Roles of prostanoids in the pathogenesis of cardiovascular diseases.

The roles of prostanoids in the pathogenesis of cardiovascular diseases and in the development of pathological conditions have been examined using mice lacking the individual, specific prostanoid receptor. Prostaglandin (PG) I2 protected the heart from ischemia-reperfusion injury in a model of acute myocardial infarction. In addition, PGI2 suppressed the development of pressure overload-induced cardiac hypertrophy. Aside from its potent vasodilatory action, PGI2 contributed critically to the development of renovascular hypertension via the activation of the renin-angiotensin-aldosterone system. Thromboxane (TX) A2 and PGF2alpha were found to be the mediators of inflammatory tachycardia under a systemic inflammatory condition induced by lipopolysaccharide. Under a septic condition leading to a vascular hypo-responsive state, TXA2 worked to maintain vascular tone by inhibiting the induction of inducible nitric oxide synthase in vascular smooth muscle cells. Mice lacking the PGE2 receptor subtype EP3 had a bleeding tendency and were resistant to thromboembolism, due to a defective activation of platelets. From these studies, the important and novel roles of prostanoids in the pathogenesis of cardiovascular diseases have been clarified.

Roles of prostaglandin I(2) and thromboxane A(2) in cardiac ischemia-reperfusion injury: a study using mice lacking their respective receptors.

BACKGROUND: Prostaglandin (PG) I(2) and thromboxane (TX) A(2), the most common prostanoids in the cardiovascular system, are produced abundantly during cardiac ischemia/reperfusion (I/R); their roles in I/R injury, however, remain undetermined. We intended to clarify these roles of PGI(2) and TXA(2) using mice lacking the PGI(2) receptor, IP(-/-) mice, or the TXA(2) receptor, TP(-/-) mice. METHODS AND RESULTS: The left anterior descending coronary artery was occluded for 1 hour and then reperfused for 24 hours. The size of myocardial infarct in IP(-/-) mice was significantly larger than that in wild-type mice, although the size of the area at risk was similar between the 2 groups of mice. In contrast, there was no such difference between TP(-/-) and wild-type mice. To further determine whether PGI(2) and TXA(2) act directly on the cardiac tissue or indirectly through their action on blood constituents, we perfused excised heart according to the Langendorff technique. The isolated heart was then subjected to global ischemia followed by reperfusion. In IP(-/-) mice, developed tension and coronary flow rate during reperfusion were significantly lower and release of creatine kinase was significantly higher than those in wild-type mice. There were no such differences, however, between TP(-/-) and wild-type mice. CONCLUSIONS: PGI(2), which was produced endogenously during cardiac I/R, exerts a protective effect on cardiomyocytes independent of its effects on platelets and neutrophils. In contrast, TXA(2) has little role in the cardiac I/R injury.

Increased bleeding tendency and decreased susceptibility to thromboembolism in mice lacking the prostaglandin E receptor subtype EP(3).

BACKGROUND: Among the prostanoids, thromboxane (TX) A(2) is a potent stimulator of platelets, whereas prostaglandin (PG) I(2) inhibits their activation. The roles of PGE(2) in the regulation of platelet function have not been established, however, and the contribution of PGE(2) in hemostasis and thromboembolism is poorly understood. The present study was intended to clarify these roles of PGE(2) by using mice lacking the PGE(2) receptor subtype 3 (EP(3)(-/-) mice). METHODS AND RESULTS: Expression of mRNAs for EP(3) in murine platelets was confirmed by quantitative reverse transcription-polymerase chain reaction. PGE(2) and AE-248, a selective EP(3) agonist, showed concentration-dependent potentiation of platelet aggregation induced by U46619, a TXA(2) receptor agonist, although PGE(2) alone could not induce aggregation. PGE(2) and AE-248 increased cytosolic calcium ion concentration ([Ca(2+)](i)), and AE-248 inhibited the forskolin-induced increase in cytosolic cAMP concentration ([cAMP](i)), suggesting G(i) coupling of EP(3). The potentiating effects of PGE(2) and AE-248 on platelet aggregation along with their effects on [Ca(2+)](i) and [cAMP](i) were absent in EP(3)(-/-) mice. In vivo, the bleeding time was significantly prolonged in EP(3)(-/-) mice. Moreover, when mice were challenged intravenously with arachidonic acid, mortality and thrombus formation in the lung were significantly reduced in EP(3)(-/-) mice. CONCLUSIONS: - PGE(2) potentiated platelet aggregation induced by U46619 via EP(3) by increasing [Ca(2+)](i), decreasing [cAMP](i), or both. This potentiating action of PGE(2) via EP(3) is essential in mediating both physiological and pathological effects of PGE(2) in vivo.

Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice.

Arachidonic acid is metabolized to prostaglandin H(2) (PGH(2)) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH(2), PGE(2) is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE(2), EP2, causes decreases in number and size of intestinal polyps in Apc(Delta 716) mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE(2) through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE(2) receptors, EP1 or EP3, did not affect intestinal polyp formation in Apc(Delta 716) mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma.

Major roles of prostanoid receptors IP and EP(3) in endotoxin-induced enhancement of pain perception.

To know the roles of prostaglandin I (IP) and prostaglandin E (EP) receptors in pain perception, we compared the acetic acid-induced writhing response in mice deficient in prostaglandin receptors, i.e. IP, EP(1,) EP(2,) EP(3,) or EP(4,) with or without lipopolysaccharide (LPS) pretreatment. Without LPS pretreatment, IP-receptor deficient mice showed a significantly smaller number of responses, as previously reported, whereas mice deficient in any of the EP-receptor subtypes showed a number of writhings similar to those of wild-type mice. When mice were pretreated with LPS for 24 hr to induce cyclooxygenase-2 expression, the wild-type as well as EP(1)-, EP(2)-, or EP(4)-receptor-deficient mice showed a similar enhanced writhing response, whereas IP- and EP(3)-receptor-deficient mice had a significantly less enhanced number of writhings. These results indicate that IP and EP(3) are the major prostaglandin receptors mediating the enhanced acetic acid-induced writhing response in mice pre-exposed to LPS, i.e. in endotoxin-enhanced inflammatory nociception.

[Pain, fever and prostanoids].

Although it has been known that prostanoids are involved in pain regulation and fever, the precise roles of their receptors and receptor subtypes are unclear. All prostanoid receptors have been cloned and mice deficient in each receptor have been developed. Recent studies using prostanoid-receptor-knockout mice are shedding some light on these issues. Nociceptive responses to an intraperitoneal injection of acetic acid and hyperalgesia induced by carrageenan were abolished by IP-receptor deficiency. In addition, the use of mice lacking prostanoid receptor is revealing an interesting role of prostanoid in neuropathic as well as inflammatory pain. With regard to pyrexia, PGE2 injected intracerebroventricularly induced the febrile response in wild-type mice, but it was without effect in mice lacking the EP3 receptor. Furthermore, febrile responses induced by IL-1 beta, an endogenous pyrogen, and LPS, an exogenous pyrogen, were specifically suppressed in mice lacking the EP3 receptor. These results indicate that PGE2 works as a common final mediator of the febrile response and that this action of PGE2 is mediated by the EP3 receptor. The determination of precise roles of prostanoids in pain and fever may provide novel targets for antipyretic analgesics with fewer side effects.

Characterization of EP receptor subtypes responsible for prostaglandin E2-induced pain responses by use of EP1 and EP3 receptor knockout mice.

Prostaglandin E2 (PGE2) is known to be the principal pro-inflammatory prostanoid and play an important role in nociception. To identify PGE receptor (EP) subtypes that mediate pain responses to noxious and innocuous stimuli, we studied them by use of EP1 and EP3 knockout (EP1(-/-) and EP3(-/-)) mice. PGE2 could induce mechanical allodynia in EP1(+/+), EP3(+/+) and EP3(-/-) mice, but not in EP1(-/-) mice. N-methyl-D-aspartate (NMDA), the substrate of nitric oxide (NO) synthase L-arginine, or the NO donor sodium nitroprusside administered intrathecal (i.t.) could induce allodynia in EP3(-/-) and EP1(-/-) mice. Activation of EP1 receptors appears to be upstream, rather than downstream, of NMDA receptor activation and NO production in the PGE2-induced allodynia. Although PGE2 produced thermal hyperalgesia over a wide range of dosages from 50 pg to 0.5 microg kg(-1) in EP3(+/+) mice, it showed a monophasic hyperalgesic action at 5 ng kg(-1) or higher doses in EP3(-/-) mice. The selective EP3 agonist, ONO-AE-248, induced hyperalgesia at 500 pg kg(-1) in EP3(+/+) mice, but not in EP3(-/-) mice. Saline-injected EP1(-/-) mice showed hyperalgesia, which was reversed by i.t. PGE2 in a dose-dependent manner. There was no significant difference in the formalin-induced behaviours between EP1(-/-) or EP3(-/-) mice and the cognate wild-type mice. These results demonstrate that spinal EP1 receptors are involved in the PGE2-induced allodynia and that spinal EP3 receptors are involved in the hyperalgesia induced by low doses of PGE2. However, the formalin-induced pain cannot be ascribed to a single EP receptor subtype EP1 or EP3.

