Yoshiteru Harada

Role of prostaglandin H synthase-2 in prostaglandin E2 formation in rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used to clarify the role of prostaglandin H synthase (PGHS)-2 in acute inflammation. Intrapleural injection of 0.2 ml of 2% lambda-carrageenin induced accumulation of exudate and infiltration of leukocytes into the pleural cavity. When PGHS-1 and -2 proteins in the pleural exudate cells were analyzed by Western blot analysis, PGHS-2 was detectable from 1 hr after carrageenin injection. Its level rose sharply, remained high from 3 to 7 hr after injection, and then fell to near the detection limit. PGHS-1 was also detected, but kept almost the same level throughout the course of the pleurisy. Levels of prostaglandin (PG) E2 and thromboxane (TX) B2 in the exudate increased from hour 3 to hour 7, and then declined. Thus, the changes of the level of PGE2 were closely paralleled those of PGHS-2. The selective PGHS-2 inhibitors NS-398, nimesulide and SC-58125 suppressed the inflammatory reaction and caused a marked decrease in the level of PGE2 but not in those of TXB2 and 6-keto-PGF 1 alpha. These results suggest that the PGHS-2 expressed in the pleural exudate cells may be involved in PGE2 formation at the site of inflammation.

Evaluation of Pharmacological Profile of Meloxicam as an Anti-Inflammatory Agent, with Particular Reference to Its Relative Selectivity for Cyclooxygenase-2 Over Cyclooxygenase-1

We studied the anti-inflammatory activity of meloxicam on rat carrageenin-induced pleurisy and its toxicity for rat gastric mucosa, relative to its in vitro inhibitory potency against partially purified cyclooxygenase (COX)-1 and COX-2 preparations in order to clarify the pharmacological profile of the compound as an anti-inflammatory agent. In rat carrageenin-induced pleurisy, the plasma exudation rate peaked at 5 h, at which time COX-2 was detectable in cells from the pleural exudate. Meloxicam and piroxicam (1 and 3 mg/kg) and NS-398 (3 mg/kg) showed almost equal anti-inflammatory potency against 5-hour pleurisy. A single oral administration of the compounds caused a dose-dependent increase in the number of rats with gastric mucosal erosion. The ED50 value for meloxicam (5.92 mg/kg) was significantly higher than that for piroxicam (1.76 mg/kg), indicating that meloxicam is safer. Indometacin showed intermediate safety (2.59 mg/kg). In in vitro experiments, indometacin inhibited COX-1 about 1.7 times more potently than COX-2. NS-398 inhibited COX-2 with an IC50 of 0.32 microM, but never affected COX-1 activity, even at 100 microM. In the same assay system, meloxicam inhibited COX-2 about 12 times more selectively than COX-1. Piroxicam, however, inhibited both isoforms almost equally. These results indicate that meloxicam is a potent anti-inflammatory agent with low gastric toxicity. One reason for its in vivo pharmacological profile may be related to its relative selectivity for COX-2 over COX-1. Thus, meloxicam may belong to a group of COX-2 selective anti-inflammatory agents with a better safety profile than conventional COX-1 and COX-2 nonselective anti-inflammatory agents.

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.

Possible background mechanisms of the effectiveness of cyclooxygenase-2 inhibitors in the treatment of rheumatoid arthritis

Abstract. Cyclooxygenase (COX)-2 was induced in an acute exudative inflammatory model (rat carrageenin-induced pleurisy) in which prostaglandin E2, 6-keto-prostaglandin F1α, and thromboxane B2 were generated in the pleural fluid. Selective COX-2 inhibitors, such as NS-398, inhibited the plasma exudation and generation of prostaglandin E2, but not that of thromboxane B2 and 6-keto-prostaglandin F1α, in the pleural fluid. In proliferative inflammatory models, COX-2 was induced, and selective COX-2 inhibitors suppressed granuloma formation, particularly, microvessel formation. COX-2 was induced during angiogenesis in a sponge model implanted into skin of rat, and the COX-2 inhibitor suppressed the angiogenesis. As induction of COX-2 was reported in osteoblasts, COX-2 was involved in most characteristic responses of acute exudative inflammation, granuloma formation, bone resorption, and pain in rheumatoid arthritis. The prevention of these COX-2 responses provides a rationale for the effectiveness of COX-2 inhibitors in the treatment of rheumatoid arthritis.

