COX-2-mediated PGD2 synthesis regulates phosphatidylcholine biosynthesis in rat renal papillary tissue

Phosphatidylcholine (PC) is the major membrane phospholipid in mammalian cells. Previous works from our laboratory demonstrated a close metabolic relationship between the maintenance of PC biosynthesis and the prostaglandins endogenously synthesized by cyclooxygenase (COX) in rat renal papilla. In the present work, we studied the COX isoform involved in papillary PC biosynthesis regulation. The incorporation of [methyl-3H]choline and [32P]orthophosphate to PC was determined in the absence and presence of SC-560 and NS-398, COX-1 and COX-2 specific inhibitors. PC synthesis was highly sensitive to COX-2 inhibition, while COX-1 inhibition only reduced PC synthesis at high SC-560 concentration. The analysis of choline-containing metabolites showed that COX-2 inhibition affected the formation of CDP-choline intermediary. The evaluation of PC biosynthetic enzymes revealed that microsomal, as well as nuclear, CTP:phosphocholine cytidylyltransferase (CCT), and nuclear-CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CTP) activities were affected by COX-2 inhibition. The addition of exogenous prostaglandin D(2) (PGD(2)) restored nuclear-CCT and -CPT activities but not microsomal CCT. Papillary synthesis of PGD(2) was only detected in nuclear fraction where it was blocked by COX-2 inhibitor NS-398, but not by COX-1 inhibitor. All together, the present results demonstrated that COX-2-mediated PGD(2) synthesis is a PC biosynthesis regulator in rat renal papilla. Considering the importance of the maintenance of PC biosynthesis for the preservation of cell membrane homeostasis to ensure cell viability, and the extensive use of COX-2 inhibitors in therapeutics, the present results could have great pharmacological implications, and can constitute a biochemical explanation for the nephrotoxic effect of non-steroidal anti-inflammatory drugs.     

Microsomal prostaglandin E synthase-1 and cyclooxygenase-2 are both required for ischaemic excitotoxicity

Background and purpose:

Although both microsomal prostaglandin E synthase (mPGES)-1 and cyclooxygenase (COX)-2 are critical factors in stroke injury, but the interactions between these enzymes in the ischaemic brain is still obscure. This study examines the hypothesis that mPGES-1 activity is required for COX-2 to cause neuronal damage in ischaemic injury.

Experimental approach:

We used a glutamate-induced excitotoxicity model in cultures of rat or mouse hippocampal slices and a mouse middle cerebral artery occlusion-reperfusion model in vivo. The effect of a COX-2 inhibitor on neuronal damage in mPGES-1 knockout (KO) mice was compared with that in wild-type (WT) mice.

Key results:

In rat hippocampal slices, glutamate-induced excitotoxicity, as well as prostaglandin (PG) E₂ production and PGES activation, was significantly attenuated by either MK-886 or NS-398, inhibitors of mPGES-1 and COX-2 respectively; however, co-application of these inhibitors had neither an additive nor a synergistic effect. The protective effect of NS-398 on the excitotoxicity observed in WT slices was completely abolished in mPGES-1 KO slices, which showed less excitotoxicity than WT slices. In the transient focal ischaemia model, mPGES-1 and COX-2 were co-localized in the infarct region of the cortex. Injection of NS-398 reduced not only ischaemic PGE₂ production, but also ischaemic injuries in WT mice, but not in mPGES-1 KO mice, which showed less dysfunction than WT mice.

Conclusion and implications:

Microsomal prostaglandin E synthase-1 and COX-2 are co-induced by excess glutamate in ischaemic brain. These enzymes are co-localized and act together to exacerbate stroke injury, by excessive PGE₂ production.     

