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Background/purpose: In a method evaluating conjunctival hyperemia using rabbits, it is common to visually grade the degree of vasodilation. However, this method is limited in evaluating consecutive value and in reproducibility. We quantified the degree of conjunctival hyperemia in rabbits as the area ratio of blood vessels by image analysis, and compared the vascular area percentage calculated by image analysis with the hyperemia score., Methods: The conjunctiva was photographed before and after the instillation of 0.1% arachidonic acid using a digital medical scope VersaCam ® (Nidek Co., Ltd.). Next, the area of the conjunctival blood vessels occupying the area of interest was calculated using hyperemia analysis software. The hyperemia score was visually graded for the degree of conjunctiva vasodilation. Furthermore, the hyperemia score and the vascular area ratio were compared., Results: Fifteen minutes after the instillation of arachidonic acid, the area ratio of the blood vessels in the conjunctiva increased significantly and gradually decreased over time. This trend correlated with the hyperemia score., Conclusion: We found that the degree of conjunctival hyperemia in rabbits can be evaluated numerically and quantitatively. This method is considered to be useful for evaluating conjunctival hyperemia in allergic conjunctival diseases.

The anti-pyretic activity of alminoprofen (AP), a non-steroidal anti-inflammatory agent, and its mode of action were investigated in conscious febrile rabbits. A fever was evoked by i.v. injection of lipopolysaccharide (LPS), intracisternal (i.c.) injection of leukocytic pyrogen (LP) or i.c. injection of arachidonic acid (AA). The amount of PGE2 or AP in the cerebrospinal fluid (CSF) after i.v. LPS was estimated using an RIA or HPLC method. AP (3-30 mg/kg, p.o.) dose-dependently inhibited the LPS (0.5 micrograms/kg, i.v.)-induced fever; AP, ibuprofen, indomethacin and pranoprofen had ED50 values of 9.64, 26.45, 4.41 and 11.91 mg/kg, p.o., respectively. PGE2 in the CSF was markedly increased during the elevation of body temperature after i.v. LPS (0.5 microgram/kg). AP (30 mg/kg, p.o.) markedly inhibited the increase in PGE2 that was observed in the CSF during fever developed in response to i.v. LPS (0.5 micrograms/kg). The AP concentration in the CSF 2 hr after AP (30 mg/kg, p.o.) was 2.86 x 10(-6) (1.15-4.57 x 10(-6) M, a concentration too low to inhibit PG synthesis. A dose-dependent fever was observed after i.c. LP (1-8 unit) or AA (10-100 micrograms). AP (30 mg/kg, p.o.) shifted the dose-response curves for the i.c. LP-induced fever to the right, but did not have any effect on the i.c. AA-induced fever. These results suggest that AP has a relatively potent anti-pyretic activity, and its mechanism of action involves competition with LP at a site in the CNS, but does not involve an inhibition of cyclooxygenase at a central site, which has been considered as an anti-pyretic mechanism of nonsteroidal anti-inflammatory drugs.

We investigated the effects of Y-20811 on chemical mediator-induced bronchoconstriction and the release of chemical mediators into lung perfusion fluid during arachidonic acid (AA)-induced bronchoconstriction in guinea pigs. Y-20811 (0.01-1 mg/kg, i.v.), like acetylsalicylic acid or indomethacin, dose-dependently suppressed arachidonic acid- and LTD4-induced bronchoconstriction, and it (1 mg/kg, i.v.) also inhibited PAF-induced bronchoconstriction in guinea pigs. However, at a dose of 1 mg/kg, i.v., it was inactive against the bronchoconstriction induced by histamine, serotonin and acetylcholine in guinea pigs. Y-20811 (0.3-10 mg/kg) administered orally also prevented the LTD4-induced bronchoconstriction in a dose-dependent manner. This protective effect of Y-20811 (10 mg/kg, p.o.) persisted for at least 24 hr. Y-20811 (10 mg/kg, p.o.) also inhibited antigen-induced bronchoconstriction in guinea pigs passively sensitized with anti-ovalbumin guinea pig serum and pretreated with mepyramine. In the perfused and ventilated guinea pig lungs, Y-20811 inhibited AA-induced bronchoconstriction, decreased the release of TXA2 (estimated as TXB2) and increased the release of PGE2 into the perfused lung fluid, significantly (TXB2 and PGE2 were measured by HPLC). Therefore, Y-20811 suppressed various stimulant-induced bronchoconstrictions through the decrease of TXA2 production and the increase of PGE2 production. Thus, Y-20811 should prove useful as an anti-asthmatic drug.