Are the anti-inflammatory effects of dexamethasone responsible for inhibition of the induction of enzymes involved in prostanoid formation in rat carrageenin-induced pleurisy?

Since anti-inflammatory steroids modulate multiple gene expression, including the expression of prostaglandin H synthase-2 and phospholipase A(2), at the molecular level, we studied the effects of dexamethasone on rat carrageenin-induced pleurisy to elucidate whether regulation of phospholipase A(2) and prostaglandin H synthase-2 expression is the primary mechanism of its anti-inflammatory action. Suppression of plasma exudation by a lower dose of dexamethasone (0.3 mg/kg) was almost equal to that by aspirin (100 mg/kg), but that by higher dexamethasone doses (3 and 30 mg/kg) was considerably stronger, suggesting the involvement of effects other than that on prostanoid formation. The lower dose of dexamethasone also significantly reduced the pleural exudate neutrophil count and prostanoid levels. However, this dose affected neither the prostaglandin H synthase-2 level nor the phospholipase A(2) activity in the exudate cells. The prostaglandin H synthase-2 level was affected only at the higher doses, while phospholipase A(2) activity was not. These results suggest that the anti-inflammatory effects of dexamethasone in acute inflammation cannot be ascribed to direct interference with prostanoid formation via suppression of phospholipase A(2) and prostaglandin H synthase-2 expression.

Potentiation of bradykinin-induced nociceptive response by arachidonate metabolites in dogs

Bradykinin was injected retrogradely into a branch of the splenic artery of lightly anesthetized dogs, and the reflex hypertensive response was used as an indicator of the nociception. The reflex hypertensive response to low doses of bradykinin (less than 1 nmol), but not to high doses (3-5 nmol), was markedly suppressed by infusion of indomethacin (0.54 mumol/min) into the splenic artery. During indomethacin infusion, the reflex hypertensive response to low doses of bradykinin was potentiated dose dependently by the following arachidonic acid metabolites (ED50): prostaglandin (PG)I2 (2.9 nmol) greater than or equal to PGH2 (4.4) greater than PGE2 (14) = thromboxane (TX)A2(15) greater than PGD2 (greater than 100). Leukotrienes B4, C4 and D4 showed practically no activity. This potentiating effect lasted up to 20 min after injection, particularly with PGE2. These results may not exclude the role of PGI2 as a major candidate for involvement in bradykinin-induced nociception, although a selective TX synthetase inhibitor (OKY-046) showed a weak analgesic effect (only 1/30 that of indomethacin).

A possible role of prostaglandins and bradykinin as a trigger of exudation in carrageenin-induced rat pleurisy.

Pleurisy was induced in rats after intrapleural injection of 2% λ-carrageenin. The volume of the pleural exudate increased rapidly between 1 and 3 h, continuing to increase until 7 h. The dye amounts exuded into the pleural cavity for 20 min after 5% pontamine sky blue (60 mg/kg, i.v.) increased markedly at 1 to 3 h, then they kept constant until 6 h, decreasing thereafter. The PGE level in the pleural fluid increased rapidly during the period from 1 to 3 h, when the exudate commenced to accumulate, and then decreased slightly until 7 h. The main PG in the pleural fluid was recognized as PGE2 on thin-layer chromatography. The pretreatment of rats with a PG synthetase inhibitor, indomethacin (5 mg/kg, i.p.), 30 min before carrageenin administration resulted in the significant reduction of dye leakage at 1 h and of the pleural fluid exudation at 1 to 3 h. The intravenous injection into rats of stem bromelain (10 mg/kg, i.v.), a SH-protease from pineapples, caused the depletion of high molecular weight kininogen in plasma without effect on the content of low molecular weight kininogen. The pretreatment of rats with bromelain 30 min before carrageenin caused a marked reduction in the dye exudation during the periods from 1 to 3 h and 5 to 6 h. The accumulation of the exudate was strongly suppressed at the same periods. Although each treatment by itself did not completely abolish the dye and fluid exudation, the treatment of rats simultaneously with indomethacin and bromelain resulted in suppression of dye leakage from 1 to 6 h and practically no fluid accumulation was observed until 4 h. These results strongly indicate that prostaglandin is released in combination with bradykinin at the period when the pleural exudate commences to accumulate.

