a potential analgesic for incision-induced mechanical hyperalgesia. We used a rat model of postoperative pain introduced by Brennan et al. (1). Withdrawal thresholds to punctate stimulation and response frequencies to nonpunctate mechanical stimulation were determined by using von Frey filaments applied adjacent to the wound and directly to the incision site of the hind paw, respectively. Mechanical hyperalgesia to punctate and nonpunctate stimuli was observed 2 and 24 h after the incision. ONO-8711 (2, 10, or 50 microg) or saline was administered subcutaneously into the hind paw on the ipsilateral side to the incision. ONO-8711 significantly (P < 0.01) increased the withdrawal thresholds to punctate mechanical stimulation and significantly (P < 0.01) decreased the response frequencies to nonpunctate mechanical stimulation in a dose- and time-dependent manner 2 and 24 h after the incision. We conclude that EP(1) receptor-mediated sensitization of sensory nerve fibers may contribute to the generation of mechanical hyperalgesia produced by incisional surgery, and that the EP(1) receptor antagonist ONO-8711 may be an option for treatment of postoperative pain, especially incident pain. Implications: The peripheral administration of an antagonist for EP(1) receptor that is a subtype of prostaglandin E receptors can inhibit the mechanical hyperalgesia induced by a surgical incision.     

IL-1 receptor antagonist inhibits monocyte chemotactic peptide 1 generation by human mesangial cells.

The elicitation of neutrophils and monocytes from the circulation into the inflamed glomerulus is a key process in the pathogenesis of proliferative glomerulonephritis. The aim of this study was to determine the factors which regulate the expression and synthesis of the monocyte specific chemotaxin, monocyte chemotactic peptide 1 (MCP-1). Mesangial cells in culture did not constitutively express MCP-1, but could be induced to express both MCP-1 mRNA and antigenic MCP-1 by either stimulation with IL-1 alpha or TNF alpha, which are also stimuli for interleukin 8 (IL-8/NAP-1) expression and release. Pre-treatment of mesangial cells with the IL-1 receptor antagonist (IL-1ra) induced dose-dependent inhibition of both the expression of MCP-1 and IL-8 mRNA as well as the release of both chemotactic peptides in response to IL-1 alpha, while the receptor antagonist had no significant effect on TNF alpha induced MCP-1 and IL-8 generation. This study demonstrates that the IL-1 receptor antagonist was four times more effective at inhibiting the IL-1 induced expression and release of IL-8 compared to that of MCP-1. These results suggest that mesangial cell-derived MCP-1 may play an important role in the recruitment of monocytes in glomerular inflammation and that an IL-1 receptor antagonist may have therapeutic potential for the treatment of glomerulonephritis.     

Crucial involvement of the EP4 subtype of prostaglandin E receptor in osteoclast formation by proinflammatory cytokines and lipopolysaccharide.

Prostaglandin E2 (PGE2) exerts its effects through the PGE receptor that consists of four subtypes (EP1, EP2, EP3, and EP4). Osteoclast formation in the coculture of primary osteoblastic cells (POB) and bone marrow cells was enhanced more by 11-deoxy-PGE1 (an EP4 and EP2 agonist) than by butaprost (an EP2 agonist) and other agonists, which suggests that EP4 is the main factor in PGE2-induced osteoclast formation. PGE2-induced osteoclast formation was not observed in the coculture of POB from EP4-deficient (EP4 k/o) mice and spleen cells from wild-type (w/t) mice, whereas osteoclasts were formed in the coculture of POB from w/t mice and spleen cells from EP4-k/o mice. In situ hybridization (ISH) showed that EP4 messenger RNA (mRNA) was expressed on osteoblastic cells but not on multinucleated cells (MNCs) in w/t mice. These results indicate that PGE2 enhances osteoclast formation through its EP4 subtype on osteoblasts. Osteoclast formation by interleukin 1alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), basic fibroblast growth factor (bFGF), and lipopolysaccharide (LPS) was hardly observed in the coculture of POB and bone marrow cells, both from EP4-k/o mice, which shows the crucial involvement of PG and the EP4 subtype in osteoclast formation by these molecules. In contrast, osteoclast formation by 1,25-hydroxyvitamin D3 (1,25(OH)2D3) was not impaired and that by parathyroid hormone (PTH) was only partially impaired in EP4-k/o mice, which may be related to the fact that EP4-k/o mice revealed no gross skeletal abnormalities. Because it has been suggested that IL-1alpha, TNF-alpha, bFGF, and LPS are involved in inflammatory bone loss, our work can be expected to contribute to an understanding of the pathophysiology of these conditions.     

The inhibitory effect of bupivacaine on prostaglandin E(2) (EP(1)) receptor functioning: mechanism of action

