Prostaglandin E2 mediates the late phase of ischemic preconditioning in the heart via its receptor subtype EP4.

Ischemic preconditioning (IPC) describes a phenomenon wherein brief ischemia of the heart induces a potent cardioprotective mechanism against succeeding ischemic insult. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostanoid biosynthesis, is upregulated in the ischemic heart and contributes to IPC. Prostaglandin E2 (PGE2) protects the heart from ischemia-reperfusion (I/R) injury via its receptor subtype EP4. We sought to clarify the role of the PGE2/EP4 system in the late phase of IPC. Mice were subjected to four IPC treatment cycles, consisting of 5 min of occlusion of the left anterior descending coronary artery (LAD). We found that COX-2 mRNA was significantly upregulated in wild-type hearts at 6 h after IPC treatment. Cardiac PGE2 levels at 24 h after IPC treatment were significantly increased in both wild-type mice and mice lacking EP4 (EP4-/-). At 24 h after IPC treatment, I/R injury was induced by 30 min of LAD occlusion followed by 2 h of reperfusion and the cardiac infarct size was determined. The infarct size was significantly reduced by IPC treatment in wild-type mice; a reduction was not observed in EP4-/- mice. AE1-329, an EP4 agonist, significantly reduced infarct size and significantly ameliorated deterioration of cardiac function in wild-type mice subjected to I/R without IPC treatment. Furthermore, AE1-329 significantly enhanced the I/R-induced activation of Akt, a pro-survival kinase. We demonstrated that the PGE2/EP4 system in the heart plays a critical role in the late phase of IPC, partly by augmenting Akt-mediated signaling. These findings clarify the mechanism of IPC and may contribute to the development of therapeutic strategies for ischemic heart disease.

Cyclooxygenase-2 is acutely induced by CCAAT/enhancer-binding protein ß to produce prostaglandin E 2 and F 2α following gonadotropin stimulation in Leydig cells.

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF 2α and PGE 2 levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein ß (C/EBPß) with cAMP-stimulation. C/EBPß expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPß via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 .

Prostaglandin I2 suppresses the development of diet-induced nonalcoholic steatohepatitis in mice.

Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of metabolic syndrome. Although the prostaglandin (PG)I2 receptor IP is expressed broadly in the liver, the role of PGI2-IP signaling in the development of NASH remains to be determined. Here, we investigated the role of the PGI2-IP system in the development of steatohepatitis using mice lacking the PGI2 receptor IP [IP-knockout (IP-KO) mice] and beraprost (BPS), a specific IP agonist. IP-KO and wild-type (WT) mice were fed a methionine- and choline-deficient diet (MCDD) for 2, 5, or 10 wk. BPS was administered orally to mice every day during the experimental periods. The effect of BPS on the expression of chemokine and inflammatory cytokines was examined also in cultured Kupffer cells. WT mice fed MCDD developed steatohepatitis at 10 wk. IP-KO mice developed steatohepatitis at 5 wk with augmented histologic derangements accompanied by increased hepatic monocyte chemoattractant protein-1 (MCP-1) and TNF-α concentrations. After 10 wk of MCDD, IP-KO mice had greater hepatic iron deposition with prominent oxidative stress, resulting in hepatocyte damage. In WT mice, BPS improved histologic and biochemical parameters of steatohepatitis, accompanied by reduced hepatic concentration of MCP-1 and TNF-α. Accordingly, BPS suppressed the LPS-stimulated Mcp-1 and Tnf-α mRNA expression in cultured Kupffer cells prepared from WT mice. PGI2-IP signaling plays a crucial role in the development and progression of steatohepatitis by modulating the inflammatory response, leading to augmented oxidative stress. We suggest that the PGI2-IP system is an attractive therapeutic target for treating patients with NASH.-Kumei, S., Yuhki, K.-I., Kojima, F., Kashiwagi, H., Imamichi, Y., Okumura, T., Narumiya, S., Ushikubi, F. Prostaglandin I2 suppresses the development of diet-induced nonalcoholic steatohepatitis in mice.