Regulation of TNFalpha and interleukin-10 production by prostaglandins I(2) and E(2): studies with prostaglandin receptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists.

To know which receptors of prostaglandins are involved in the regulation of TNFalpha and interleukin 10 (IL-10) production, we examined the production of these cytokines in murine peritoneal macrophages stimulated with zymosan. The presence of PGE(2) or the PGI(2) analog carbacyclin in the medium reduced the TNFalpha production to one-half, whereas IL-10 production increased several fold; and indomethacin caused the reverse effects, suggesting that endogenous prostaglandins may have a regulatory effect on the cytokine production. Among prostaglandin E (EP) receptor-selective synthetic agonists, EP2 and EP4 agonists caused down-regulation of the zymosan-induced TNFalpha production, but up-regulation on the IL-10 production; while EP1 and EP3 agonists showed no effect. Macrophages harvested from prostaglandin I (IP) receptor-deficient mice showed the up- and down-regulatory effects on the cytokine production by the EP2 and EP4 agonists or PGE(2), but no effect was obtained by carbacyclin. On the contrary, macrophages from EP2-deficient mice showed the effect by PGE(2), carbacyclin, and the EP4 agonist, but not by the EP2 agonist; and the cells from EP4-deficient mice showed the effect by PGE(2), carbacyclin, and EP2 agonist, but not by the EP4 agonist. These functional effects of prostaglandins well accorded with the mRNA expression of TNFalpha and IL-10 when such expression was examined by the RT-PCR method. The peritoneal macrophages from normal mice expressed IP, EP2, and EP4 receptors, but not EP1 and EP3, when examined by RT-PCR. Thus the results suggest that PGI(2) and PGE(2) generated simultaneously with cytokines by macrophages treated with zymosan may influence the cytokine production through IP, EP2, and EP4 receptors.

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats.

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.

Impaired bone resorption by lipopolysaccharide in vivo in mice deficient in the prostaglandin E receptor EP4 subtype.

In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions.

Advanced glycation end products-induced gene expression of scavenger receptors in cultured human monocyte-derived macrophages.

We investigated the effects of advanced glycation end products (AGEs) on the expression of oxidized low-density lipoprotein (OxLDL) receptors in human monocyte-derived macrophages and THP-1 cells treated with PMA. Both RT-PCR procedure and Northern blot analysis revealed that AGEs induced not only the gene expression of two major OxLDL receptors, macrophage scavenger receptor (MSR) class A and CD36, but also MSR-B I and lectin-like oxidized low-density lipoprotein receptor 1. Also, as a result of gel shift assay, AGEs increased transcriptional activities of AP-1, NF-kappaB, and peroxisome proliferator-activated receptor gamma. These findings indicate that AGEs-induced enhancement of these transcriptional activities might be involved in increased levels of mRNA for some of OxLDL receptors in THP-1-cells treated with PMA. The upregulated surface expression of these receptors on macrophage membranes was closely associated with increased uptake of modified LDL, and culminated in enhanced foam cell transformation. Thus, AGEs may be involved in the cause of variable levels of foam cell formation via the increased numbers of OxLDL receptors in accelerated atherosclerotic lesions of individuals with diabetes.

Characterization of prostanoid receptors mediating contraction of the gastric fundus and ileum: studies using mice deficient in prostanoid receptors.

