A pyrroloquinazoline derivative with anti-inflammatory and analgesic activity by dual inhibition of cyclo-oxygenase-2 and 5-lipoxygenase.

In a previous study, we reported a new pyrroloquinazoline derivative, 3-(4'-acetoxy-3',5'-dimethoxy)benzylidene-1,2-dihydropyrrolo[2,1-b]quinazoline-9-one (PQ), which inhibited human purified 5-lipoxygenase activity and prostaglandin E2 release in lipopolysaccharide-stimulated RAW 264.7 cells. In the present work, we show that PQ inhibits cyclo-oxygenase-2 activity in intact cell assays (human monocytes) and purified enzyme preparations (ovine isoenzymes) without affecting cyclo-oxygenase-1 activity. This behaviour was confirmed in vivo by using the zymosan-injected mouse air pouch model, where PQ caused a marked reduction in cell migration and leukotriene B4 levels at 4 h, as well as inhibition of prostaglandin E2 levels without affecting cyclo-oxygenase-2 expression at 24 h after zymosan stimulation. In addition, oral administration of this compound significantly reduced carrageenan-induced mouse paw oedema and phenyl-p-benzoquinone-induced writhings in mice. These results indicate that oral PQ exerts analgesic and anti-inflammatory effects, which are related to dual inhibition of cyclo-oxygenase-2 and 5-lipoxygenase activities.     

Hypocholesterolemic activity of dipyridamole: effects on chick plasma and lipoprotein composition and arachidonic acid levels

We have studied the effects of dipyridamole treatment on chick plasma and lipoprotein composition in postprandial and fasting (12 h) conditions. Plasma cholesterol levels were higher in fasted than in fed chicks, whereas triglycerides declined during starvation. Dipyridamole treatment reduced plasma cholesterol content, mainly of the free cholesterol fraction. In postprandial conditions, total cholesterol content of high and low density lipoproteins decreased in a similar proportion to that observed in plasma. However, cholesterol and other chemical constituents of intermediate and very low density lipoproteins were more drastically reduced by dipyridamole than in plasma. Total amounts of these lipoprotein fractions were also reduced about 50%. The effects of dipyridamole in fasted animals were not significant. To our knowledge, this is one of the first reports about the response of lipoprotein cholesterol to dipyridamole treatment. A strong decrease was also found in the arachidonic acid content of plasma phospholipids and cholesterol esters fractions.     

Inhibition of the arachidonic acid cascade by norathyriol via blockade of cyclooxygenase and lipoxygenase activity in neutrophils

Recent studies have suggested that dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LO) may be more beneficial in the treatment of inflammatory diseases in which platelet-leukocyte interaction dominates the underlying inflammatory process, than inhibitors of COX or LO alone. In this study, we examined oxygenated xanthones, shown previously to inhibit platelet and neutrophil activation, with respect to the potency of COX inhibition. 1,3,6,7-Tetrahydroxyxanthone (norathyriol) was the most potent. Norathyriol suppressed thromboxane B₂ (TXB₂) and leukotriene B₄ (LTB₄) formation in calcium ionophore (A23187)- and formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils. Norathyriol was 3–4 times more active against LTB₄ formation than against TXB₂ formation (IC₅₀ about 2.8 vs. 10 µM, respectively). Norathyriol also inhibited prostaglandin D₂ (PGD₂) formation in A23187-stimulated rat mast cells (IC₅₀ 3.0±1.2 µM) and in arachidonic acid (AA)-activated mast cell lysate. Norathyriol was a more effective inhibitor of 5-LO activity than of COX, as shown also by analyses of enzyme activities in a cell-free system, of COX and 5-LO metabolic capacity in neutrophils and of ex vivo TXB₂ and LTB₄ formation in A23187-stimulated neutrophils. Moreover, norathyriol inhibited COX-2 and 12-LO with IC₅₀ values (19.6±1.5 and 1.2±0.1 µM, respectively) similar to those required for the inhibition of COX-1 and 5-LO (16.2±1.5 and 1.8±0.4 µM, respectively). Inhibition of 15-LO by norathyriol was slightly less active. Norathyriol had no effect on A23187-induced AA release from neutrophils and did not affect phospholipase A₂ (PLA₂) activity in a cell-free system. These results indicate that norathyriol inhibits the formation of PGs and LTs in neutrophils probably through direct blockade of COX and 5-LO activities. Norathyriol, a single molecule with multiple targets, might provide a potential therapeutic benefit in the treatment of inflammatory diseases.     