The role of arachidonic acid in patients with acute myocardial infarction (AMI) was investigated. Plasma levels of thromboxane (TX) B2, 6-keto-prostaglandin F1 alpha (6KPG), leukotriene (LT) B4, LTC4, peptideLTs (LTC4 + LTD4 + LTE4) were evaluated in 19 AMI patients, 2 unstable angina (UA) patients and 12 normal controls. Blood samples were obtained from the femoral and pulmonary arteries. Further, plasma levels of LTC4 from the antecubital vein were determined in 14 AMI patients on admission and 10 healthy volunteers. Plasma levels of TXB2, LTB4, LTC4, peptideLTs from arterial blood significantly increased in AMI patients at the acute stage and one day after an attack of AMI compared with those of normal controls. TXB2, LTB4, LTC4, peptideLTs levels in UA patients were higher than those of normal controls. TXB2, LTC4 and peptideLTs levels on week after an attack of AMI significantly decreased compared with those at the acute stage, and these values decreased to normal control levels one month after an attack of AMI. LTB4 levels did not decrease one week after an attack of AMI but they significantly decreased one month after an attack compared with those at the acute stage. These values from the pulmonary artery were not significantly different from the values from the femoral artery obtained at the equivalent phases. Plasma levels of 6KPG did not show significant serial changes either. But the patients showing high levels of 6KPG in the acute stage showed lower levels of CPK and lower Wanger's QRS score than the patients showing low levels of 6KPG. Plasma LTC4 levels in the venous blood of 14 AMI patients were also significantly higher than those of healthy volunteers. Serum levels of lipid peroxide, alpha-tocopherol and superoxide dismutase activity were also measured but did not show significant serial changes in AMI patients. These results in AMI patients suggest that LTB4, LTC4 and peptideLTs, which are lipoxygenase metabolites of arachidonic acid, play an important role in the pathogenesis of AMI attacks as well as TXB2 and 6KPG, which are cyclooxygenase metabolites of arachidonic acid.

Using helical strips of the bovine middle cerebral arteries, changes in vascular tension were measured during isometric contractions induced by endothelin. 1) Both Ca(++)-free media and Ca(++)-antagonists depressed the endothelin-induced contractions only by 40% of the control, suggesting the involvement of both Ca(++)-entry from outside the muscle cell and intracellular Ca(++)-release from the sarcoplasmic reticulum. 2) Endothelin-induced contractions were significantly depressed by 1 microgram/ml tetrodotoxin (TTX). Relative size of depression by TTX was practically the same as that observed in Na(+)-free media without TTX. These results indicated a partial involvement of Na(+)-entry through TTX-sensitive Na(+)-channels. 3) Endothelin-induced contractions were effectively depressed by NCDC, an inhibitor of phospholipase C, suggesting the involvement of PI-turnover in the contraction. 4) Protein kinase inhibitors such as H-7 and H-8 effectively depressed endothelin-induced contractions. This result suggested the phosphorylation of a certain protein by protein kinase C as a cause of long lasting contractions. 5) A phospholipase A2 (PL A2) inhibitor, quinacrine, significantly depressed the endothelin-induced contractions, suggesting a possible involvement of PL A2. However, neither the cyclooxygenase inhibitor nor the lipoxygenase inhibitor depressed endothelin-induced contractions.

Much evidence indicates that human tissues produce prolactin. However, little is known as to which agents regulate its production in decidual tissues. Therefore, it is of interest to examine the effect of arachidonic acid on the production of prolactin, since it is present in considerably large amounts in decidual tissues. The addition of arachidonic acid to culture media resulted in a significant reduction in the amount of prolactin secreted from incubated decidual tissues in a dose-related fashion. Other poly-unsaturated fatty acids, i.e. linoleic and gamma-linolenic acid also inhibited its secretion, whereas saturated fatty acid and mono-unsaturated fatty acids, such as oleic acid, were without effect. The addition of phospholipase A2 and phospholipase C, enzymes to liberate arachidonic acid from phospholipids, also suppressed the secretion of prolactin. Indomethacin and BW-755C, inhibitors of cyclooxygenase and lipoxygenase, respectively, had essentially no effect on the secretion of prolactin. Translatable mRNA for prolactin was detected in decidual tissues. Arachidonic did not alter either the amount of mRNA in prolactin of the amount of its mRNA as a percentage of total mRNA. Thus, it appears that arachidonic acid possibly plays a role as a physiological regulator of prolactin secretion in human decidual tissues. The action of arachidonic acid is not mediated by cyclooxygenase or lipoxygenase products, suggesting that arachidonic acid itself, or its metabolites other than prostaglandin and leukotriene, may be involved in the regulation of prolactin secretion.

