Structural and Chemical Biology of the Interaction of Cyclooxygenase with Substrates and Non-Steroidal Anti-Inflammatory Drugs.

Cyclooxgenases are key enzymes of lipid signaling. They carry out the first step in the production of prostaglandins, important mediators of inflammation, pain, cardiovascular disease, and cancer, and they are the molecular targets for nonsteroidal anti-inflammatory drugs, which are among the oldest and most chemically diverse set of drugs known. Homodimeric proteins that behave as allosterically modulated, functional heterodimers, the cyclooxygenases exhibit complex kinetic behavior, requiring peroxide-dependent activation and undergoing suicide inactivation. Due to their important physiological and pathophysiological roles and keen interest on the part of the pharmaceutical industry, the cyclooxygenases have been the focus of a vast array of structural studies, leading to the publication of over 80 crystal structures of the enzymes in complex with substrates or inhibitors supported by a wealth of functional data generated by site-directed mutation experiments. In this review, we explore the chemical biology of the cyclooxygenases through the lens of this wealth of structural and functional information. We identify key structural features of the cyclooxygenases, break down their active site into regional binding pockets to facilitate comparisons between structures, and explore similarities and differences in the binding modes of the wide variety of ligands (both substrates and inhibitors) that have been characterized in complex with the enzymes. Throughout, we correlate structure with function whenever possible. Finally, we summarize what can and cannot be learned from the currently available structural data and discuss the critical intriguing questions that remain despite the wealth of information that has been amassed in this field.

Pharmaco-Genetic Screen To Uncover Actin Regulators Targeted by Prostaglandins During Drosophila Oogenesis.

Prostaglandins (PGs) are lipid signaling molecules with numerous physiologic functions, including pain/inflammation, fertility, and cancer. PGs are produced downstream of cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs). In numerous systems, PGs regulate actin cytoskeletal remodeling, however, their mechanisms of action remain largely unknown. To address this deficiency, we undertook a pharmaco-genetic interaction screen during late-stage Drosophila oogenesis. Drosophila oogenesis is as an established model for studying both actin dynamics and PGs. Indeed, during Stage 10B, cage-like arrays of actin bundles surround each nurse cell nucleus, and during Stage 11, the cortical actin contracts, squeezing the cytoplasmic contents into the oocyte. Both of these cytoskeletal properties are required for follicle development and fertility, and are regulated by PGs. Here we describe a pharmaco-genetic interaction screen that takes advantage of the fact that Stage 10B follicles will mature in culture and COX inhibitors, such as aspirin, block this in vitro follicle maturation. In the screen, aspirin was used at a concentration that blocks 50% of the wild-type follicles from maturing in culture. By combining this aspirin treatment with heterozygosity for mutations in actin regulators, we quantitatively identified enhancers and suppressors of COX inhibition. Here we present the screen results and initial follow-up studies on three strong enhancers - Enabled, Capping protein, and non-muscle Myosin II Regulatory Light Chain. Overall, these studies provide new insight into how PGs regulate both actin bundle formation and cellular contraction, properties that are not only essential for development, but are misregulated in disease.

Structure and Dynamics of the Acyl Chains in the Membrane Trafficking and Enzymatic Processing of Lipids.