Diltiazem potentiates pentobarbital-induced hypnosis via 5-HT1A and 5-HT2A/2C receptors: role for dorsal raphe nucleus

It has been reported that the sedative component of pentobarbital is mediated by GABA receptors in an endogenous sleep pathway and the ventrolateral preoptic area (VLPO)-tuberomammillary nucleus (TMN) or VLPO-dorsal raphe nucleus (DRN) neural circuit is important in the sedative response to pentobarbital. Our previous findings indicated that the VLPO-TMN neuronal circuit may play crucial part in the augmentative effect of diltiazem on pentobarbital sleep and the serotonergic system may be involved. This study was designed to investigate the role of DRN and the serotonergic receptors 5-HT_1A and 5-HT_2A/2C in the augmentative effect of diltiazem on pentobarbital-induced hypnosis in rats. The results showed that diltiazem (5 mg/kg, i.g.) significantly reversed pentobarbital-induced (35 mg/kg, i.p.) reduction of c-Fos expression in 5-HT neurons of DRNV (at − 7.5 mm Bregma), DRND, DRNVL and MRN (at − 8.0 mm Bregma). However it did not influence this reducing effect of pentobarbital on non-5-HT neurons either in DRN or in MRN. Moreover, the effect of diltiazem (1 or 2 mg/kg, i.g.) on pentobarbital-induced (35 mg/kg, i.p.) hypnosis was significantly inhibited by 5-HT_1A agonist 8-OH-DPAT (0.5 mg/kg, i.p.) and 5-HT_2A/2C agonist DOI (0.5 mg/kg, i.p.), and potentiated by 5-HT_1A antagonist p-MPPI (2 mg/kg, i.p.) and 5-HT_2A/2C antagonist ritanserin (2 mg/kg, i.p.), respectively. From these results, it should be presumed that the augmentative effect of diltiazem on pentobarbital-induced sleep may be related to 5-HT_1A and 5-HT_2A/2C receptors, and DRN may be involved. In addition, it also suggested that the DRN may play a multi-modulating role in sleep-wake regulation rather than being recognized simply as arousal nuclei.     

In vivo control of 5-hydroxytryptamine release by terminal autoreceptors in rat brain areas differentially innervated by the dorsal and median raphe nuclei

Selective serotonin reuptake inhibitors (SSRIs) reduce the 5-HT release in vivo. This effect is due to the activation of somatodendritic 5-HT_1A receptors and it displays a regional pattern comparable to that of selective 5-HT_1A agonists, i.e., preferentially in forebrain areas innervated by the dorsal raphe nucleus (DRN). However, despite a comparatively lower 5-HT_1A-mediated inhibition of 5-HT release and a greater density of serotonergic uptake sites in hippocampus, the net elevation produced by the systemic administration of SSRIs is similar in various forebrain areas, regardless of the origin of serotonergic fibres. As terminal autoreceptors may also limit the SSRI-induced elevations of 5-HT in the extracellular brain space, we reasoned that a differential control of 5-HT release by terminal autoreceptors in DRN- and median raphe-innervated areas might be accountable. To examine this possibility, we have conducted a regional microdialysis study in the DRN, MRN and four forebrain regions preferentially innervated either by the DRN (frontal cortex, striatum) or the median raphe nucleus (MRN; dorsal and ventral hippocampus) using freely moving rats. Dialysis probes were perfused with 1 μM of the SSRI citalopram to augment the endogenous tone on terminal 5-HT autoreceptors. The non-selective 5-HT₁ antagonist methiothepin (10 and 100 μM, dissolved in the dialysis fluid) increased extracellular 5-HT in frontal cortex and dorsal hippocampus in a concentration-dependent manner. The 5-HT_1B/1D antagonist GR 127935 was ineffective at 10 μM and tended to reduce 5-HT in dorsal hippocampus at 100 μM. The local infusion of 100 μM methiothepin significantly elevated the extracellular 5-HT concentration to 142–173% of baseline (mean values of 260 min post-administration) in the DRN, MRN, frontal cortex, striatum and hippocampus (dorsal and ventral). Comparable elevations were noted in the four forebrain regions examined. As observed in frontal cortex and dorsal hippocampus, the perfusion of 10 μM GR 127935 did not elevate 5-HT in DRN, MRN, striatum or ventral hippocampus. Because the stimulated 5-HT release in the DRN has been suggested to be under control of 5-HT_1B/1D receptors, we examined the possible contribution of these receptor subtypes to the effects of methiothepin in the DRN. The perfusion of sumatriptan (0.01–10 μM) or GR 127935 (0.01–10 μM) did not significantly modify the 5-HT concentration in dialysates from the DRN. Thus, the present data suggest that the comparable effects of SSRIs in DRN- and MRN-innervated forebrain regions are not explained by a preferential attenuation of 5-HT release by terminal 5-HT_1B autoreceptors in hippocampus, an area with a low inhibitory influence of somatodendritic 5-HT_1A receptors. Methiothepin-sensitive autoreceptors (possibly 5-HT_1B) appear to play an important role not only in the projection areas but also with respect to the control of 5-HT release in the DRN and MRN. In addition, our findings indicate that GR 127935 is not an effective antagonist of the actions of 5-HT at rat terminal autoreceptors. Received: 27 February 1998 / Accepted: 12 June 1998     

