Akinori Ueno

Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2.

Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE2 biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathioneS-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE2 generation. Arg110, a residue well conserved in the microsomal GSHS-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A2 allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE2 biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.

Altered pain perception and inflammatory response in mice lacking prostacyclin receptor

Prostanoids are a group of bioactive lipids working as local mediators and include D, E, F and I types of prostaglandins (PGs) and thromboxanes. Prostacyclin (PGI2) acts on platelets and blood vessels to inhibit platelet aggregation and to cause vasodilatation, and is thought to be important for vascular homeostasis. Aspirin-like drugs, including indomethacin, which inhibit prostanoid biosynthesis, suppress fever, inflammatory swelling and pain, and interfere with female reproduction, suggesting that prostanoids are involved in these processes,, although it is not clear which prostanoid is the endogenous mediator of a particular process. Prostanoids act on seven-transmembrane-domain receptors which are selective for each type. Here we disrupt the gene for the prostacyclin receptor in mice by using homologous recombination. The receptor-deficient mice are viable, reproductive and normotensive. However, their susceptibility to thrombosis is increased, and their inflammatory and pain responses are reduced to the levels observed in indomethacin-treated wild-type mice. Our results establish that prostacyclin is an antithrombotic agent in vivo and provide evidence for its role as a mediator of inflammation and pain.

Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice.

We found that intraperitoneal injection of organic acids, such as propionic and lactic acid, are able to develop writhing responses in mice similarly as that of acetic acid. These acid-induced writhing reactions were significantly attenuated by capsazepine, a VR1 receptor-specific antagonist, but the phenylbenzoquinone-induced one was not, suggesting that the acids but not phenylbenzoquinone activate the VR1 receptor, which is involved in polymodal pain perception. Hoe 140, a bradykinin B2 receptor antagonist, also suppressed the acid-induced writhing response. Furthermore, these writhing responses were significantly suppressed after neonatal treatment with capsaicin, which treatment is known to destroy peripheral sensory afferent C-fibers. Capsazepine and Hoe 140 did not further attenuate the already reduced writhing responses of capsaicin-treated mice, suggesting that the acids stimulate the VR1 and the bradykinin B2 receptor in the pathway comprising sensory afferent C-fibers. On the other hand, indomethacin further significantly suppressed the writhing number of the capsaicin-treated animals, suggesting that the acid-induced pain perception requires prostanoid receptors not only in the pathway via capsaicin-sensitive C-fibers but also in other sensory pathways. These results provide the first evidence for the involvement of the vanilloid receptor in the acid-induced inflammatory pain perception via sensory C-fibers in addition to the known mediators bradykinin, neurokinins, and prostanoids.

Regulation of TNFα and interleukin-10 production by prostaglandins I2 and E2: 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.

Species difference in increased vascular permeability by synthetic leukotriene C4 and D4.

The activity of synthetic LTC4 was tested in guinea-pig ileum and was 200 times more potent than histamine in contraction of the ileum (3 x 10(-11) M- 3 x 10(-9) M). The activities of LTC4 and LTD4 in increased vascular permeability in guinea pigs, rats and rabbits were compared with those of histamine, bradykinin and prostaglandin (PG) E2. LTC4 was approximately equipotent to bradykinin on a molar basis in guinea pigs and rats and 5-100 times more potent than histamine. LTD4 was about 10 times more potent than LTC4 in guinea pigs and was equipotent to LTC4 in rats. On the contrary, in rabbits, neither LTC4 (up to 30 nmole/site) nor LTD4 (1 nmole/site) induced the dye exudation. These results show that species difference is present in activity of LTC4 and LTD4 in vascular permeability. Furthermore, in guinea pigs, the vascular permeability increased by LTC4 was not affected after pretreatment with pyrilamine (2.5 mg/kg, i.v.), and LTC4 and LTD4 did not potentiate the activity of bradykinin in vascular permeability.

Major roles of prostanoid receptors IP and EP3 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.

Possible involvement of thromboxane in bronchoconstrictive and hypertensive effects of LTC4 and LTD4 in guinea pigs

The actions of leukotriene (LT) C4 and D4 on the systemic arterial pressure and the insufflation pressure in guinea pigs and rabbits were examined. In guinea pigs, 0.3 - 3 nmole/kg of LTC4 and 0.1 - 1.0 nmole/kg of LTD4 administrated from left jugular vein caused dose-dependent increase of the airway resistance measured by the Konzett-Rössler method and a triphasic blood pressure response; an initial hypotension, a secondary hypertension and a third long-lasting hypotension. All of the hypertensive phase and 100 - 150% of the increase of the airway resistance by LTC4 and LTD4 were inhibited by a selective thromboxane synthetase inhibitor, OKY-1581 (10 mg/kg, i.v.) and only the hypotension was observed. Indomethacin (10 mg/kg, i.p.) also inhibited not only the airway resistance increase, but also the prolonged hypotension by LTC4 and shortened the duration of the hypotension by LTD4. It is suggested that thromboxane might be involved in bronchoconstriction and hypertensive effects by LTC4 and LTD4 and that hypotensive prostaglandin might be involved in the hypotensive phase after LTC4 and LTD4. In rabbits, the increase of the airway resistance by LTC4 and LTD4 (upto 100 nmole/kg, i.v.) was negligible and only the hypotension was observed.

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.

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of lambda-carrageenin into the pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B2 were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethylisopropylsilyl (DMiPS) ether or ME-methoxime-DMiPS ether derivatives, 6-keto-PGF1 alpha reached the maximum at 1 hr after carrageenin, then PGE2 and TXB2 showed peaks at 3 hr and waned off before 9 hr. The PGF/ alpha level was kept low, but PGD2, PGE1 and PGF1 alpha were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB2 levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE2, definitely play an important role in the exudation during the first 5 hr.

ACTIVATION OF PLASMA KALLIKREIN-KININ SYSTEM AND ITS SIGNIFICANT ROLE IN PLEURAL FLUID ACCUMULATION OF RAT CARRAGEENIN-INDUCED PLEURISY

Rat pleurisy was induced by intrapleural injection of λ-carrageenin. The pleural exudate began to be accumulated 1–3 h after carrageenin administration and showed a peak at 19 h. The levels of prekallikrein and high-molecular-weight (HMW), but not low-molecular-weight (LMW), kininogen in the pleural fluid were markedly decreased when compared with those in plasma. Prekallikrein in rat plasma was activated by incubation with carrageenin in vitro. Captopril increased the plasma exudation significantly at 1–5 h. Depletion of prekallikrein and HMW kininogen in rat plasma by preadministration of bromelain caused marked inhibition of the plasma exudation at 1–24 h. The rest of the plasma exudation after bromelain was further decreased by simultaneous pretreatment of rats with both bromelain and aspirin. These results clearly indicate that plasma prekallikrein was activated in the pleural cavity and bradykihin released was responsible for plasma exudation during the entire course of this pleurisy.