European Journal of Pharmacology (v.663, #1-3)

A residue W756 in the P-loop segment of the sodium channel is critical for primaquine binding by Eduardo M. Salinas-Stefanon; Evelyn Martinez-Morales; Thomas F. Scior; Lourdes Millan-PerezPeña (1-8).
Our study on the wild-type and mutants of the voltage-dependent sodium channel in the rat skeletal muscle Nav 1.4 was to examine the possible binding site of primaquine PQ by using an experimental approach. We used a standard voltage-clamp in oocytes. Previously, we had demonstrated that PQ blocks the voltage-dependent sodium current in rat myocytes and that this blocking is concentration-dependent and voltage-independent. The direct-site mutagenesis in the P-loop segment W402C, W756C, W1239C, W1531A at the outer tryptophan-rich lip, and D400C, E758C, K1237C, A1529C of the DEKA locus helped us to identify residues playing a key role in aminoquinoline binding. In full agreement with our computed results, where a 1000-fold reduction of inhibition was measured, the tryptophan 756 is crucial for the reversible modulating effects of PQ. The W756C decreased the blocking effect of PQ in voltage-clamp assays. This new binding site may be important to the development of new drugs that modulate sodium inward currents.
Keywords: Sodium channel; Primaquine; Oocyte; Xenopus laevis; Voltage-clamp; Site mutagenesis; Nav 1.4 isoform;

The effect of topiramate on tumor-related angiogenesis and on the serum proteome of mice bearing Lewis lung carcinoma by Bing Ma; Yan Pan; Qianliu Song; Lu Tie; Ye Zhang; Yuan Xiao; Jianzhao Zhang; Jing Han; Yan Xu; Yang Xiang; He-Ming Yu; Xue-Jun Li (9-16).
Topiramate has been used in patients with brain tumors who develop epilepsy. In our previous research we found topiramate could inhibit tumor metastases of Lewis lung carcinoma in C57BL/6 mice. In this study we aimed to assess the antimetastatic activity of topiramate and determine its mechanism of action. After confirming the effects of topiramate on Lewis lung carcinoma in C57BL/6 mice, we assessed the mRNA expression of carbonic anhydrases II and IX, and the vascular endothelial growth factor (VEGF) distribution in tumor tissue. We studied the role of topiramate on primary angiogenesis using a chicken embryo chorioallantoic membrane angiogenesis model, and analyzed the protein profile of serum from mice treated with or without topiramate by two-dimensional electrophoresis. We found that topiramate significantly reduced the primary tumor growth (P  < 0.05) and the degree of damage to the lung alveoli caused by metastatic tumor deposits. The two-dimensional electrophoresis revealed changes that occurred with topiramate treatment and four down-regulated protein spots were clearly identified as tropomyosin, osteopontin, transthyretin, and serum amyloid A-1. The mRNA and protein expression of serum amyloid A-1, osteopontin and its receptor, integrin αvβ3 in tumor tissue were reconfirmed. The results suggest that topiramate has antitumor and antimetastatic effects on Lewis lung carcinoma. Its mechanism of action may be related to its inhibition of angiogenesis by down-regulation of osteopontin, VEGF and carbonic anhydrase II.
Keywords: Tumor metastasis; Angiogenesis; Topiramate; Osteopontin; VEGF; Carbonic anhydrase II;

Sustained activation of ERK and Cdk2/cyclin-A signaling pathway by pemetrexed leading to S-phase arrest and apoptosis in human non-small cell lung cancer A549 cells by Tsung-Ying Yang; Gee-Chen Chang; Kun-Chieh Chen; Hsiao-Wen Hung; Kuo-Hsuan Hsu; Gwo-Tarng Sheu; Shih-Lan Hsu (17-26).
Pemetrexed, a multitargeted antifolate with the ability to inhibit several enzymes involved in purine and pyrimidine syntheses, has demonstrated clinical activity in non-small cell lung cancer cells, as well as in a broad array of other solid tumors. In this study, we show that inducing cell cycle S-phase arrest and apoptosis in human lung adenocarcinoma A549 cells with pemetrexed is associated with increased cyclin-A and cyclin-dependent kinase 2 (Cdk2) protein and Cdk2/cyclin-A kinase activity. Knockdown of cyclin-A using small interfering RNA (siRNA), and inhibiting Cdk2 activity with flavopiridol, strikingly reduced S-phase arrest and apoptosis. Moreover, pemetrexed induced sustained activation of extracellular signal-regulated kinase1/2 (ERK1/2). Knockdown of ERK1/2 using specific siRNA, as well as known inhibitors (PD98059 and U0126), effectively suppressed the expression of cyclin-A and Cdk2, and reduced S-phase arrest and apoptosis induced by pemetrexed. These data provide the first evidence that pemetrexed-induced S-phase arrest and apoptosis is associated with an increase in Cdk2 and cyclin-A expression and activation, which is ERK-dependent and upstream of caspase-3. Our findings suggest that the ERK-mediated Cdk2/cyclin-A signaling pathway is an important regulator of pemetrexed-induced S-phase arrest and apoptotic cell death.
Keywords: Pemetrexed; S phase arrest; Apoptosis; ERK; CDK2; Cyclin-A;