原花青素对脂多糖诱导RAW2647细胞COX-2酶活性、mRNA及蛋白表达的影响

目的探讨原花青素对脂多糖(LPS)诱导小鼠巨噬细胞株RAW264.7细胞COX-2酶活性及蛋白表达的影响。方法放射免疫法检测COX-2酶活性，RT-PCR检测COX-2 mRNA表达，Western blotting检测COX-2蛋白表达。结果原花青素(0.8，4和20 mg·L^-1)不影响LPS诱导RAW264.7细胞COX-2酶活性，可下调LPS诱导RAW264.7细胞COX-2 mRNA表达；原花青素(4和20 mg·L^-1)下调LPS诱导RAW264.7细胞COX-2蛋白表达。结论原花青素不影响LPS诱导RAW2647细胞COX-2酶活性，但对LPS诱导RAW264.7细胞COX-2 mRNA及蛋白表达抑制作用明显。     

Influence of AMPA/kainate receptors on extracellular 5-hydroxytryptamine in rat midbrain raphe and forebrain

1. The regulation of 5-hydroxytryptamine (5-HT) release by excitatory amino acid (EAA) receptors was examined by use of microdialysis in the CNS of freely behaving rats. Extracellular 5-HT was measured in the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), nucleus accumbens, hypothalamus, frontal cortex, dorsal and ventral hippocampus.
2. Local infusion of kainate produced increases in extracellular 5-HT in the DRN and MRN. Kainate infusion into forebrain sites had a less potent effect.
3. In further studies of the DRN and nucleus accumbens, kainate-induced increases in extracellular 5-HT were blocked by the EAA receptor antagonists, kynurenate and 6,7-dinitroquinoxaline-2,3-dione (DNQX).
4. The effect of infusing kainate into the DRN or nucleus accumbens was attenuated or abolished by tetrodotoxin (TTX), suggesting that the increase in extracellular 5-HT is dependent on 5-HT neuronal activity. In contrast, ibotenate-induced lesion of intrinsic neurones did not attenuate the effect of infusing kainate into the nucleus accumbens. Thus, the effect of kainate in the nucleus accumbens does not depend on intrinsic neurones.
5. Infusion of α-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate (AMPA) into the DRN and nucleus accumbens induced nonsignificant changes in extracellular 5-HT. Cyclothiazide and diazoxide, which attenuate receptor desensitization, greatly enhanced the effect of AMPA on 5-HT in the DRN, but not in the nucleus accumbens.
6. In conclusion, AMPA/kainate receptors regulate 5-HT in the raphe and in forebrain sites.

     

Cyclooxygenase-2 and tissue inhibitor of matrix metalloproteinases-1 confer the antimigratory effect of cannabinoids on human trabecular meshwork cells