Receptors mediating prostanoid-induced contractions of longitudinal sections of gastric fundus and ileum were characterized by using tissues obtained from mice deficient in each type and subtype of prostanoid receptors. The fundus and ileum from mice deficient in either EP(3) (EP(3)(-/-) mice), EP(1) (EP(1)(-/-) mice) and FP (FP(-/-) mice) all showed decreased contraction to PGE(2) compared to the tissues from wild-type mice, whereas contraction of the fundus slightly increased in EP(4)(-/-) mice. 17-phenyl-PGE(2) also showed decreased contraction of the fundus from EP(3)(-/-), EP(1)(-/-) and FP(-/-) mice. Sulprostone showed decreased contraction of the fundus from EP(3)(-/-) and FP(-/-) mice, and decreased contraction of the ileum to this compound was seen in tissues from EP(3)(-/-), EP(1)(-/-) and FP(-/-) mice. In DP(-/-) mice, sulprostone showed increased contraction. DI-004 and AE-248 caused the small but concentration-dependent contraction of both tissues, and these contractions were abolished in tissues obtained from EP(1)(-/-) and EP(3)(-/-) mice, respectively, but not affected in other mice. Contractions of both fundus and ileum to PGF(2)alpha was absent at lower concentrations (10(-9) to 10(-7) M), and suppressed at higher concentrations (10(-6) to 10(-5) M) of the agonist in the FP(-/-) mice. Suppression of the contractions at the higher PGF(2)alpha concentrations was also seen in the fundus from EP(3)(-/-), EP(1)(-/-) and TP(-/-) mice and in the ileum from EP(3)(-/-) and TP(-/-) mice. Contraction of the fundus to PGD(2) was significantly enhanced in DP(-/-) mice, and contractions of the fundus and ileum to this PG decreased in FP(-/-) and EP(3)(-/-) mice. Contractions of both tissues to I-BOP was absent at 10(-9) to 10(-7) M and much suppressed at higher concentrations in TP(-/-) mice. Slight suppression to this agonist was also observed in the tissues from EP(3)(-/-) mice. PGI(2) induced small relaxation of both tissues from wild-type mice. These relaxation reactions were much potentiated in EP(3)(-/-) mice. On the other hand, significant contraction to PGI(2) was observed in both tissues obtained from IP(-/-) mice. These results show that contractions of the fundus and ileum induced by each prostanoid agonist are mediated by actions of this agonist on multiple types of prostanoid receptors and in some cases modified by its action on relaxant receptors.

Roles of prostanoids revealed from studies using mice lacking specific prostanoid receptors.

The actions of prostanoids in various physiological and pathophysiological conditions have been being examined using mice lacking different prostanoid receptors. Prostaglandin (PG) I2 worked not only as a mediator of inflammation but also as an antithrombotic agent. PGF2alpha was found to be an essential inducer of labor. Several important actions of PGE2 are exerted via each of the four PGE2 receptor subtypes: EP1, EP2, EP3 and EP4. PGE2 participated in colon carcinogenesis via the EP1. PGE2 also participates in ovulation and fertilization and contributes to the control of blood pressure under high-salt intake via the EP2. PGE2 worked as a mediator of febrile responses to both endogenous and exogenous pyrogens and as a regulator of bicarbonate secretion induced by acid-stimulation in the duodenum via the EP3. It regulated the closure of ductus arteriosus and showed bone resorbing action via the EP4. PGD2 was found to be a mediator of allergic asthma. These studies have revealed important roles of prostanoids, some of which had not previously been known.

Ranolazine attenuates palmitoyl-L-carnitine-induced mechanical and metabolic derangement in the isolated, perfused rat heart.

The effect of ranolazine, a novel anti-ischaemic drug that stimulates the activity of pyruvate dehydrogenase, on palmitoyl-L-carnitine-induced mechanical dysfunction and metabolic derangement in isolated perfused rat hearts has been studied and compared with the effect of dichloroacetate, an activator of pyruvate dehydrogenase. Rat hearts paced electrically were perfused aerobically at constant flow by the Langendorff technique. Palmitoyl-L-carnitine (4 microM) increased left ventricular end-diastolic pressure and reduced left ventricular developed pressure (i.e. induced mechanical dysfunction); it also reduced tissue levels of adenosine triphosphate and increased tissue levels of adenosine monophosphate (i.e. induced metabolic derangement). These functional and metabolic alterations induced by palmitoyl-L-carnitine were attenuated by ranolazine (5, 10, and 20 microM) in a concentration-dependent manner. In contrast, dichloroacetate (1 and 10 mM) did not attenuate palmitoyl-L-carnitine-induced mechanical and metabolic derangement. In the normal (palmitoyl-L-carnitine-untreated) heart, however, ranolazine did not modify mechanical function and energy metabolism. These results suggest that ranolazine attenuates palmitoyl-L-carnitine-induced mechanical and metabolic derangement in the rat heart, and that the beneficial action of ranolazine is not because of the energy-sparing effect or activation of pyruvate dehydrogenase.