The regulatory chemical mechanisms of lipid trafficking and degradation are involved in many pathophysiological processes, being implicated in severe pain, inflammation, and cancer. In addition, the processing of lipids is also relevant for industrial and environmental applications. However, there is poor understanding of the chemical features that control lipid membrane trafficking and allow lipid-degrading enzymes to efficiently select and hydrolyze specific fatty acids from a complex cellular milieu of bioactive lipids. This is particularly true for lipid acyl chains, which have diverse structures that can critically affect the many complex reactions needed to elongate, desaturate, or transport fatty acids. Building upon our own contributions in this field, we will discuss how molecular simulations, integrated with experimental evidence, have revealed that the structure and dynamics of the lipid tail are actively involved in modulating membrane trafficking at cellular organelles, and enzymatic reactions at cell membranes. Further evidence comes from recent crystal structures of lipid receptors and remodeling enzymes. Taken together, these recent works have identified those structural features of the lipid acyl chain that are crucial for the regioselectivity and stereospecificity of essential desaturation reactions. In this context, we will first illustrate how atomistic and coarse-grained simulations have elucidated the structure-function relationships between the chemical composition of the lipid's acyl chains and the molecular properties of lipid bilayers. Particular emphasis will be given to the prominent chemical role of the number of double carbon-carbon bonds along the lipid acyl chain, that is, discriminating between saturated, monounsaturated, and polyunsaturated lipids. Different levels of saturation in fatty acid molecules dramatically influence the biophysical properties of lipid assemblies and their interaction with proteins. We will then discuss the processing of lipids by membrane-bound enzymes. Our focus will be on lipids such as anandamide and 2-arachidonoylglycerol. These are the main molecules that act as neurotransmitters in the endocannabinoid system. Specifically, recent findings indicate a crucial interplay between the level of saturation of the lipid tail, its energetically and sterically favored conformations, and the hydrophobic accessory cavities in lipid-degrading enzymes, which help form catalytically active conformations of the selected substrate. This Account will emphasize how the specific chemical structure of acyl chains affects the molecular mechanisms for modulating membrane trafficking and selective hydrolysis. The results examined here show that, by using molecular simulations to investigate lipid plasticity and substrate flexibility, researchers can enrich their interpretation of experimental results about the structure-function relationships of lipids. This could positively impact chemical and biological studies in the field and ultimately support protein engineering studies and structure-based drug discovery to target lipid-processing enzymes.

Synergistically dual-functional nano eye-drops for simultaneous anti-inflammatory and anti-oxidative treatment of dry eye disease.

We have developed a rapid and straightforward topical treatment method for dry eye disease (DED) using poly(catechin) capped-gold nanoparticles (Au@Poly-CH NPs) carrying amfenac [AF; a nonsteroidal anti-inflammatory drug (NSAID)] through effective attenuation of ocular surface tissue damage in dry eyes. A dual-targeted strategy based on ocular therapeutics was adopted to simultaneously block the cyclooxygenase enzymes-induced inflammation and reactive oxygen species (ROS)-induced oxidative stress, the primary two causes of DED. The self-assembled core-shell Au@Poly-CH NPs synthesized via a simple reaction between tetrachloroaurate(iii) and catechin possess a poly(catechin) shell (∼20 nm) on the surface of each Au NP (∼60 nm). The anti-oxidant and anti-inflammatory properties of AF/Au@Poly-CH NPs were evaluated by DCFH-DA and prostaglandin E2/VEGF assays, respectively. Our results demonstrate that Au@Poly-CH NPs not only act as an anti-oxidant to suppress ROS-mediated processes, but also serve as a drug carrier of AF for a synergistic effect on anti-inflammation. In vivo biocompatibility studies show good tolerability of AF/Au@Poly-CH NPs for potential use in the treatment of ocular surface pathologies. The dual-targeted therapeutic effects of AF/Au@Poly-CH NPs lead to rapid recovery from DED in a rabbit model. Au@Poly-CH NPs loaded with NSAIDs is a promising multifunctional nanocomposite for treating various inflammation- and oxidative stress-related diseases.

Optimization of structures, biochemical properties of ketorolac and its degradation products based on computational studies.

BACKGROUND: Ketorolac (KTR) is used as an analgesic drug with an efficacy close to that of the opioid family. It is mainly used for the short term treatment of post-operative pain. It can inhibit the prostaglandin synthesis by blocking cyclooxygenase (COX). METHODS: In this investigation, the inherent stability and biochemical interaction of Ketorolac (KTR) and its degradation products have been studiedon the basis of quantum mechanical approaches. Density functional theory (DFT) with B3LYP/ 6-31G (d) has been employed to optimize the structures. Thermodynamic properties, frontier molecular orbital features, dipole moment, electrostatic potential, equilibrium geometry, vibrational frequencies and atomic partial charges of these optimized structureswere investigated. Molecular docking has been performed against prostaglandin H2 (PGH2) synthase protein 5F19 to search the binding affinity and mode(s). ADMET prediction has performed to evaluate the absorption, metabolism and carcinogenic properties. RESULTS: The equilibrium geometry calculations support the optimized structures. Thermodynamic results disclosed the thermal stability of all structures. From molecular orbital data, all the degradents are chemically more reactive than parent drug (except K3). However, the substitution of carboxymethyl radicalin K4 improved the physicochemical properties and binding affinity. ADMET calculations predict the improved pharmacokinetic and non-carcinogenic properties of all degradents. CONCLUSION: Based on physicochemical, molecular docking, and ADMET calculation, this study can be helpful to understand the biochemical activities of Ketorolac and its degradents and to design a potent analgesic drug.