Cannabinoids have received considerable attention as potential antiglaucomatous drugs. Recently, prostaglandins (PG) have been suggested to contribute to this effect. Within the factors conferring the development of glaucoma, depletion of the aqueous humor outflow-regulating trabecular meshwork (TM) cells elicited by migration from the outflow system is considered to play a pivotal role. This study therefore investigates the impact of two cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), on the migration of human TM cells and the involvement of the PG-synthesizing enzyme cyclooxygenase-2 (COX-2) and one of its potential downstream targets, the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), to this response. Using Boyden chamber assays cannabinoids were shown to elicit an antimigratory effect that was reversed by antagonists for CB₁ as well as CB₂ receptors and accompanied by upregulation of COX-2 and TIMP-1 expression and PGE₂ synthesis. Knockdown of cannabinoid-induced COX-2 or TIMP-1 expression by siRNA or inhibition of COX-2 activity by NS-398 led to a significant suppression of this antimigratory action. Migration was also diminished by the major COX-2 product PGE₂ and by recombinant TIMP-1. Experiments using selective E prostanoid (EP) receptor agonists and antagonists revealed that decreased migration by PGE₂, THC and MA was mediated via EP₂ and EP₄ receptors. Finally, the cannabinoid-mediated increases of TIMP-1 levels were abolished by NS-398, and PGE₂ was shown to elicit a concentration-dependent increase of TIMP-1. Collectively, this data demonstrate a COX-2-dependent upregulation of TIMP-1 conferring the antimigratory action of cannabinoids. A decreased migration reducing TM cell loss in glaucoma might be involved in the antiglaucomatous action of cannabinoids.     

角叉菜诱导大鼠胸膜炎中环氧酶-2的生成及NS-398对其选择性抑制作用

目的：检测角叉菜诱导的大鼠胸膜炎渗出细胞中COX-2的存在并观察NS-398对其活性的影响。方法：用SDS-PAGE和Western blot分析及PGHS-1和PGHS-2抗血清,识别并检测胸膜炎渗出细胞中的COX-2。酶活性用TLC法测定。结果：致炎后5 h, PGHS-2出现, 19 h时消失,而PGHS-1在注射角叉菜前后均出现。此外,在胸膜炎大鼠和正常大鼠的肺、胃、肾微粒体中,只检测到PGHS-1, 未检测到PGHS-2。新的非甾体抗炎药NS-398,可明显抑制COX-2活性（IC₅₀=4.5 μmol.L^-1 ）。 结论：COX-2仅存在于角叉菜诱导的炎症部位;NS-398可选择性抑制COX-2,并呈剂量依赖性。     

Somatodendritic 5-HT1A receptors are critically involved in the anxiolytic effects of 8-OH-DPAT

In the rat shock-induced ultrasonic vocalization test, the anxiolytic effects of the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) obtained after systemic (IP) and intracerebral injection into the dorsal raphe nucleus (DRN) were selectively abolished by pretreatment with the 5-HT_1A receptor antagonist WAY-100635 [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclo-hexanecarboxamide trihydrochloride]. This blockade was demonstrated both after systemic and DRN application of WAY-100635. Therefore, it is concluded that the anxiolytic effects of 8-OH-DPAT are mediated by activation of somatodendritic 5-HT_1A receptors.     

N-(p-Coumaryol)-Tryptamine Suppresses the Activation of JNK/c-Jun Signaling Pathway in LPS-Challenged RAW264.7 Cells

N-(p-Coumaryol) tryptamine (CT), a phenolic amide, has been reported to exhibit anti-oxidant and anti-inflammatory activities. However, the underlying mechanism by which CT exerts its pharmacological properties has not been clearly demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of CT in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells. CT significantly inhibited LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and PGE₂, and protein expressions of iNOS and COX-2. In addition, CT significantly suppressed LPS-induced secretion of pro-inflammatory cytokines such as TNF-α and IL-1β. To elucidate the underlying anti-inflammatory mechanism of CT, involvement of MAPK and Akt signaling pathways was examined. CT significantly attenuated LPS-induced activation of JNK/c-Jun, but not ERK and p38, in a concentration-dependent manner. Interestingly, CT appeared to suppress LPS-induced Akt phosphorylation. However, JNK inhibition, but not Akt inhibition, resulted in the suppression of LPS-induced responses, suggesting that JNK/c-Jun signaling pathway significantly contributes to LPS-induced inflammatory responses and that LPS-induced Akt phosphorylation might be a compensatory response to a stress condition. Taken together, the present study clearly demonstrates CT exerts anti-inflammatory activity through the suppression of JNK/c-Jun signaling pathway in LPS-challenged RAW264.7 macrophage cells.     