Effect of linoleic acid, linoleic acid anilide, and arachidonic acid on the expression of adhesion molecules on human neutrophils

The effects of linoleic acid, linoleic acid anilide, and arachidonic acid on the expression of CD11b/CD18, CD11c/CD18 integrins and l-selectin on human neutrophils were studied by flow cytometry in a whole blood assay. None of these compounds had any effect on the basal expression of CD11b, CD11c, or l-selectin in the concentration range of 20–100 μM. However, linoleic acid at a concentration of 1000 μM slightly up-regulated CD11b and CD11c by a factor of 2.1 and 1.7, respectively. Linoleic acid, linoleic acid anilide, and arachidonic acid did not affect the formyl-methionyl-leucyl-phenylalanine induced up-regulation of CD11b or CD11c. However, linoleic acid and linoleic acid anilide slightly inhibited the phorbol myristate acetate (PMA)-induced expression of CD11b, which was decreased by 27 and 21% at concentrations of 100 and 1000 μM, respectively. Likewise, arachidonic acid at 40 μM inhibited the PMA-induced expression of CD11b by 19%. Our results suggest that linoleic acid, linoleic acid anilide, and arachidonic acid do not dramatically affect the expression of leukocyte adhesion molecules in a whole blood assay. Received: 17 February 1997  / Accepted: 5 May 1997     

Blockade by metal complexing agents and by catalase of the effects of arachidonic acid on platelets: relevance to the study of anti-inflammatory mechanisms.

Metal-chelating agents inhibited platelet aggregation and the accompanying generation of rabbit aorta contracting and PG-like activities, when platelets were challenged with arachidonic acid. Inhibition required the presence of the chelating agents in the medium, and was insured by reagents avid for free or protein-bound copper. Catalase also prevented aggregation and generation of pharmacologically active substances; its activity was reversed by aminothiol agents and by Cu2+ and Zn2+, shown previously to potentiate the platelet effects of arachidonic acid. Inhibition by indomethacin was not prevented by amino-thiol drugs nor by Cu2+ or Zn2+. The catalase-induced inhibition was not affected by scavenging of thiol groups; this rules out, as a mechanism of action of catalase, the increased destruction of popoperoxides by glutathione peroxidase, which requires reduced glutathione as hydrogen donor. The results are compatible with the hypothesis that the agent that mediates platelet aggregation by arachidonic acid is a popoperoxide, requiring the presence either of H2O2 or of a similarly catalase-sensitive substance to be generated.     

甘草次酸衍生物的抗炎活性及水钠潴留作用的研究

目的 对合成的系列甘草次酸衍生物进行抗炎活性,水钠潴留作用的评价.方法 构建了急性及慢性炎症模型,评价合成的6个甘草次酸衍生物的抗炎活性及水钠潴留作用.结果 脱氧甘草次酸、脱氧甘草次酸甲酯、甘草次酸甲酯-3-O-乙酸酯及甘草次酸甲酯均表现出较好的急性炎症拮抗活性,甘草次酸乙酯-3-O-乙酸酯及甘草次酸也具有一定的活性.脱氧甘草次酸、脱氧甘草次酸甲酯、甘草次酸甲酯-3-O-乙酸酯及甘草次酸甲酯对棉球肉芽肿模型大鼠24 h尿量、尿钾离子含量、血清钠离子含量和钾离子含量均未见明显影响.结论 脱氧甘草次酸、脱氧甘草次酸甲酯、甘草次酸甲酯-3-O-乙酸酯及甘草次酸甲酯对急性炎症(渗出反应为主)有明显的拮抗作用,甘草次酸乙酯-3-O-乙酸酯及甘草次酸对急性炎症也有一定抑制作用.4种具有显著急性炎症拮抗作用的衍生物未表现出明显水钠潴留作用.     

Acute hypotension due to carrageenan, arachidonic acid and slow reacting substance C in the rabbit: role of platelets and nature of pharmacological antagonism.