Could Aspirin and Diets High in Fiber Act Synergistically to Reduce the Risk of Colon Cancer in Humans?

Early inhibition of inflammation suppresses the carcinogenic process. Aspirin is the most commonly used non-steroid anti-inflammatory drugs (NSAIDs), and it irreversibly inhibits cyclooxygenase-1 and -2 (COX1, COX2). Multiple randomized clinical trials have demonstrated that aspirin offers substantial protection from colon cancer mortality. The lower aspirin doses causing only minimal gastrointestinal disturbance, ideal for long-term use, can achieve only partial and transitory inhibition of COX2. Aspirin's principal metabolite, salicylic acid, is also found in fruits and vegetables that inhibit COX2. Other phytochemicals such as curcumin, resveratrol, and anthocyanins also inhibit COX2. Such dietary components are good candidates for combination with aspirin because they have little or no toxicity. However, obstacles to using phytochemicals for chemoprevention, including bioavailability and translational potential, must be resolved. The bell/U-shaped dose-response curves seen with vitamin D and resveratrol might apply to other phytochemicals, shedding doubt on 'more is better'. Solutions include: (1) using special delivery systems (e.g., nanoparticles) to retain phytochemicals; (2) developing robust pharmacodynamic biomarkers to determine efficacy in humans; and (3) selecting pharmacokinetic doses relevant to humans when performing preclinical experiments. The combination of aspirin and phytochemicals is an attractive low-cost and low-toxicity approach to colon cancer prevention that warrants testing, particularly in high-risk individuals.

Ruthenium(ii) arene NSAID complexes: inhibition of cyclooxygenase and antiproliferative activity against cancer cell lines.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a group of molecules which have been found to be active against cancer cells with chemopreventive properties by targeting cyclooxygenase (COX-1 and COX-2) and lipoxygenase (LOX), commonly upregulated (particularly COX-2) in malignant tumors. Arene ruthenium(ii) complexes with a pseudo-octahedral coordination environment containing different ancillary ligands have shown remarkable activity against primary and metastatic tumors as reported earlier. This work describes the synthesis of four novel ruthenium(ii)-arene complexes viz. [Ru(η6-p-cymene)(nap)Cl] 1 [Hnap = naproxen or (S)-2-(6-methoxy-2-naphthyl)propionic acid], [Ru(η6-p-cymene)(diclo)Cl] 2 [Hdiclo = diclofenac or 2-[(2,6-dichlorophenyl)amino] benzeneacetic acid, [Ru(η6-p-cymene)(ibu)Cl] 3 [Hibu = ibuprofen or 2-(4-isobutylphenyl)propanoic acid] and [Ru(η6-p-cymene)(asp)Cl] 4 [Hasp = aspirin or 2-acetoxy benzoic acid] using different NSAIDs as chelating ligands. Complexes 1-3 have shown promising antiproliferative activity against three different cell lines with GI50 (concentration of drug causing 50% inhibition of cell growth) values comparable to adriamycin. At the concentration of 50 µM, complex 3 is more effective in the inhibition of cyclooxygenase and lipooxygenase enzymes, followed by complex 2 and complex 1 in comparison to their respective free NSAID ligands indicating a possible correlation between the inhibition of COX and/or LOX and anticancer properties. Molecular docking studies with COX-2 reveal that complexes 1 and 2 having naproxen and diclofenac ligands exhibit stronger interactions with COX-2 than their respective free NSAIDs and these results are in good agreement with their relative experimentally observed COX inhibition as well as anti-proliferative activities.