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE₂ synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC₅₀ = 16.5 ± 3.8 nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE₂ synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE₂ synthesis (IC₅₀ in the range of 0.5-5 μM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF_1α (PGF_1α) and PGF_2α. In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE₂, PGF_1α and PGF_2α synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.     

Dopamine D(2) receptor-induced COX-2-mediated production of prostaglandin E(2) in D(2)-transfected Chinese hamster ovary cells without simultaneous administration of a Ca(2+)-mobilizing agent

We have earlier demonstrated that dopamine stimulates the liberation of the prostaglandin E(2) (PGE(2)) precursor, arachidonic acid, in Chinese hamster ovary cells transfected with the rat dopamine D(2) receptor (long isoform), also without concomitant administration of a Ca(2+)-releasing agent [Nilsson et al., Br J Pharmacol 1998;124:1651-8]. In the present report, we show that dopamine, under the same conditions, also induces a concentration-dependent increase in the production of PGE(2), with a maximal effect of 235% at approximately 100 microM, and with an EC(50) of 794 nM. The effect was counteracted by the D(2) antagonist eticlopride, pertussis toxin, the inhibitor of intracellular Ca(2+) release TMB-8, incubation in Ca(2+)-free experimental medium, and PKC desensitization obtained by chronic pretreatment with the phorbol ester TPA. It was also antagonized by the non-specific cyclooxygenase (COX) inhibitor, indomethacin, and by the selective COX-2 inhibitor, NS-398, but not by the specific COX-1 inhibitor, valeryl salicylate. Both the non-specific phospholipase A(2) inhibitor, quinacrine, and an inhibitor of cPLA(2) and iPLA(2), AACOF3, counteracted the effect; in contrast, a selective iPLA(2) inhibitor, BEL, and a selective sPLA(2) inhibitor, TAPC, were ineffective. No effects of dopamine were obtained in control cells mock-transfected with the p3C vector only. The results reinforce previous assumptions that dopamine may interact with eicosanoid metabolism by means of D(2) receptor activation, and implicate an involvement of cPLA(2) and COX-2 in this effect. It is suggested that measurement of dopamine-induced PGE(2) production may serve as a convenient way to study D(2) receptor function in vitro.     

Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 µM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A₄ (LTA₄) hydrolase and leukotriene C₄ (LTC₄) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA₄ hydrolase and LTC₄ synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.     

Anti-inflammatory activity of bark of Dioscorea batatas DECNE through the inhibition of iNOS and COX-2 expressions in RAW264.7 cells via NF-κB and ERK1/2 inactivation

We identified a bioactive herbal medicine with anti-inflammatory activity from an ethanol extract derived from the bark of Dioscorea batatas DECNE (BDB) in RAW264.7 cells. We examined the effects of BDB on nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in LPS-induced RAW264.7 cells. BDB consistently inhibited both NO and PGE₂ production in a dose-dependent manner, with an IC₅₀ of 87–71 μg/ml, respectively. The reduction of NO and PGE₂ production were accompanied by a reduction in iNOS and COX-2 protein expression, as evaluated by Western blotting. To evaluate the action mode of BDB and its ability to inhibit iNOS and COX-2 protein expression, we assessed the effects of BDB on nuclear factor-κB (NF-κB) DNA-binding activity, NF-κB-dependent reporter gene activity, inhibitory factor-κB (IκB) phosphorylation and degradation, and p65 nuclear translocation. BDB suppressed DNA-binding activity and reporter gene activity as well as translocation of the NF-κB p65 subunit. BDB also down-regulated IκB kinase (IKK), thus inhibiting LPS-induced both phosphorylation and the degradation of IκBα. In addition, BDB also inhibited the LPS-induced activation of ERK1/2.     