Thromboxane A2 is Involved in Itch-associated Responses in Mice with Atopic Dermatitis-like Skin Lesions.

To investigate the mechanisms underlying itching in atopic dermatitis, we examined whether thromboxane (TX) A2, an arachidonic acid metabolite, is involved in spontaneous scratching, an itch-related response, in NC mice with atopic dermatitis-like skin lesions. The TXA2 receptor (TP) antagonist ONO-3708 inhibited the spontaneous scratching. The mRNA expression of TX synthase (TXSyn) distributed mainly in epidermis and the concentration of TXB2, a metabolite of TXA2, were increased in lesional skin. Scratching caused by the PAR2 agonist SLIGRL-NH2 was suppressed by ONO-3708. SLIGRL-NH2-induced scratching decreased approximately 75% in TP-deficient mice, compared to wild-type mice. In primary cultures of mouse keratinocytes, SLIGRL-NH2 induced the production of TXA2, as evidenced by the increased TXB2, which was inhibited by the TXSyn inhibitor sodium ozagrel and a PAR2-neutralizing antibody. Taken together, these results suggest that epidermal TXA2, which may be produced via PAR2 activation, is involved in itching in atopic dermatitis.

The novel prostaglandin I2 mimetic ONO-1301 escapes desensitization in an antiplatelet effect due to its inhibitory action on thromboxane A2 synthesis in mice.

ONO-1301 [(E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylidene-aminooxy]ethyl]-7,8-dihydronaphthalene-1-yloxy]acetic acid] is a novel prostaglandin (PG) I2 mimetic with inhibitory activity on the thromboxane (TX) A2 synthase. Interestingly, ONO-1301 retains its inhibitory effect on platelet aggregation after repeated administration, while beraprost, a representative agonist for the PGI2 receptor (IP), loses its inhibitory effect after repeated administration. In the present study, we intended to clarify the mechanism by which ONO-1301 escapes desensitization of an antiplatelet effect. In platelets prepared from wild-type mice, ONO-1301 inhibited collagen-induced aggregation and stimulated cAMP production in an IP-dependent manner. In addition, ONO-1301 inhibited arachidonic acid-induced TXA2 production in platelets lacking IP. Despite the decrease in stimulatory action on cAMP production, the antiplatelet effect of ONO-1301 hardly changed after repeated administration for 10 days in wild-type mice. Noteworthy, beraprost could retain its antiplatelet effect after repeated administration in combination with a low dose of ozagrel, a TXA2 synthase inhibitor. Therefore, we hypothesized that chronic IP stimulation by beraprost induces an increase in TXA2 production, leading to reduction in the antiplatelet effect. As expected, repeated administration of beraprost increased the plasma and urinary levels of a TXA2 metabolite, while ONO-1301 did not increase them significantly. In addition, beraprost could retain the ability to inhibit platelet aggregation after repeated administration in mice lacking the TXA2 receptor (TP). These results indicate that TP-mediated signaling participates in platelet desensitization against IP agonists and that simultaneous inhibition of TXA2 production confers resistance against desensitization on IP agonists.

Prostacyclin stimulated integrin-dependent angiogenic effects of endothelial progenitor cells and mediated potent circulation recovery in ischemic hind limb model.

BACKGROUND: Prostacyclin (PGI2) enhances angiogenesis, especially in cooperation with bone marrow (BM)-derived endothelial progenitor cells (EPCs). However, the mechanisms of PGI2 in EPC-mediated angiogenesis in vivo remain unclear. The purpose of this study was to clarify the role of PGI2 in EPC-mediated angiogenesis using BM-specific IP deletion mice. METHODS AND RESULTS: Hind limb ischemia (HLI) was induced in wild-type (WT) mice transplanted with IP-deleted BM (WT/BM(IP(-/-)). Recovery of blood flow (RBF) in WT/BM(IP(-/-)) was impaired for 28 days after HLI, whereas RBF in IP(-/-)/BM(WT) was attenuated for up to 7 days compared with WT/BM(WT). The impaired RBF in WT/BM(IP(-/-)) was completely recovered by intramuscular injection of WT EPCs but not IP(-/-) EPCs. The impaired effects of IP(-/-) EPCs were in accordance with reduced formation of capillary and arterioles in ischemic muscle. An ex vivo aortic ring assay revealed that microvessel formation was enhanced by accumulation/adhesion of EPCs to perivascular sites as pericytes. IP(-/-)EPCs, in which expression of integrins was decreased, had impaired production of angiogenic cytokines, adhesion to neovessels and their angiogenic effects. The small-interfering RNA (siRNA)-mediated knockdown of integrin ß1 in WT EPCs attenuated adhesion to microvessels and their in vivo and in vitro angiogenic effects. CONCLUSIONS: PGI2 may induce persistent angiogenic effects in HLI through adhesion of EPCs to perivascular sites of neovessels via integrins in addition to paracrine effects.