Carrageenan injected i.v. to rabbits induced thrombocytopenia, hypotension and death. The latter two phenomena, but not the former, were prevented by aspirin and by indomethacin. Immune platelet depletion protected against the effects of carrageenan, but failed to interfere with hypotension by arachidonic acid (AA) and by slow reacting substance C. Inhibition by aspirin of hypotension due to AA was short lasting (< 1 h for 5 mg/kg), whereas inhibition of AA-induced platelet aggregation lasted more than 7 h. It thus appears that neither hypotension due to AA nor its inhibition by aspirin, depend upon a platelet effect. In contrast, hypotension by carrageenan is platelet-dependent. Generation of prostaglandin (PG)-like activity and of rabbit aorta contracting activity in blood incubated with AA or with carrageenan was suppressed after i.v. injection of aspirin. Aggregation by AA was also inhibited, wheras aggregation by carrageenan was only partly delayed. Prostaglandin synthetase was not inhibited by salicylic acid, although this compound delayed aggregation by carrageenan as potently as did aspirin. Salicylic acid failed to interfere with in vivo effects of AA or of carrageenan, and prevented aspirin-indcued inhibition of in vivo and in vitro effects of AA. Salicylic acid inhibits transport and/or binding of aspirin to PG synthetase-related sites, but does not interfere with the mechanisms through which aspirin inhibits platelet aggregation by carrageenan. Release of platelet mediators by carrageenan requires platelet integrity, supporting the concept of a multistep/multicompartmental process. Hypotension and death due to carrageenan result from this release of platelet mediators, which is suppressed by aspirin and by indomethacin, despite concurrent aspirin-resistant platelet aggregation.     

Efficacy of a phenol derivative,isopropyl vanillate,as an anti-inflammatory agent: A new small molecule inhibitor of COX and neutrophil migration

Inflammation is the response of the body to noxious stimuli such as infections, trauma, or injury. Experimental studies have shown that vanillic acid has anti-inflammatory effects. The objective of this study was to investigate the anti-inflammatory and antipyretic properties of the derivative of vanillic acid, isopropyl vanillate (ISP-VT), in mice. The results of this study indicated that ISP-VT reduced paw edema induced by carrageenan, dextran sulfate (DEX), compound 48/80, serotonin, bradykinin (BK), histamine (HIST), and prostaglandin E2 (PGE2). Furthermore, ISP-VT reduced recruitment of leukocytes and neutrophils and reduced its adhesion and rolling, and decreased myeloperoxidase enzyme activity (MPO), cytokine levels (tumor necrosis factor-α and interleukin-6), and vascular permeability. ISP-VT also significantly reduced the expression of cyclooxygenase-2 (COX-2) in subplantar tissue of mice. ISP-VT inhibited COX-2 selectively compared to the standard drug. Our results showed that although ISP-VT binds to COX-1, it is less toxic than indomethacin, as evidenced by MPO analysis of gastric tissue. Treatment with the ISP-VT significantly reduced rectal temperature in yeast-induced hyperthermia in mice. Our results showed that the main mechanism ISP-VT-induced anti-inflammatory activity is by inhibition of COX-2. In conclusion, our results indicate that ISP-VT has potential as an anti-inflammatory and antipyretic therapeutic compound.     

Effects of non-steroidal anti-inflammatory agents on human neutrophil functions in vitro and in vivo

Human blood neutrophils exposed to appropriate stimuli aggregate, degranulate and generate superoxide anion (O2-). These responses are anteceded by mobilization of membrane-associated calcium, monitored as a decrease in fluorescence of cells preloaded with chlortetracycline (CTC). We studied the effects, both in vitro and in vivo, of non-steroidal anti-inflammatory agents (aspirin, indomethacin, ibuprofen and piroxicam) on these neutrophil responses to three stimuli: a chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP); a tumor promotor, phorbol myristate acetate (PMA); and a lectin, concanavalin A (Con A). The effects of these drugs were compared with those of two polyenoic inhibitors of arachidonate metabolism: eicosatrienoic acid (ETI) and eicosatetraynoic acid (ETYA). The pattern of inhibition of neutrophil functions varied both with inhibitor and the nature of the stimulus. Thus, aspirin, piroxicam, ETYA and ETI inhibited neutrophil aggregation, degranulation, and O2- generation in response to FMLP, whereas ibuprofen inhibited only aggregation and degranulation and indomethacin only inhibited aggregation. None of the agents inhibited aggregation or degranulation induced by PMA or Con A: only piroxicam inhibited O2- generation in response to PMA or Con A. ETI and ibuprofen inhibited decrements of CTC fluorescence induced by FMLP, but whereas ETI inhibited the CTC response to PMA or Con A, ibuprofen was without effect. The agents had varying effects on binding of the stimulus [( 3H]FMLP, [3H]Con A), but these did not correlate with neutrophil responses to the ligands. Neutrophils from subjects taking therapeutic doses of ibuprofen, indomethacin, or piroxicam showed profiles of inhibited responses to FMLP similar to those observed with these agents in vitro. These data suggest that, although non-steroidal anti-inflammatory agents may inhibit discrete neutrophil functions both in vitro and in vivo, their effects do not duplicate those of polyenoic inhibitors of arachidonate metabolism. Moreover, since the susceptibility of neutrophils differed not only with respect to each inhibitor, but also to the stimulus, it is unlikely that all neutrophil responses are necessarily linked by a common pathway that is blocked by inhibitors of arachidonic acid metabolism.     