Anticancer Potential of Thiazole Derivatives: A Retrospective Review.

This review brings forth the potential of thiazole derivatives for their anticancer activities. The emphasis is placed on the structural diversity of thiazole derivatives, responsible for their specific anticancer activity. Multiple classes of thiazole derivatives such as Schiff base, mono-, di-, tri-, and heterocyclic substituents that possess anticancer activity have been exemplified. Molecular modelling of compounds that predicts enhanced anticancer activity of the modified structures has also been elaborated in the review. Significant advancements in synthetic chemistry related to cytotoxicity can now better position the drug discovery team to undertake thiazoles as valuable leads. The beneficial thiazole derivatives possessing anticancer activity will reignite the interest of medicinal chemists in thiazole and their derivatives.

Old drugs with new skills: fenoprofen as an allosteric enhancer at melanocortin receptor 3.

The efficiency of drug research and development has paradoxically declined over the last decades despite major scientific and technological advances, promoting new cost-effective strategies such as drug repositioning by systematic screening for new actions of known drugs. Here, we performed a screening for positive allosteric modulators (PAMs) at melanocortin (MC) receptors. The non-steroidal anti-inflammatory drug fenoprofen, but not the similar compound ibuprofen, presented PAM activity at MC3, MC4, and MC5 receptors. In a model of inflammatory arthritis, fenoprofen afforded potent inhibition while ibuprofen was nearly inactive. Fenoprofen presented anti-arthritic actions on cartilage integrity and synovitis, effects markedly attenuated in Mc3r-/- mice. Fenoprofen displayed pro-resolving properties promoting macrophage phagocytosis and efferocytosis, independently of cyclooxygenase inhibition. In conclusion, combining repositioning with advances in G-protein coupled receptor biology (allosterism) may lead to potential new therapeutics. In addition, MC3 PAMs emerged as a viable approach to the development of innovative therapeutics for joint diseases.

Linoleic acid-derived lipid mediators increase in a female-dominated subphenotype of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality; however, the role of inflammatory mediators in its pathobiology remains unclear. The aim of this study was to investigate the influence of gender in COPD on lipid mediator levels.Bronchoalveolar lavage fluid (BALF) and serum were obtained from healthy never-smokers, smokers and COPD patients (Global Initiative for Chronic Obstructive Lung Disease stage I-II/A-B) (n=114). 94 lipid mediators derived from the cytochrome-P450, lipoxygenase, and cyclooxygenase pathways were analysed by liquid chromatography-mass spectrometry.Multivariate modelling identified a 9-lipid panel in BALF that classified female smokers with COPD from healthy female smokers (p=6×10(-6)). No differences were observed for the corresponding male population (p=1.0). These findings were replicated in an independent cohort with 92% accuracy (p=0.005). The strongest drivers were the cytochrome P450-derived epoxide products of linoleic acid (leukotoxins) and their corresponding soluble epoxide hydrolase (sEH)-derived products (leukotoxin-diols). These species correlated with lung function (r=0.87; p=0.0009) and mRNA levels of enzymes putatively involved in their biosynthesis (r=0.96; p=0.003). Leukotoxin levels correlated with goblet cell abundance (r=0.72; p=0.028).These findings suggest a mechanism by which goblet cell-associated cytochrome-P450 and sEH activity produce elevated leukotoxin-diol levels, which play a putative role in the clinical manifestations of COPD in a female-dominated disease sub-phenotype.

Mimosine Dipeptide Enantiomsers: Improved Inhibitors against Melanogenesis and Cyclooxygenase.