Characterization of the PGI2 producing enzyme system (P.E.S.) in pregnancy induced hypertension (PIH) was carried out by comparing its conversion from arachidonic acid (A.A.) or PGH2 to PGI2 using enzyme preparations from endothelial cells of the umbilical vein in normal pregnancy (N) and mild (M) and severe (S) types of PIH. 1) The conversion rate from A.A. to PGI2 in N was significantly (p less than 0.05) higher than in M and S. 2) The conversion rate from PGH2 to PGI2 in N was higher than in M and that in M was higher than in S. 3) The apparent Vmax values (nM/mg protein) (mean +/- S.E.M.) for P.E.S. in N (0.88 +/- 0.21) were significantly (p less than 0.05) lower than in M (2.76 +/- 0.71) and S (1.63 +/- 0.18). The apparent Km values (microM) (mean +/- S.E.M.) for P.E.S. in N (0.76 +/- 0.25) were significantly (p less than 0.05) higher than in M (0.29 +/- 0.07) and significantly (p less than 0.05) lower than in S (3.26 +/- 0.78). 4) The apparent Vmax values for PGI2 synthetase (P.S.) in N (0.44 +/- 0.09) and M (0.61 +/- 0.10) were significantly (p less than 0.05) higher than in S (0.11 +/- 0.06). The apparent Km values for P.S. in N, M and S were 0.12 +/- 0.07, 0.13 +/- 0.06 and 0.16 +/- 0.04, respectively. The present study showed that the symptoms in M may be kept from becoming aggravated by the production of large amounts of PGI2, due mainly to activation of cyclooxygenase, whereas progressive disorder of the regulation of blood pressure in S may be caused by low production of PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)

To assess the role of arachidonic acid metabolites in pathogenesis of bronchial asthma, we measured thromboxane B2 (TxB2), 6-keto PGF1 alpha, leukotriene (LT) C4, and LTB4 in venous blood plasma in patients with bronchial asthma and in controls. The level of TxB2 was significantly higher in 18 asthmatics with attacks than that in 11 controls (77.3 +/- 48.8 pg/ml vs 48.8 +/- 9.4 pgml). The levels of 6-keto PGF1 alpha in both asthmatics with attacks (17.8 +/- 6.7 pg/ml) and without an attack (16.4 +/- 9.7) were significantly higher than that in controls (11.6 +/- 3.9 pg/ml). The levels of LTC4 in asthmatics with attacks (0.84 +/- 0.11 ng/ml, n = 11) were significantly higher than that in controls, furthermore the level of LTC4 in asthmatics without an attack. The level of LTB4 was significantly higher in asthmatics with attacks (295.0 +/- 160.7 pg/ml, n = 26) than that in controls (161.7 +/- 25.3 pg/ml, n = 12) and asthmatics without an attack (182.4 +/- 97.9 pg/ml, n = 22). These findings suggest that the arachidonic acid metabolites are related to the asthma attack and are associated with the pathogenesis of bronchial asthma.