The intrinsic prostaglandin E2-EP4 system of the renal tubular epithelium limits the development of tubulointerstitial fibrosis in mice.

Inflammatory responses in the kidney lead to tubulointerstitial fibrosis, a common feature of chronic kidney diseases. Here we examined the role of prostaglandin E(2) (PGE(2)) in the development of tubulointerstitial fibrosis. In the kidneys of wild-type mice, unilateral ureteral obstruction leads to progressive tubulointerstitial fibrosis with macrophage infiltration and myofibroblast proliferation. This was accompanied by an upregulation of COX-2 and PGE(2) receptor subtype EP(4) mRNAs. In the kidneys of EP(4) gene knockout mice, however, obstruction-induced histological alterations were significantly augmented. In contrast, an EP(4)-specific agonist significantly attenuated these alterations in the kidneys of wild-type mice. The mRNAs for macrophage chemokines and profibrotic growth factors were upregulated in the kidneys of wild-type mice after ureteral obstruction. This was significantly augmented in the kidneys of EP(4)-knockout mice and suppressed by the EP(4) agonist but only in the kidneys of wild-type mice. Notably, COX-2 and MCP-1 proteins, as well as EP(4) mRNA, were localized in renal tubular epithelial cells after ureteral obstruction. In cultured renal fibroblasts, another EP(4)-specific agonist significantly inhibited PDGF-induced proliferation and profibrotic connective tissue growth factor production. Hence, an endogenous PGE(2)-EP(4) system in the tubular epithelium limits the development of tubulointerstitial fibrosis by suppressing inflammatory responses.

Thromboxane A2 and prostaglandin F2alpha mediate inflammatory tachycardia.

Systemic inflammation induces various adaptive responses including tachycardia. Although inflammation-associated tachycardia has been thought to result from increased sympathetic discharge caused by inflammatory signals of the immune system, definitive proof has been lacking. Prostanoids, including prostaglandin (PG) D(2), PGE(2), PGF(2alpha), PGI(2) and thromboxane (TX) A(2), exert their actions through specific receptors: DP, EP (EP(1), EP(2), EP(3), EP(4)), FP, IP and TP, respectively. Here we have examined the roles of prostanoids in inflammatory tachycardia using mice that lack each of these receptors individually. The TXA(2) analog I-BOP and PGF(2alpha) each increased the beating rate of the isolated atrium of wild-type mice in vitro through interaction with TP and FP receptors, respectively. The cytokine-induced increase in beating rate was markedly inhibited in atria from mice lacking either TP or FP receptors. The tachycardia induced in wild-type mice by injection of lipopolysaccharide (LPS) was greatly attenuated in TP-deficient or FP-deficient mice and was completely absent in mice lacking both TP and FP. The beta-blocker propranolol did not block the LPS-induced increase in heart rate in wild-type animals. Our results show that inflammatory tachycardia is caused by a direct action on the heart of TXA(2) and PGF(2alpha) formed under systemic inflammatory conditions.

Facilitation of colonic T cell immune responses is associated with an exacerbation of dextran sodium sulfate-induced colitis in mice lacking microsomal prostaglandin E synthase-1.