Exploring the influence of steric, electronic and lipophilic descriptors of 1,3-diarly propenones on their anti-inflammatory activity

BACKGROUND AND THE PURPOSE OF THE STUDY: Various compounds from natural and synthetic origins containing the 1,3-diarylpropenone structure have been reported to produce a variety of biological activities like anti-microbial, anti-inflammatory, vascular muscle relaxant, etc. A systematic analysis of the structural features responsible for anti-inflammatory activity and a possible mode of their actions were proposed to be evaluated by synthesizing a set of compounds, screening them for anti-inflammatory activity and developing a QSAR model. METHODS: Two types of 1,3-diarylpropenone derivatives were synthesized employing the Claisen-Schmidt condensation. These compounds were then screened for their in vivo anti- inflammatory activity by the carrageenin induced rat paw edema method and also for in vitro cyclooxygenase-2 (COX-2) inhibition activity using a colorimetric kit for COX (ovine) inhibitor screening assay. These derivatives and their anti-inflammatory activity data were employed for QSAR analysis on Vlife MDS 3.5 software. The molecules were divided into training and test sets based on observed activity and QSAR models were generated for the training set and validated. The activity of the molecules of the test set was predicted according to the QSAR equation fit. Possible correlation between observed anti-inflammatory activity and in vitro cyclooxygenase-2 inhibition was also studied. RESULTS AND CONCLUSION: Insignificant difference between the observed and predicted biological activity revealed that the selected electronic, steric and lipophilic parameters have a significant correlation (r(2)=0.85) with anti-inflammatory activity of the selected class of compounds. On the basis of results it may be suggested that the 1,3-diaryl-2-propen-1-ones framework is an attractive template for structural optimization to achieve better potency of anti-inflammatory activity. Similarly, the relatively low correlation between anti-inflammatory activity and cyclooxygenase-2 inhibition indicates that other modes of actions may also be responsible for the anti-inflammatory activity of the tested compounds.     

2-乙酰氧基-5-乙酰氨基苯甲酸的合成及镇痛抗炎作用

目的：合成2-乙酰氧基-2-乙酰氨基苯甲酸并进行镇痛、抗炎作用的初步研究。方法：以水杨酸和苯胺为原料,经重氮、偶合、还原及酰化合成目标化合物,化学结构经元素分析、IR和^1H-NMR确定;采用小鼠醋酸扭体、耳部肿胀法研究镇痛和抗炎作用。结果：目标化合物的熔点为170-173℃,中和法测量99.2％,收率42.7％（以水杨酸计）。小鼠灌胃后抑制醋酸所致疼痛的ED50为1.2mmol/kg,抑制二甲苯所致耳廓炎症的ED50 为2.3mmol/kg,LD50为2598mg/kg。结论：2-乙酰氧基-5-乙酰氨基苯甲酸合成简单,收率稳定,具有比阿司匹林和对乙酰氨基酚更强的镇痛和抗炎作用。     

Effect of NC-1900, an active fragment analog of arginine vasopressin, and inhibitors of arachidonic acid metabolism on performance of a passive avoidance task in mice