Melanogenesis plays an important role in the protection of skin against UV through production of melanin pigments, but abnormal accumulation of this pigment causes unaesthetic hyperpigmentation. Much effort is being made to develop effective depigmenting agents. Here, we show for the first time that a small library of mimosine dipeptide enantiomers (Mi-L/D-amino acid) inhibit the melanogenesis in B16F10 melanoma cells by down-regulating the cellular tyrosinase with little effect on their growth or viability. Two of them, Mi-D-Trp and Mi-D-Val, turned out to be the most potent inhibitors on melanin content and cellular tyrosinase in B16F10 melanoma cells. In addition, most of the mimosine dipeptides were more potent than mimosine for inhibiting cyclooxygenase 1 (COX-1) with IC50 of 18-26 µM. Among them, Mi-L-Val and Mi-L-Trp inhibited cyclooxygenase 2 (COX-2) more potently than indomethacin, with IC50 values of 22 and 19 µM, respectively. Taken together, our results suggest the possibility that mimosine dipeptides could be better candidates (than mimosine) for anti-melanogenic (skin hyperpigmentation treatment) and cyclooxygenase (COX) inhibition.

Epigenetic regulations of inflammatory cyclooxygenase-derived prostanoids: molecular basis and pathophysiological consequences.

The potential relevance of prostanoid signaling in immunity and immunological disorders, or disease susceptibility and individual variations in drug responses, is an important area for investigation. The deregulation of Cyclooxygenase- (COX-) derived prostanoids has been reported in several immunoinflammatory disorders such as asthma, rheumatoid arthritis, cancer, and autoimmune diseases. In addition to the environmental factors and the genetic background to diseases, epigenetic mechanisms involved in the fine regulation of prostanoid biosynthesis and/or receptor signaling appeared to be an additional level of complexity in the understanding of prostanoid biology and crucial in controlling the different components of the COX pathways. Epigenetic alterations targeting inflammatory components of prostanoid biosynthesis and signaling pathways may be important in the process of neoplasia, depending on the tissue microenvironment and target genes. Here, we focused on the epigenetic modifications of inflammatory prostanoids in physiological immune response and immunological disorders. We described how major prostanoids and their receptors can be functionally regulated epigenetically and consequently the impact of these processes in the pathogenesis inflammatory diseases and the development of therapeutic approaches that may have important clinical applications.

The ambidextrous cyclooxygenase: an enduring target.

With the discovery of COX, pain has not been treated in a relentless manner. Numbers of drugs with fewer side effects were discovered and are used successfully for treatment of pain. With new research coming into being since the discovery of COX, it was established that COX can be targeted not only for treatment of pain but for curing various diseases like cancer, Alzheimer's, Parkinson's disease, Glaucoma etc. With the sighting of COX-3, another isoform of COX, new diseases are being targeted for better treatment.

Current concepts in colorectal cancer prevention with cyclooxygenase inhibitors.

Colorectal cancer is one of the commonest malignancies worldwide. Recently, there has been much speculation regarding the role of cyclooxygenase-2 (COX-2) suppression in chemoprevention. Drugs with the ability to inhibit COX-2 expression include aspirin, nonsteroidal anti-inflammatory drugs (NSAID) and selective COX-2 inhibitors. Any strategy for chemoprevention must be able to quantify how effective the potential treatment is likely to be and which drugs will be most useful. We would also need to know for how long the agent could be taken safely and if any side-effects could preclude long-term use. Evidence from observational studies and recent updates of randomised controlled trials have been very encouraging - at least indicating benefit from the long term use of aspirin, even at low dose, with greatest impact on prevention of proximal colon cancers and adenomas. Most studies do, however, also warn that risks of gastrointestinal bleeding increase with long-term use of aspirin and related drugs. The risk-to-benefit ratio of a chemoprevention regimen using these medications needs to be carefully examined.

Platelets, cyclooxygenases, and colon cancer.