Effects of protizinic acid (PRT) on prostaglandins (PG) and the production of oxygen radicals were compared with those of other non-steroidal anti-inflammatory agents. Oral administration of 30 mg/kg of PRT, indomethacin (IM), or ibuprofen (IB) significantly inhibited arachidonic acid-induced erythema in guinea pigs. Although 30 mg/kg of PRT significantly inhibited PGE2-induced erythema, IM and IB did not significantly inhibit it. PRT inhibited phospholipase A2 (PLA2) activity, and the IC50 value was 2.1 X 10-4 M. On the other hand, IM and IB exerted no effect on the PLA2 activity at 3 X 10-4 M. These results suggest that PRT possesses a broader pharmacological activity on the PG system than IM and IB. As for effects on the production of oxygen radicals, in order of relative inhibitory potency was PRT greater than metiazinic acid (MA) = IM greater than IB = phenylbutazone (PB) in the xanthine oxidase assay, PB great than IM greater than PRT greater than MA = IB in the rabbit neutrophil myeloperoxidase assay, and IM greater than PB greater than PRT greater than MA greater than IB in the guinea pig macrophage assay. In the rabbit neutrophil and aggregated IgG-bound micropore filter assay, the order was PRT greater than MA greater than PB greater than IM = IB. Thus, the inhibitory effects of PRT was verified in all experiments on the production of oxygen radicals in contrast to IB. In particular, it could be especially meaningful that PRT showed the most potent activity in the aggregated IgG-bound micropore filter assay which has been reported to be a good model for studying the pathogenesis of inflammatory diseases believed to be caused by immune complexes.

In order to study prostanoid biosynthesis, pregnant myometrium was taken from eleven patients undergoing cesarean section. Homogenates of myometrium were incubated with 14C labelled arachidonic acid and biosynthetic products were studied using silicic acid column chromatography, thin layer chromatography and gas-liquid chromatography. There were two metabolites assumed to be 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) or hydroxy acid. Concerning the prostaglandins, pregnant myometrium produced 6-keto PGF1 alpha, PGF2 alpha and PGE2. But the conversion rates to each prostaglandin has no relation to the gestational week. Moreover, the conversion rate to each prostaglandin was not higher in the samples taken from the patients with pains than that from the patients with no pains. The total conversion rate to prostaglandins also has no relation to the gestational week or pains. However the PGE2/PGF2 alpha (p less than 0.001) and 6-keto PGF1 alpha/PGF2 alpha (p less than 0.05) ratios were significantly higher in the patients with pains.

(6)-Shogaol, a pungent component of ginger, which is contained in semi-dried ginger but is rarely found in fresh ginger inhibited carrageenin-induced swelling of hind paw in rats and arachidonic acid (AA)-induced platelet aggregation in rabbits. Moreover, (6)-shogaol prevented prostaglandin I2 (PGI2) release from the aorta of rats when tested as an inhibitor of platelet aggregation. These results suggest that (6)-shogaol may have an inhibitory action on the cyclo-oxygenases in both platelets and aorta. Examination of the effects of (6)-shogaol on cyclo-oxygenases in rabbit platelets and microsome fractions of rat aorta indicated that (6)-shogaol inhibited cyclo-oxygenase activities of both tissues in a concentration-dependent manner. Furthermore, when we examined the effect of (6)-shogaol on 5-lipoxygenase from RBL-1 cells, (6)-shogaol exhibited an inhibitory action on 5-lipoxygenase activity. Therefore, it seems that the inhibitory effects of (6)-shogaol on the carrageenin-induced paw edema, AA-induced platelet aggregation and PGI2 production of aorta may be caused by the inhibition of cyclo-oxygenase activity.

Damaging effects on the gastrointestinal tract of proglumetacin maleate (PGM), a new indomethacin (IND) derivative, were examined in comparison with those of IND. Gastric and small intestinal lesions were maximum 4 and 24 hr after a single oral administration of PGM, respectively. Ulcerogenic effects of PGM on the gastric mucosa were approximately 1/7 and 1/10 times as potent as those of IND on a molar ratio 4 hr after single oral dosing to fasting and feeding rats, respectively. PGM was also less active on the small intestinal mucosa 24 hr after single oral dosing. After repeated dosing for 7 days, ulcerogenic effects of PGM were about 1/3 and 1/2 times more potent than those of IND on the gastric and the small intestinal mucosa, respectively. The weak ulcerogenicity of PGM appears to be due to the fact that it has little direct action on the gastrointestinal mucosa. On the other hand, protective effects of PGM on diarrhea induced by arachidonic acid in mice were about 1/2 times as potent as those of IND in both 1 and 4 hr pretreatments. So PGM must have less inhibitory effects on prostaglandin biosynthesis in the intestine than IND. PGM is a safer drug than IND because it has less damaging effect on the gastrointestinal mucosa. Therefore, it may be much more useful for the treatment of chronic inflammatory disorders like rheumatoid arthritis.