In this study, we investigated the effect of administration of inhibitors of each of the arachidonic acid metabolism pathways and the effect of co-administration of these inhibitors with NC-1900, a fragment analog of arginine vasopressin, on step-through passive avoidance task performance. All drugs were administered just after the acquisition trial in the passive avoidance task. Intracerebroventricular (i.c.v.) administration of nordihydroguaiaretic acid (NDGA, 1 and 10 microg), a phospholipase A2 (PLA2) and lipoxygenase (LOX) inhibitor, and of arachidonyl trifluoromethyl ketone (ATK, 1 and 10 microg), a specific PLA2 inhibitor caused reductions in latency on the retention trial. The i.c.v. administration of either of baicalein (0.1-10 microg), a 12-LOX inhibitor, or AA-861 (0.1-10 microg), a 5-LOX inhibitor, did not influence the latency. Intraperitoneal administration of indomethacin (20 mg/kg), a non-specific COX inhibitor, or NS-398 (10 mg/kg), a specific COX-2 inhibitor, impaired performance on the retention trial in the task, while piroxicam (20 mg/kg), a specific COX-1 inhibitor, did not. Subcutaneous administration of NC-1900 (0.1 ng/kg) ameliorated the reduction of latency caused by NDGA, ATK, indomethacin, or NS-398. These results suggested that the COX-2 pathway of arachidonic acid metabolism may be important for learning and/or memory in the passive avoidance task in mice, and that the ameliorating effect of NC-1900, in part, is due to mimicking of the effects of metabolites of the COX-2 pathway.     

The effects of the protein synthesis inhibitor anisomycin on the febrile responses to intracerebroventricular injections of bacterial pyrogen,arachidonic acid and prostaglandin E2

Summary 1. Anisomycin (15 mg/kg) was administered s. c. to cats at ambient temperatures of 5°C, 20°C and 38°C. It produced biphasic effects on body temperature at 5°C and 20°C, an initial fall in temperature followed by a rise in body temperature, and a rise in body temperature of long latency at 38°C. 2. Anisomycin (15 mg/kg) attenuated the hyperthermic responses to centrally injected PGE₂ (1 g) at all ambient temperatures studied and also completely abolished the hyperthermic response to arachidonic acid (100 ng i. c. v.) at 20°C. 3. Shigella dysenteriae (100 ng i. c. v.) raised the body temperature of cats by increasing heat production and reducing heat loss at 5°C and 20°C, and by increasing heat conservation at 38°C. Anisomycin (15 mg/kg s. c.) pretreatment did not affect the temperature responses to the pyrogen at 20°C and 38°C, but did reduce the responses to Shigella dysenteriae (100 ng and 1 g i. c. v.) at 5°C. 4. Anisomycin (15 mg/kg s. c.) was administered to cats, 90 min after the injection of Shigella dysenteriae (100 ng i.e. v.), at 20°C at the onset of hyperthermia inn control experiments. Under these conditions, no hyperthermia was observed over a 2 h period following anisomycin injection. 5. It is concluded that anisomycin interferes with pyrogen induced fever by acting at a site after PGE₂ in the pathway to fever. Send offprint requests to: A. S. Milton     

A comparison of the effect of timegadine, levamisole, and D-penicillamine on human neutrophil metabolism of endogenous arachidonic acid and chemotaxis

The effect of timegadine, a novel experimental antirheumatic drug, on human neutrophil (PMN) 5-lipoxygenase activity and leukotriene B4 (LTB4) chemotaxis was compared with that of two second-line antiinflammatory drugs, D-penicillamine and levamisole. 1-14C-Arachidonic acid (AA) was incorporated into the purified cells until steady state conditions were obtained. After preincubation with serial dilutions of the three drugs, AA release and metabolism was stimulated with calcium ionophore A23187. The radioactive eicosanoids released were extracted and separated by thin-layer chromatography, followed by autoradiography and quantitative laser densitometry. Chemotaxis of PMNs towards LTB4 was measured in a modified Boyden chamber. Timegadine showed dose-dependent inhibition of both the 5-lipoxygenase pathway (IC50 3.4 x 10(-5) M), and of chemotaxis (IC50 3 x 10(-4) M). Inhibition of the release of AA from phospholipids, however, occurred only at therapeutically irrelevant doses (millimolar concentrations). Levamisole and D-penicillamine did not inhibit any of the cell functions investigated. Inhibition of both neutrophil motility and cellular synthesis of pro-inflammatory eicosanoids, may thus contribute to the clinical effects of timegadine in rheumatoid arthritis.     