BACKGROUND: Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme that acts downstream of cyclooxygenase and plays a major role in inflammation by converting prostaglandin (PG) H2 to PGE2. The present study investigated the effect of genetic deletion of mPGES-1 on the development of immunologic responses to experimental colitis induced by dextran sodium sulfate (DSS), a well-established model of inflammatory bowel disease (IBD). METHODS: Colitis was induced in mice lacking mPGES-1 (mPGES-1-/- mice) and wild-type (WT) mice by administering DSS for 7 days. Colitis was assessed by body weight loss, diarrhea, fecal bleeding, and histological features. The colonic expression of mPGES-1 was determined by real-time PCR, western blotting, and immunohistochemistry. The impact of mPGES-1 deficiency on T cell immunity was determined by flow cytometry and T cell depletion in vivo. RESULTS: After administration of DSS, mPGES-1-/- mice exhibited more severe weight loss, diarrhea, and fecal bleeding than WT mice. Histological analysis further showed significant exacerbation of colonic inflammation in mPGES-1-/- mice. In WT mice, the colonic expression of mPGES-1 was highly induced on both mRNA and protein levels and colonic PGE2 increased significantly after DSS administration. Additionally, mPGES-1 protein was localized in the colonic mucosal epithelium and infiltrated inflammatory cells in underlying connective tissues and the lamina propria. The abnormalities consistent with colitis in mPGES-1-/- mice were associated with higher expression of colonic T-helper (Th)17 and Th1 cytokines, including interleukin 17A and interferon-γ. Furthermore, lack of mPGES-1 increased the numbers of Th17 and Th1 cells in the lamina propria mononuclear cells within the colon, even though the number of suppressive regulatory T cells also increased. CD4+ T cell depletion effectively reduced symptoms of colitis as well as colonic expression of Th17 and Th1 cytokines in mPGES-1-/- mice, suggesting the requirement of CD4+ T cells in the exacerbation of DSS-induced colitis under mPGES-1 deficiency. CONCLUSIONS: These results demonstrate that mPGES-1 is the main enzyme responsible for colonic PGE2 production and deficiency of mPGES-1 facilitates the development of colitis by affecting the development of colonic T cell-mediated immunity. mPGES-1 might therefore impact both the intestinal inflammation and T cell-mediated immunity associated with IBD.

Thromboxane receptor activation enhances striatal dopamine release, leading to suppression of GABAergic transmission and enhanced sugar intake.

The extracellular dopamine level is regulated not only by synaptic inputs to dopamine neurons but also by local mechanisms surrounding dopaminergic terminals. However, much remains to be investigated for the latter mechanism. Thromboxane A(2) is one of the cyclooxygenase products derived from arachidonic acid, and acts on its cognate G protein-coupled receptor [thromboxane receptor (TP)]. We show here that TP in the striatum locally facilitates dopamine overflow. Intrastriatal injection of a TP agonist increased extracellular dopamine levels in the striatum as measured by in vivo microdialysis. TP stimulation also augmented electrically evoked dopamine overflow from striatal slices. Conversely, TP deficiency reduced dopamine overflow evoked by N-methyl-d-aspartic acid (NMDA) and acetylcholine in striatal slices. TP immunostaining showed that TP is enriched in vascular endothelial cells. Pharmacological blockade of nitric oxide (NO) synthesis and genetic deletion of endothelial NO synthase (eNOS) suppressed NMDA/acetylcholine-induced dopamine overflow. This involvement of NO was abolished in TP-deficient slices, suggesting a role for eNOS-derived NO synthesis in TP-mediated dopamine overflow. As a functional consequence of TP-mediated dopamine increase, a TP agonist suppressed GABAergic inhibitory postsynaptic currents in medium spiny neurons through a D2-like receptor-dependent mechanism. Finally, TP is involved in sucrose intake, a dopamine-dependent motivational behavior. These data suggest that TP stimulation in the striatum locally facilitates dopamine overflow evoked by synaptic inputs via NO synthesis in endothelial cells.

Prostaglandin I2 promotes recruitment of endothelial progenitor cells and limits vascular remodeling.