A growing body of evidence supports the central role of platelets in early events of tumorigenesis and metastasis. Activated platelets, in response to tissue damage, induce a proinflammatory program involving the aberrant expression of cyclooxygenase (COX)-2, which leads to increased tissue concentrations of the proinflammatory and protumorigenic prostaglandin E2. The central role of platelet activation in cancer development is sustained by the analysis of clinical studies with aspirin showing an anti-cancer efficacy by the drug, even at the low doses used for the prevention of atherothrombosis. Low-dose aspirin acts as an antiplatelet agent by causing an irreversible inactivation of platelet COX-1 activity and the synthesis of thromboxane A2. Further experimental and clinical studies are ongoing to confirm the central role of platelets in the development of inflammation and cancer. The corroboration of this hypothesis will open new opportunities for the prevention and treatment of cancer. In addition to the possible use of traditional antithrombotic agents, the recent identification of novel molecular determinants involved in the cross-talk between platelets and other cellular player of tumorigenesis and metastasis has led to the suggestion of novel therapeutic strategies in oncology.

Pharmacological evaluation and docking studies of α,ß-unsaturated carbonyl based synthetic compounds as inhibitors of secretory phospholipase A2, cyclooxygenases, lipoxygenase and proinflammatory cytokines.

Arachidonic acid and its metabolites have generated high level of interest among researchers due to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as synergistic anti-inflammatory effect. A series of novel α,ß-unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory activity on secretory phospholipase A2 (sPLA2), cyclooxygenases (COX), soybean lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-α. Six α,ß-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of sPLA2 activity, with IC50 values in the range of 2.19-8.76 µM. Nine compounds 1-4 and 10-14 displayed inhibition of COX-1 with IC50 values ranging from 0.37 to 1.77 µM (lower than that of reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The compounds 10-14 exhibited strong inhibitory activity against LOX enzyme. All compounds were evaluated for the inhibitory activities against LPS-induced TNF-α and IL-6 release in the macrophages. On the basis of screening results, five active compounds 3, 4, 12, 13 and 14 were found strong inhibitors of TNF-α and IL-6 release in a dose-dependent manner. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX and LOX inhibitory activities of the investigated compounds. Present findings increases the possibility that these α,ß-unsaturated carbonyl based compounds might serve as beneficial starting point for the design and development of improved anti-inflammatory agents.

Chemoprevention of urothelial cell carcinoma growth and invasion by the dual COX-LOX inhibitor licofelone in UPII-SV40T transgenic mice.

Epidemiologic and clinical data suggest that use of anti-inflammatory agents is associated with reduced risk for bladder cancer. We determined the chemopreventive efficacy of licofelone, a dual COX-lipoxygenase (LOX) inhibitor, in a transgenic UPII-SV40T mouse model of urothelial transitional cell carcinoma (TCC). After genotyping, six-week-old UPII-SV40T mice (n = 30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm licofelone for 34 weeks. At 40 weeks of age, all mice were euthanized, and urinary bladders were collected to determine urothelial tumor weights and to evaluate histopathology. Results showed that bladders of the transgenic mice fed control diet weighed 3 to 5-fold more than did those of the wild-type mice due to urothelial tumor growth. However, treatment of transgenic mice with licofelone led to a significant, dose-dependent inhibition of the urothelial tumor growth (by 68.6%-80.2%, P < 0.0001 in males; by 36.9%-55.3%, P < 0.0001 in females) compared with the control group. The licofelone diet led to the development of significantly fewer invasive tumors in these transgenic mice. Urothelial tumor progression to invasive TCC was inhibited in both male (up to 50%; P < 0.01) and female mice (41%-44%; P < 0.003). Urothelial tumors of the licofelone-fed mice showed an increase in apoptosis (p53, p21, Bax, and caspase3) with a decrease in proliferation, inflammation, and angiogenesis markers (proliferating cell nuclear antigen, COX-2, 5-LOX, prostaglandin E synthase 1, FLAP, and VEGF). These results suggest that licofelone can serve as potential chemopreventive for bladder TCC.