局部应用环孢素A聚乳酸微球的性状及释药研究

目的对局部应用环孢素A聚乳酸微球的性状及其在体外、体内释放进行研究。方法通过采用O/W型乳化-溶剂挥发法制备环孢素A聚乳酸微球。观察微球分散度、粒径及外观形态及体外释药特性,对比全身与局部给药后全血中及气管组织中的环孢素A药物浓度。结果环孢素A聚乳酸微球的形态圆整,平均粒径18.234μm,跨距:1.131,粒径在9.525～32.400μm者占总数的80%以上。包封率为(86.1±0.8)%,载药量为(34.5±0.6)%。环孢素A聚乳酸微球的体外释药情况:30d的累积释药量为40.8%,采用局部埋植微球后前2周可以维持较高的血药浓度,2周后也可以维持在200ng/ml的药物浓度。结论环孢素A聚乳酸微球具有较好的缓释性能。局部应用可获得有效的血药浓度,在局部气管组织中的药物浓度高于全身用药组。     

紫锥菊花中药效成分的提取工艺及抗炎活性研究

目的 研究临沂引种紫锥菊花中药效成分的最佳提取工艺及其抗炎活性。方法 采用L₉正交试验法筛选,通过高效液相色谱仪,用提取物中菊苣酸含量为量化指标进行量化,确定最佳提取工艺。耳肿胀和足跖肿胀试验确定紫锥菊花的抗炎活性。结果 紫锥菊花中药效成分的最佳提取工艺为70 ℃时用20倍量 60%乙醇提取120 min提取2次,菊苣酸得率为2.34%。紫锥菊花的乙醇提取物在试验剂量表现出较好抗炎活性。结论 该方法提取紫锥菊的药效成分的工艺简便,污染少,可用于工业化生产。紫锥菊花的乙醇提取物具有较好的抗炎活性。     

Inhibition of arachidonic acid release and cytosolic phospholipase A2 alpha activity by D-erythro-sphingosine

Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide can mediate many cellular events including apoptosis, stress responses and growth arrest. Although ceramide stimulates arachidonic acid metabolism in several cells, the effects of sphingosine and its endogenous analogs have not been established. We investigated the effects of D-erythro-sphingosine and its metabolites on arachidonic acid release in the two cells and on the activity of cytosolic phospholipase A2alpha. C2-Ceramide (N-acetyl-D-erythro-sphingosine, 100 microM) alone stimulated [3H]arachidonic acid release and enhanced the ionomycin-induced release from the prelabeled PC12 cells and L929 cells. In contrast, exogenous addition of D-erythro-sphingosine inhibited the responses in a concentration-dependent manner in the two cell lines. D-erythro-sphingosine, D-erythro-N,N-dimethylsphingosine (D-erythro-DMS) and D-erythro-dihydrosphingosine (D-erythro-DHS) significantly inhibited mastoparan-, but not Na3VO4-, stimulated arachidonic acid release in PC12 cells. D-erythro-S1P and DL-threo-DHS showed no effect on the responses. Production of prostaglandin F2alpha was also enhanced by C2-ceramide (20 microM) and suppressed by D-erythro-sphingosine (10 microM) in PC12 cells. An in vitro study revealed that D-erythro-sphingosine, D-erythro-DMS and D-erythro-DHS directly inhibited cytosolic phospholipase A2alpha activity. These findings suggest that ceramide and D-erythro-analogs of sphingosine have opposite effects on phospholipase A2 activity and thus regulate arachidonic acid release from cells.     

Effects of lysophosphatidylcholine and arachidonic acid on the regulation of intracellular Ca2+ transport

Summary The role of lysophosphatidylcholine and arachidonic acid in signal transduction was investigated using subcellular organelles and permeabilized cells from liver. Both substances can be generated intracellularly by the action of phospholipase A₂ on phosphatidy1choline. Lysophosphatidylcholine as well as arachidonic acid raised the free Ca²⁺ concentration in the incubation media of permeabilized cells, isolated mitochondria and microsomes. The half maximally effective concentrations for Ca²⁺ release from mitochondria were 78 ± 1 mol/l for lysophosphatidylcholine and 80 ± 11 mol/l for arachidonic acid. Though isolated microsomes released Ca²⁺ in response to both agents, the combined presence of mitochondria and microsomes did not exhibit a synergism in Ca²⁺ release in response to arachidonic acid; the increase in the free Ca²⁺ concentration in response to lysophosphatidylcholine was even smaller than with mitochondria alone. It is concluded that the two reaction products of phospholipase A₂ can raise the cytoplasmic Ca²⁺ concentration and therefore may participate in cellular signal transduction. Send offprint requests to I. Rustenbeck at the above address     