OBJECTIVE: Endothelial progenitor cells (EPCs) play an important role in the self-healing of a vascular injury by participating in the reendothelialization that limits vascular remodeling. We evaluated whether prostaglandin I(2) plays a role in the regulation of the function of EPCs to limit vascular remodeling. METHODS AND RESULTS: EPCs (Lin(-)cKit(+)Flk-1(+) cells) were isolated from the bone marrow (BM) of wild-type (WT) mice or mice lacking the prostaglandin I(2) receptor IP (IP(-/-) mice). Reverse transcription-polymerase chain reaction analysis showed that EPCs among BM cells specifically express IP. The cellular properties of EPCs, adhesion, migration, and proliferation on fibronectin were significantly attenuated in IP-deficient EPCs compared with WT EPCs. In contrast, IP agonists facilitated these functions in WT EPCs, but not in IP-deficient EPCs. The specific deletion of IP in BM cells, which was performed by transplanting BM cells of IP(-/-) mice to WT mice, accelerated wire injury-mediated neointimal hyperplasia in the femoral artery. Notably, transfused WT EPCs, but not IP-deficient EPCs, were recruited to the injured vessels, participated in reendothelialization, and efficiently rescued the accelerated vascular remodeling. CONCLUSIONS: These findings clearly indicate that the prostaglandin I(2)-IP system is essential for EPCs to accomplish their function and plays a critical role in the regulation of vascular remodeling.

Prostacyclin in vascular diseases. - Recent insights and future perspectives -.

Prostacyclin (PGI(2)) is one of the important vascular prostanoids, the effects of which counteract those of thromboxane (TXA(2)), and these 2 prostanoids provide an important balance in cardiovascular homeostasis. The clinical experience of COX-2 selective inhibitors having unexpected adverse effects in patients with cardiovascular risk has opened up a debate about the role of COX-2-derived prostanoids in vascular pathophysiology. PGI(2) is a major anti-atherogenic prostanoid produced by COX-2 in vascular cells, including endothelial and vascular smooth muscle cells. The balance between COX-2-derived PGI(2), COX-1-derived TXA(2), and other COX-2-mediated atherogenic prostanoids is a crucial factor in determining pathophysiological outcomes. Recent studies using stable PGI(2) analogs and genetically deficient mice have revealed novel effects of PGI(2) on its target cells, such as endothelial and endothelial progenitor cells. The role PGI(2) in the physiology and pathophysiology of vascular diseases is reviewed and the recent findings linking PGI(2), COX-2 and atherothrombosis are summarized.

Evaluation of 17ß-hydroxysteroid dehydrogenase activity using androgen receptor-mediated transactivation.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) catalyze the reduction of 17-ketosteroids and the oxidation of 17ß-hydroxysteroids to regulate the production of androgens and estrogens. Among them, 17ß-HSD type 3 (HSD17B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of 17ß-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of 17ß-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD17B1 and HSD17B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B3. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins.

Prostaglandin F2α Facilitates Platelet Activation by Acting on Prostaglandin E2 Receptor Subtype EP3 and Thromboxane A2 Receptor TP in Mice.

Platelets play an important role in both physiological hemostasis and pathological thrombosis. Thromboxane (TX) A2 and prostaglandin (PG) I2 are well known as a potent stimulator and an inhibitor of platelet function, respectively. Recently, PGE2 has also been reported to regulate platelet function via PGE2 receptor subtypes. However, the effect of PGF2α on platelet function remains to be determined. The aim of the present study was to clarify the effect of PGF2α on murine platelet function both in vitro and in vivo. Platelets prepared from wild-type mice (WT platelets) expressed several types of prostanoid receptors, including the PGE2 receptor subtype EP3 and the TXA2 receptor TP, while expression of the PGF2α receptor FP was not detected. In WT platelets, PGF2α potentiated adenosine diphosphate-induced aggregation in a concentration-dependent manner, while PGF2α alone did not induce aggregation. In platelets prepared from mice lacking FP, however, PGF2α-induced potentiation was not significantly different from that in WT platelets. Interestingly, the potentiation was significantly blunted in platelets lacking EP3 or TP and disappeared completely in platelets lacking both EP3 and TP. Accordingly, PGF2α decreased the cyclic adenosine monophosphate level via EP3 and increased the inositol triphosphate level via TP in WT platelets. Intravenously administered PGF2α significantly shortened the bleeding time and aggravated arachidonic acid-induced acute thromboembolism in WT mice, suggesting that PGF2α works as a platelet stimulator also in vivo. In conclusion, PGF2α potentiates platelet aggregation in vitro via EP3 and TP but not FP. Accordingly, PGF2α facilitates hemostasis and thromboembolism in vivo.