Increased migration of neutrophils to granulocyte-colony stimulating factor in rat carrageenin-induced pleurisy: Roles of complement, bradykinin, and inducible cyclooxygenase-2

Administration of human recombinant granulocyte colony-stimulating factor (G-CSF, 100 μg/kg/day, s.c) to rats for 4 days significantly increased circulating neutrophil counts (by 1130%), together with an increase in mononuclear leukocyte counts (by 119%). Infiltrated pleural neutrophil counts in G-CSF-treated rats (G-CSF-r) 5h after the intrapleural injection of zymosanactivated serum were significantly higher (by 155%) than those in control rats (Vehicle-r). In carrageenin-induced pleurisy, counts of infiltrated pleural neutrophils in G-CSF-r 5 and 7h after carrageenin were significantly higher (by 119% and 116%) than those in Vehicle-r. G-CSF treatment increased the volume of pleural exudate and the plasma exudation rate by 122% and 226%, compared to values in Vehicle-r 5h after carrageenin. Cobra venom factor (75 μg/kg, i.v.) significantly reduced pleural neutrophil migration in G-CSF-r (by 53%) and Vehicle-r (by 49%). Bromelain (10 mg/kg, i.v.) and aspirin (100 mg/kg, p.o.) reduced pleural neutrophil migration and reduced exudate volume and plasma exudation. Intrapleural bradykinin-(1–5) and prostaglandin E2 levels were significantly higher in G-CSF-r than in Vehicle-r. The increased neutrophil migration in G-CSF-r may be atributed to enhanced activation of the complement system facilitated by increased plasma exudation due to bradykinin and prostaglandins.

Concurrent evolution and resolution in an acute inflammatory model of rat carrageenin‐induced pleurisy

Granulocyte apoptosis and subsequent clearance by phagocytes are critical for the resolution of inflammation. However, no studies have addressed how the resolution proceeds in the inflammatory site. We studied the time course of neutrophil apoptosis and the following ingestion by mononuclear leukocytes in rat carrageenin‐induced pleurisy, detecting DNA fragmentation by the deoxyuridine triphosphate‐biotin nick‐end labeling (TUNEL) method, by acridine orange staining, and from the DNA ladder pattern on electrophoresis. Neutrophil accumulation started 3–5 h after carrageenin injection and then maintained a plateau until 24 h. Neutrophils decreased steeply between days 1 and 3. Mononuclear leukocytes started to accumulate at 5 h and reached a peak at day 2. TUNEL‐positive bodies and acridine orange‐positive bodies first became detectable in the cytoplasm of the mononuclear leukocytes from 24 h and 9 h, respectively. Both methods indicated that mononuclear leukocytes containing fragmented DNA increased rapidly on days 1 and 2 and reached a peak at day 3. The characteristic ladder pattern of neutrophil DNA was observed from 5 h. Tumor necrosis factor α was detectable on the start, and the levels of interleukin‐10 and transforming growth factor‐β1 rose together with signs of neutrophil apoptosis and the following ingestion by mononuclear leukocytes. These results indicate that neutrophils start to undergo apoptosis just after the beginning of their accumulation in the inflammation site. Thus, evolution and resolution processes may proceed concurrently in acute inflammation.