Amino acid residues conferring ligand binding properties of prostaglandin I and prostaglandin D receptors. Identification by site-directed mutagenesis.

Using chimeras of the mouse prostaglandin (PG) I receptor (mIP) and the mouse PGD receptor (mDP), we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, whereas that of mDP binds the D type of PGs alone (Kobayashi, T., Kiriyama, M., Hirata, T., Hirata, M., Ushikubi, F., and Narumiya, S. (1997) J. Biol. Chem. 272, 15154-15160). In the present study, we performed a more detailed chimera analysis, and narrowed the domain for the ring recognition to a region from the first transmembrane domain to the first extracellular loop. One chimera with the replacement of the second transmembrane domain and the first extracellular loop of mDP with that of mIP bound only iloprost. The amino acid substitutions in this chimera suggest that Ser(50) in the first transmembrane domain of mIP confers the broad ligand recognition of mIP and that Lys(75) and Leu(83) in the second transmembrane domain of mDP confer the high affinity to PGD(2) and the strict specificity of ligand binding of mDP, respectively.

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice.

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.

The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs.

PGE2 functions as a potent stimulator of bone resorption. The action of PGE2 is thought to be mediated by some PGE receptor subtypes present in osteoblastic cells. In this study, we examined the involvement of PGE receptor subtypes, EP1, EP2, EP3, and EP4, in PGE2-induced bone resorption using specific agonists for the respective EPs. In mouse calvaria cultures, EP4 agonist markedly stimulated bone resorption, but its maximal stimulation was less than that induced by PGE2. EP2 agonist also stimulated bone resorption, but only slightly. EP1 and EP3 agonists did not stimulate it at all. RT-PCR showed that osteoblastic cells isolated from newborn mouse calvaria expressed all of the EPs messenger RNA (mRNA). Both EP2 agonist and EP4 agonist induced cAMP production and the expression of osteoclast differentiation factor (ODF) mRNA in osteoblastic cells. Simultaneous addition of EP2 and EP4 agonists cooperatively induced cAMP production and ODF mRNA expression. In mouse bone marrow cultures, EP2 and EP4 agonists moderately induced osteoclast formation, but the simultaneous addition of the two agonists cooperatively induced it, similar to that by PGE2. In calvaria culture from EP4 knockout mice, a marked reduction in bone resorption to PGE2 was found. In EP4 knockout mice, EP4 agonist failed to induce bone resorption, but EP2 agonist slightly, but significantly, induced bone resorption. These findings suggest that PGE2 stimulates bone resorption by a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2.

Intrinsic prostacyclin contributes to exudation induced by bradykinin or carrageenin: a study on the paw edema induced in IP-receptor-deficient mice.

To prove that prostaglandin I2 (PGI2) is a major prostaglandin involved in bradykinin-induced exudation, we examined carrageenin- or bradykinin-induced paw edema in prostacyclin receptor-deficient mice (IPKO). Paw volume of wild-type mice (IPWT) increased gradually 5-6 hr after the carrageenin injection in a similar manner as in ICR mice, but the swelling in IPKO mice was significantly smaller (about 60% of the IPWT volume). Indomethacin, at 10 mg/kg, suppressed the swelling of the IPWT paw to the level of the non-pretreated IPKO, which was not affected by indomethacin, confirming the previous result that PGI2 is a major prostaglandin involved in the swelling. The paw edema of IPWT and IPKO was significantly attenuated by the nonpeptide bradykinin B2-receptor antagonist FR173657, at 30 mg/kg, to the same level of swelling, indicating kinin involvement. Injection of bradykinin (1.2 nmole) into the paw caused rapid edema, which peaked around 15 min in both mice. However, the edema induced in IPKO was smaller and almost at the same level as that elicited in the indomethacin-treated IPWT, suggesting that edema induced by bradykinin includes the intrinsic effect of PGI2. Concomitant injection of carbacyclin with bradykinin caused enhancement of edema in IPWT mice but not in IPKO mice, indicating that intrinsic PGI2 could cause enhancement of bradykinin- or even carrageenin-induced edema formation. These results clearly demonstrate that bradykinin released by carrageenin may be a key mediator to induce PGI2 formation, and both autacoids work together to induce enhanced inflammatory exudation.

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.