Prostaglandin I2 plays a key role in zymosan-induced mouse pleurisy.

Zymosan, the cell wall of Saccharomyces cerevisiae, induces innate immune responses involving prostanoid production and complement activation. However, the roles of prostanoids in zymosan-induced inflammation and their interaction with the complement system remain to be determined. To clarify these issues, we examined zymosan-induced pleurisy in mice lacking receptors for prostaglandin (PG) E(2) (EP(-/-) mice) or PGI(2) (IP(-/-) mice). Zymosan-induced exudate formation was significantly reduced in IP(-/-) mice compared with wild-type (WT) mice, whereas none of the EP(-/-) mice (EP(1)(-/-), EP(2)(-/-), EP(3)(-/-), and EP(1)(-/-)(4) mice) showed any significant difference from WT mice. Furthermore, indomethacin, an inhibitor of prostanoid biosynthesis, suppressed exudate formation in WT mice to almost the same level as that of IP(-/-) mice. Accordingly, significant production of PGI(2) in the pleural cavity, suggested to be cyclooxygenase-2-dependent, was observed after zymosan injection. Complement activation in the pleural cavity after zymosan injection was confirmed, and preinjection of cobra venom factor (CVF), to deplete blood complement C3, was significantly suppressed after zymosan-induced exudate formation in WT mice. Simultaneous treatment with indomethacin and CVF further suppressed exudate formation in WT mice compared with each treatment alone. Because, some degree of exudate formation was still observed, other factor(s) seem to be involved. However, platelet-activating factor, a promising candidate as one such factor, was not involved in zymosan-induced exudate formation. These results clearly indicate that the PGI(2)-IP system together with the complement system plays a key role in exudate formation in zymosan-induced pleurisy.

Decreased susceptibility to renovascular hypertension in mice lacking the prostaglandin I2 receptor IP.

Persistent reduction of renal perfusion pressure induces renovascular hypertension by activating the renin-angiotensin-aldosterone system; however, the sensing mechanism remains elusive. Here we investigated the role of PGI2 in renovascular hypertension in vivo, employing mice lacking the PGI2 receptor (IP-/- mice). In WT mice with a two-kidney, one-clip model of renovascular hypertension, the BP was significantly elevated. The increase in BP in IP-/- mice, however, was significantly lower than that in WT mice. Similarly, the increases in plasma renin activity, renal renin mRNA, and plasma aldosterone in response to renal artery stenosis were all significantly lower in IP-/- mice than in WT mice. All these parameters were measured in mice lacking the four PGE2 receptor subtypes individually, and we found that these mice had similar responses to WT mice. PGI2 is produced by COX-2 and a selective inhibitor of this enzyme, SC-58125, also significantly reduced the increases in plasma renin activity and renin mRNA expression in WT mice with renal artery stenosis, but these effects were absent in IP-/- mice. When the renin-angiotensin-aldosterone system was activated by salt depletion, SC-58125 blunted the response in WT mice but not in IP-/- mice. These results indicate that PGI2 derived from COX-2 plays a critical role in regulating the release of renin and consequently renovascular hypertension in vivo.

Cigarette Smoke Extract Inhibits Platelet Aggregation by Suppressing Cyclooxygenase Activity.

The results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A 2 receptor agonist, and that induced by collagen with respective IC 50 values of 1.05 ± 0.14% and 1.34 ± 0.19%. A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid-induced TXA 2 production in murine platelets with an IC 50 value of 7.32 ± 2.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA 2 receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I 2 receptor and PGE 2 receptor subtypes EP 2 and EP 4 , were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA 2 production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC 50 value of 1.07 ± 0.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA 2 production in platelets, leading to inhibition of platelet aggregation.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary.

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.