Prostaglandins (PGs) play an important role in cervical ripening. It is known that the hydrolytic release of arachidonic acid from phospholipids regulates the rate of PG formation. To study the PG biosynthesis in cervical tissue, phosphatidylcholine containing 14C-arachidonic acid in Sn-2 position was incubated with the 800 X g supernatant of cervical tissue obtained from pregnant women at delivery. The only recognizable radiolabeled metabolite, 14C-arachidonic acid, was found on an autoradiogram of TLC, which corresponded to authentic arachidonic acid. Therefore, phospholipase A2 activity was calculated as the rate of the release of arachidonic acid from phosphatidylcholine under the conditions used. The optimal pH of phospholipase A2 activity in 800 X g supernatant was found to be 7.0. It was found that the addition of Ca2+ increased the enzyme activity. It was demonstrated that the concentrations of DHA-sulfate (DHA-S) and conjugated estrogens were higher in ripened cervical tissue than in non-ripened tissue and that PGI2 and PGE2 production increased following the addition of DHA-S. The effects of steroids mainly derived from feto-placental unit, cortisol, pregnenolone, 20 alpha-dihydroprogesterone, pregnenolone-sulfate, DHA, DHA-S, estrone, estradiol and estriol on arachidonic acid release were also studied in vitro. After the onset of labor. DHA-S was administered to the patients in vivo and their cervical tissues were collected at delivery. It was found that steroids including DHA-S did not affect phospholipase A2 activity under the conditions used. These results indicate that cervical tissue possesses the ability to release arachidonic acid from phospholipid, although this step in PG formation might not be affected by steroids including DHA-S.

The inhibitory effects of anti-inflammatory drugs on th erythema induced by arachidonic acid in guinea pigs were studied. Values of ED50 (mg/kg, p.o.) were; diclofenac sodium (19.9), indomethacin (28.1), naproxen (52.4), phenylbutazone (67.1), ibuprofen (81.3), aspirin (92.6). These drugs, however, were ineffective on the erythema induced by prostaglandin E2. Basic nonsteroidal and steroidal anti-inflammatory drugs and anti-oxidants were negative in the inhibition of arachidonic acid-induced erythema. Cyproheptadine as an anti-histamine and -serotonin agent and morphine as a CNS acting agent were also negative. Erythema did not occur following treatment with linoleic acid, linolenic acid and gamma-linolenic acid. Bishomo-gamma-linolenic acid-induced erythema was inhibited by diclofenac sodium. The inhibition of acidic nonsteroidal anti-inflammatory drugs in this system seemed to be the result of a block of the enzymatic conversion of arachidonic acid to prostaglandins. The arachidonic acid-induced erythema in guinea pigs can serve as a new animal model for evaluating prostaglandins biosynthesis inhibitors such as anti-inflammatory drugs.

Arachidonic acid metabolites, prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), are classified as a type of autacoids. They are not stored in the cells, but stored as a precursor acid, arachidonic acid, in membrane phospholipids. Various physiological stimuli activate phospholipase A2 and release arachidonic acid, which is converted to 1 or 2 products by related enzymes within a few minutes, depending upon individual cell functions. The metabolites are readily inactivated in aqueous solution and in the body. The structures of the various metabolites are quite similar, but their pharmacological actions vary from metabolite to metabolite and some exert opposite actions to those of others. The metabolites play some pivotal roles in physiological or pathophysiological responses such as pain sensation, fever, plasma leakage and skin erythema. Thus, non-steroidal anti-inflammatory drugs, like aspirin, exert their actions through cyclooxygenase inhibition at low doses. PGE2 can be detected in the exudate of acute inflammatory models, and the simultaneous release of PGE2 with bradykinin induces a large increase in plasma leakage and triggers exudate accumulation. LTB4 induces extravasation of PMN leukocytes at the microcirculatory level and is generated in the reperfused area of infarcted cardiac tissue after ligation of rat coronary artery, but in the latter case, the leukocyte migration was not solely induced by LTB4, which was replaced by a complement component (C5a). LTC4 may be involved in ethanol injury of the gastric mucosa and endogenous PGE2 prevents this injury. The real roles of individual arachidonic acid metabolites have been gradually disclosed, but most of the roles are still yet to be clarified.