白桦脂酸衍生物的合成修饰及其抗菌活性研究

目的：设计白桦脂酸（BA）的新型衍生物结构，研究其及衍生物的体外抗菌活性。方法：通过Jones氧化反应、Claisen Schmidt缩合反应等得到目标化合物BA-01；采用96孔板的琼脂稀释法测定化合物对5种细菌的最小抑菌浓度（MIC）。结果：合成了一种具有新型结构的BA衍生物，且为首次报道的新化合物，其结构通过¹H NMR，¹³C NMR和MS（ESI）等表征分析确定。体外抗菌活性测试结果表明，目标化合物对3种革兰阳性菌均有显著的抑菌活性，其中对金黄色葡萄球菌的抑菌活性最高，其最小抑菌浓度（MIC）为12.5 μmol·L^-1，与BA相比显著提高。结论：合成修饰的新型结构中C-3位羟基的修饰以及C-2位上苯环的连接对其生物活性具有重要影响，值得进一步深入探究。     

A comparison of the effects of L-NAME, 7-NI and L-NIL on carrageenan-induced hindpaw oedema and NOS activity

1. Intraplantar injection of carrageenan (150 μl, 1–3% w/v) in the rat resulted in a dose-related increase in hindpaw weight (oedema) characterized by a rapid ‘early'' phase (up to 2.5 h) response followed by a more sustained ‘late'' phase (2–6 h) response. No change in weight of either the contralateral (i.e. noninjected) hindpaw or hindpaws injected with saline was observed.
2. Six hours after intraplantar injection of carrageenan (1–3% w/v) hindpaw constitutive (i.e. calcium-dependent) nitric oxide synthase (cNOS) activity (determined ex vivo as the conversion of radiolabelled L-arginine to radiolabelled citrulline) was increased (e.g. 2% w/v; 0.64±0.08 pmol citrulline mg⁻¹ protein 15 min⁻¹ c.f. 0.08±0.04 pmol citrulline mg⁻¹ protein 15 min⁻¹ in saline-injected, control animals, n=4, P<0.05). Carrageenan injection also resulted in the appearance in hindpaw homogenates of inducible (i.e. calcium-independent) nitric oxide synthase (iNOS, e.g. 2% w/v; 0.67±0.14 pmol citrulline mg⁻¹ protein 15 min⁻¹, n=4). Hindpaw cyclic GMP concentration was also significantly increased 6 h after intraplantar injection of carrageenan (e.g. 2% w/v; 379.6±26.8 fmol mg⁻¹ protein c.f. 261.8±42.2 fmol mg⁻¹ protein, in saline-injected, control animals, n=4, P<0.05).
3. Pretreatment (5–25 mg kg⁻¹, i.p., 30 min before carrageenan, 2% w/v) of animals with L-N^G nitro arginine methyl ester (L-NAME; isoform nonselective inhibitor of NOS) or 7-nitro indazole (7-NI; inhibitor of neuronal NOS, nNOS) caused dose-related inhibition of both the early (2 h) and late (6 h) phase hindpaw oedema, associated with reduced hindpaw iNOS and cNOS activity and cyclic GMP concentration in animals killed at 6 h. Administration of 7-NI (5–25 mg kg⁻¹, i.p.) to animals 2.5 h after intraplantar carrageenan (2% w/v) injection (i.e. at the end of the early phase oedema response) produced dose-related inhibition of the late phase response.
4. Pretreatment (5–25 mg kg⁻¹, i.p., 30 min before carrageenan, 2% w/v) of animals with L-N6-iminoethyllysine (L-NIL, selective inhibitor of iNOS) (5–25 mg kg⁻¹) failed to affect the early phase hindpaw oedema response but did produce a dose-related inhibition of the late phase oedema. L-NIL pretreatment also inhibited the carrageenan-induced increase in both hindpaw iNOS and cNOS activity as well as the rise in hindpaw cyclic GMP concentration.
5. The present experiments demonstrate an anti-inflammatory effect of 7-NI as evidenced by inhibition of carrageenan-induced hindpaw oedema in the rat. Inhibition of nNOS (early phase) and iNOS (late phase) at the site of inflammation most probably accounts for the anti-inflammatory activity observed. These data suggest a role for nitric oxide synthesized by the nNOS isoform (most probably within sensory nerves) in this model of inflammation.