Interaction of nonsteroidal anti-inflammatory drugs with membranes: in vitro assessment and relevance for their biological actions.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world due to their anti-inflammatory, analgesic and antipyretic properties. Nevertheless, the consumption of these drugs is still associated with the occurrence of a wide spectrum of adverse effects. Regarding the major role of membranes in cellular events, the hypothesis that the biological actions of NSAIDs may be related to their effect at the membrane level has triggered the in vitro assessment of NSAIDs-membrane interactions. The use of membrane mimetic models, cell cultures, a wide range of experimental techniques and molecular dynamics simulations has been providing significant information about drugs partition and location within membranes and also about their effect on diverse membrane properties. These studies have indeed been providing evidences that the effect of NSAIDs at membrane level may be an additional mechanism of action and toxicity of NSAIDs. In fact, the pharmacokinetic properties of NSAIDs are closely related to the ability of these drugs to interact and overcome biological membranes. Moreover, the therapeutic actions of NSAIDs may also result from the indirect inhibition of cyclooxygenase due to the disturbing effect of NSAIDs on membrane properties. Furthermore, increasing evidences suggest that the disordering effects of these drugs on membranes may be in the basis of the NSAIDs-induced toxicity in diverse organ systems. Overall, the study of NSAIDs-membrane interactions has proved to be not only important for the better understanding of their pharmacological actions, but also for the rational development of new approaches to overcome NSAIDs adverse effects.

Novel indomethacin action: selective and direct activation of protein kinase C-ε.

BACKGROUND/AIMS: In our earlier study, indomethacin potentiated α7 acetylcholine (ACh) receptor responses by activating protein kinase C (PKC). The present study was conducted to gain further insight into the indomethacin action on PKC. METHODS: PKC activity was assayed in PC-12 cells or under the cell-free conditions. PKC-ε was knocked-down using the siRNA to silence the PKC-ε-targeted gene. A fluorescein-conjugated indomethacin was synthesized to examine the interaction of indomethacin with PKC-ε. RESULTS: In the in situ PKC assay, indomethacin activated PKC in PC-12 cells in a concentration (1-100 µM)-dependent manner, and the activation was suppressed by knocking-down PKC-ε. In the cell-free PKC assay, indomethacin (100 µM) activated PKC-ε in the absence of diacylglycerol, phosphatidylserine, and calcium, but other PKC isozymes such as α, ßΙ, ßΙΙ, γ, δ, ι, and ζ were not activated. In the indomethacin binding assay using a fluorescent-conjugated indomethacin on blue native-polyacrylamide gel electrophoresis (blue native-PAGE), a fluorescent signal was detected at the site consistent with PKC-ε protein and the signal was attenuated by adding non-conjugated indomethacin or eliminated by pretreatment with non-conjugated indomethacin. CONCLUSION: The results of the present study show that indomethacin has the potential to selectively activate PKC-ε through its direct binding, independently of cyclooxygenase (COX) inhibition.

Pharmacological differences of endothelin receptors-mediated modulation in cultured interstitial cells of Cajal from the murine small and large intestine.

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal smooth muscle. Under current clamping, ICCs had a mean resting membrane potential of -58 ± 3 mV and externally applied ET produced membrane depolarization in a dosedependent manner. These effects were reduced by intracellular GDP beta S. A comparison of the concentration-dependent membrane depolarizations on pacemaker potentials to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1≥ET-2≥ET-3 in cultured murine small intestinal ICCs. The pretreatment with Ca(2+)-free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the ET-1 induced membrane depolarizations. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the ET-1 induced effects on pacemaker potentials. Pretreatment with BQ-123 (ET(A )receptor antagonist) or BQ-788 (ET(B )receptor antagonist) blocked the ET-1 induced effects on pacemaker potentials in cultured murine small intestinal ICCs. However, pretreatment with BQ-788 selectively did not block the ET-1 induced effects on pacemaker potentials in cultured murine large intestinal ICCs. Also, only externally applied selective ET(B )receptor agonist, IRL 1620 did not show any influence on pacemaker potentials in cultured murine large intestine ICCs. RT-PCR results indicated the presence of the ET(A )and ET(B )receptor in ICCs. These results suggested that ET-1 modulates pacemaker potentials through ET(A )and ET(B )receptor activation in murine small intestinal ICCs and ET(A )receptor activation in murine large intestinal ICCs by external Ca(2+) influx and internal Ca(2+) release via protein kinase C or cyclooxygenase-independent mechanism. Therefore, the ICCs are targets for ET and their interaction can affect intestinal motility.