Melatonin modulates nitric oxide-regulated WNK-SPAK/OSR1-NKCC1 signaling in dorsal raphe nucleus of rats

The sleep-wake cycle is regulated by the alternating activity of sleep- and wake-promoting neurons. The dorsal raphe nucleus (DRN) secretes 5-hydroxytryptamine (5-HT, serotonin), promoting wakefulness. Melatonin secreted from the pineal gland also promotes wakefulness in rats. Our laboratory recently demonstrated that daily changes in nitric oxide (NO) production regulates a signaling pathway involving with-no-lysine kinase (WNK), Ste20-related proline alanine rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1), and cation-chloride co-transporters (CCC) in rat DRN serotonergic neurons. This study was designed to investigate the effect of melatonin on NO-regulated WNK-SPAK/OSR1-CCC signaling in wake-inducing DRN neurons to elucidate the mechanism underlying melatonin’s wake-promoting actions in rats. Ex vivo treatment of DRN slices with melatonin suppressed neuronal nitric oxide synthase (nNOS) expression and increased WNK4 expression without altering WNK1, 2, or 3. Melatonin increased phosphorylation of OSR1 and the expression of sodium-potassium-chloride co-transporter 1 (NKCC1), while potassium-chloride co-transporter 2 (KCC2) remained unchanged. Melatonin increased the expression of tryptophan hydroxylase 2 (TPH2, serotonin-synthesizing enzyme). The present study suggests that melatonin may promote its wakefulness by modulating NO-regulated WNK-SPAK/OSR1-KNCC1 signaling in rat DRN serotonergic neurons.     

COX-2 inhibitors block chemotherapeutic agent-induced apoptosis prior to commitment in hematopoietic cancer cells

Enzymatic inhibitors of pro-inflammatory cyclooxygenase-2 (COX-2) possess multiple anti-cancer effects, including chemosensitization. These effects are not always linked to the inhibition of the COX-2 enzyme. Here we analyze the effects of three COX-2 enzyme inhibitors (nimesulide, NS-398 and celecoxib) on apoptosis in different hematopoietic cancer models. Surprisingly, COX-2 inhibitors strongly prevent apoptosis induced by a panel of chemotherapeutic agents. We selected U937 cells as a model of sensitive cells for further studies. Here, we provide evidence that the protective effect is COX-independent. No suppression of the low basal prostaglandin (PG)E₂ production may be observed upon treatment by COX-2 inhibitors. Besides, the non-active celecoxib analog 2,5-dimethyl-celecoxib is able to protect from apoptosis as well. We demonstrate early prevention of the stress-induced apoptotic signaling, prior to Bax/Bak activation. This preventive effect fits with an impairment of the ability of chemotherapeutic agents to trigger apoptogenic stress. Accordingly, etoposide-induced DNA damage is strongly attenuated in the presence of COX-2 inhibitors. In contrast, COX-2 inhibitors do not exert any anti-apoptotic activity when cells are challenged with physiological stimuli (anti-Fas, TNFα or Trail) or with hydrogen peroxide, which do not require internalization and/or are not targeted by chemoresistance proteins. Altogether, our findings show a differential off-target anti-apoptotic effect of COX-2 inhibitors on intrinsic vs. extrinsic apoptosis at the very early steps of intracellular signaling, prior to commitment. The results imply that an exacerbation of the chemoresistance phenomena may be implicated.     

当归A_3活性部位对小鼠巨噬细胞环氧化酶-2活性及基因表达的影响

目的研究当归A3活性部位对小鼠巨噬细胞RAW264.7环氧化酶-2(cyclooxygenase-2,COX-2)活性及基因表达的影响。方法采用酶联免疫法(enzyme-line immu-nosorbnent assay,ELISA)检测前列腺素E2(prostaglandin E2,PGE2)产量及COX-2活性,采用RT-PCR和Western blot法检测COX-2 mRNA及蛋白表达水平。结果 A3(20、40、80mg.L-1)剂量依赖性地抑制LPS诱导的RAW264.7细胞PGE2产量、COX-2活性、COX-2 mRNA及蛋白表达水平增高。结论 A3能够直接抑制PGE2产量,此作用可能与抑制COX-2基因表达有关。