Prostaglandin E(2) receptors, subtype EP(1) (PGE(2)EP(1)) have been linked to several physiologic responses, such as fever, inflammation, and mechanical hyperalgesia. Local anesthetics modulate these responses, which may be due to direct interaction of local anesthetics with PGE(2)EP(1) receptor signaling. We sought to characterize the local anesthetic effects on PGE(2)EP(1) signaling and elucidate mechanisms of anesthetic action. In Xenopus laevis oocytes, recombinant expressed PGE(2)EP(1) receptors were functional (half maximal effect concentration, 2.09 +/- 0.98 x 10(-6) M). Bupivacaine, after incubation for 10 min, inhibited concentration-dependent PGE(2)EP(1) receptor functioning (half-maximal inhibitory effect concentration, 3.06 +/- 1.26 x 10(-6) M). Prolonged incubation in bupivacaine (24 h) inhibited PGE(2)-induced calcium-dependent chloride currents (I(Cl(Ca))) even more. Intracellular pathways were not significantly inhibited after 10 min of incubation in bupivacaine. But I(Cl(Ca)) activated by intracellular injection of GTPgammaS (a nonhydrolyzable guanosine triphosphate [GTP] analog that activates G proteins, irreversible because it cannot be dephosphorylated by the intrinsic GTPase activity of the alpha subunit of the G protein) was reduced after 24 h of incubation in bupivacaine, indicating a G protein-dependent effect. However, inositol 1,4,5-trisphosphate- and CaCl(2)- induced I(Cl(Ca)) were unaffected by bupivacaine at any time points tested. Therefore, bupivacaine's effect is at phospholipase C or at the G protein or the PGE(2)EP(1) receptor. All inhibitory effects were reversible. We conclude that bupivacaine inhibited PGE(2)EP(1) receptor signaling at clinically relevant concentrations. These effects could, at least in part, explain how local anesthetics affect physiologic responses such as fever, inflammation, and hyperalgesia during the perioperative period.     

Mechanisms and urodynamic effects of a potent and selective EP4 receptor antagonist,MF191, on cyclophosphamide and prostaglandin E2‐induced bladder overactivity in rats

Study Type – Aetiology (case control) Level of Evidence 3b What's known on the subject? and What does the study add? Recent evidence has suggested that up‐regulation of the prostaglandin E₂ (PGE₂) receptor subtype 4 (EP4) receptor in the bladder is involved in bladder overactivity. The present study found that MF191, a selective EP4 receptor antagonist, may have effects on the bladder urothelium and inflammatory cells and suppress CYP‐ or PGE₂‐induced bladder overactivity. Systemic or intravesical MF191 administration for the treatment of overactive bladder may merit clinical study.

OBJECTIVE

• ? To investigate the mechanisms and urodynamic effects of a potent and selective prostaglandin E₂ (PGE₂) receptor subtype 4 (EP4) antagonist, MF191, on cyclophosphamide (CYP) or PGE₂‐induced bladder overactivity in rats.

MATERIALS AND METHODS

• ? Experimental and control rats were injected with CYP (200 mg/kg i.p.) or saline on day 1. Continuous cystometrogram (CMGs) were performed on day 3.
• ? In group 1, MF191 (vehicle 0.1 and 1 mg/kg) was given i.v. The bladder was then harvested for histology and immunohistochemistry. Some bladders were harvested for analysis of EP4 expression by Western blotting without a CMG study.
• ? In group 2, MF191 (vehicle 10 nM, and 100 nM) was continuously infused into the bladder.
• ? In group 3, bladder overactivity was induced by intravesical instillation of PGE₂ (200 uM) and vehicle or MF191 (1 mg/kg) was given i.v.

RESULTS

• ? CYP induced bladder inflammation, overactivity and EP4 upregulation. The CYP effects were suppressed by MF191 (1 mg/kg i.v.; intercontraction interval [ICI]: 39.4% increase, and reduced inflammatory cells infiltration, and EP4 expression).
• ? Intravesical instillation of MF191 (100 nM) suppressed CYP‐induced bladder overactivity (ICI: 71.8% increase).
• ? PGE₂‐induced bladder overactivity was suppressed by MF191 (ICI: 43.2% increase).
• ? MF191 had no significant effects on other CMG variables or on control rats.

CONCLUSIONS

• ? MF191 might affect the bladder urothelium and inflammatory cells and suppresses CYP‐ or PGE₂‐induced bladder overactivity.
• ? Systemic or intravesical MF191 administration for the treatment of overactive bladder merits clinical study.

     

Augmentation of bone morphogenetic protein-induced bone mass by local delivery of a prostaglandin E EP4 receptor agonist

Recombinant human bone morphogenetic protein (rhBMP) is viewed as a therapeutic cytokine because of its ability to induce bone. However, the high doses of rhBMP required for bone induction in humans remain a major hurdle for the therapeutic application of this protein. The development of a methodology that would effectively overcome the weak responsiveness to human BMP is highly desired. In the present study, we investigate the ability of a prostaglandin E EP4 receptor selective agonist (EP4A) to augment the bone-inducing ability of BMP in a biodegradable delivery system. A block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG, polymer) was used as the delivery system. Polymer discs containing rhBMP-2 and EP4A were implanted into the left dorsal muscle pouch of mice to examine the dose-dependent effects of EP4A. Fifty mice were divided into 5 groups based on the contents of rhBMP and EP4 in the polymer (group 1; BMP 5 microg EP4A 0 microg, group 2; BMP 5 microg EP4 3 microg, group 3; BMP 5 microg EP4 30 microg, group 4; BMP 5 microg EP4 300 microg, group 5; BMP 0 microg EP4 30 microg, n=10 each). All implants were harvested, examined radiologically, and processed for histological analysis 3 weeks after surgery. On dual-energy X-ray absorptiometry (DXA) analysis, the bone mineral content (BMC) of the ossicles was 6.52+/-0.80 (mg), 9.36+/-1.89, 14.21+/-1.27, and 18.75+/-2.31 in groups 1, 2, 3, and 4 respectively. In terms of BMC, the values of groups 3 and 4 were significantly higher than those of group 1. The mean BMC value of group 4 was approximately 3 times higher than that of group 1. No significant difference in body weight was noted among the groups during the experimental period. In summary, the presence of a prostaglandin E EP4 receptor selective agonist in the carrier polymer enhanced the bone-inducing capacity of rhBMP-2 with no apparent systemic adverse effects.