Acidosis promoted tartaric acid-resistant acid phosphatase-positive multinuclear cell (TRAP + MNC) or osteoclast formation. Large osteoclast or TRAP + LMNC formation was observed far more in an acidosis environment than in a physiologically neutral environment. One of the major action points of acidosis was determined to be located in the last phase of preosteoclast differentiation using a co-culture system and a soluble RANKL-dependent bone marrow cell culture system. On-going osteoclast formation in an acidosis environment markedly deteriorated when the medium was replaced with physiologically neutral medium within the first 6 h; however, bone marrow cells previously stimulated in an acidosis environment for 9 h differentiated into TRAP + LMNC in pH 7.4 medium. Messenger RNA (mRNA) expression levels of DC-STAMP, a key molecule in cell fusion, and NFATc1 did not increase in the acidosis environment compared with those under physiologically neutral conditions. Ruthenium red, a general TRP antagonist, deteriorated acidosis-promoted TRAP + LMNC formation. 4-Alpha-PDD, a TRPV4-specific agonist, added in the last 21 h of preosteoclast differentiation, potentiated TRAP + LMNC formation in a mild acidosis environment, showing synergism between TRPV4 activation and acidosis. RN1734, a TRPV4-specific antagonist, partly inhibited acidosis-promoted TRAP + LMNC formation. We thus narrowed down the major action points of acidosis in osteoclast formation and elucidated the characteristics of this system in detail. Our results show that acidosis effectively uses TRPV4 to drive large-scale cell fusion and also utilizes systems independently of TRPV4.
Keywords: Acidosis; Osteoclast formation; Cell fusion; Proton sensing; TRP channel;

Characterization of 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl)-amine (JNJ-39729209) as a novel TRPV1 antagonist by Michael P. Maher; Anindya Bhattacharya; Hong Ao; Nadia Swanson; Nyan-Tsz Wu; Jamie Freedman; Mena Kansagara; Brian Scott; Dong H. Li; William A. Eckert; Yi Liu; Kia Sepassi; Michele Rizzolio; Anne Fitzgerald; Jing Liu; Bryan J. Branstetter; Jason C. Rech; Alec D. Lebsack; J. Guy Breitenbucher; Alan D. Wickenden; Sandra R. Chaplan (40-50).
As an integrator of multiple nociceptive and/or inflammatory stimuli, TRPV1 is an attractive therapeutic target for the treatment of various painful disorders. Several TRPV1 antagonists have been advanced into clinical trials and the initial observations suggest that TRPV1 antagonism may be associated with mild hyperthermia and thermal insensitivity in man. However, no clinical efficacy studies have been described to date, making an assessment of risk:benefit impossible. Furthermore, it is not clear whether these early observations are representative of all TRPV1 antagonists and whether additional clinical studies with novel TRPV1 antagonists are required in order to understand optimal compound characteristics. In the present study we describe 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl)-amine (JNJ-39729309) as a novel, TRPV1 antagonist. JNJ-39729209 displaced tritiated resiniferotoxin binding to TRPV1 and prevented TRPV1 activation by capsaicin, protons and heat. In-vivo, JNJ-39729209 blocked capsaicin-induced hypotension, induced a mild hyperthermia and inhibited capsaicin-induced hypothermia in a dose dependent manner. JNJ-39729209 showed significant efficacy against carrageenan- and CFA-evoked thermal hyperalgesia and exhibited significant anti-tussive activity in a guinea-pig model of capsaicin-induced cough. In pharmacokinetic studies, JNJ-39729209 was found to have low clearance, a moderate volume of distribution, good oral bioavailability and was brain penetrant. On the basis of these findings, JNJ-39729209 represents a structurally novel TRPV1 antagonist with potential for clinical development. The advancement of JNJ-39729209 into human clinical trials could be useful in further understanding the analgesic potential of TRPV1 antagonists.
Keywords: Ion channels; TRPV1; Vanilloid receptor; Capsaicin; Pain; Cough;

The present investigation was designed to study the cardioprotective effects of nicorandil and pioglitazone preconditioning in myocardial ischemia/reperfusion-induced hemodynamic, biochemical and histological changes in rats. Oral doses of nicorandil (3 or 6 mg/kg) and pioglitazone (10 or 20 mg/kg) were administered once daily for 5 consecutive days. Rats were then subjected to myocardial ischemia/reperfusion (40 min/10 min). Heart rate and ventricular arrhythmias were recorded during ischemia/reperfusion progress. At the end of reperfusion, plasma creatine kinase-MB activity and total nitrate/nitrite were determined. In addition, lactate, adenine nucleotides, thiobarbituric acid reactive substances, reduced glutathione and myeloperoxidase activity were estimated in the heart left ventricle. Finally, histological examination was performed to visualize the protective cellular effects of different pretreatments. Nicorandil (3 or 6 mg/kg) was effective in attenuating the ischemia/reperfusion-induced ventricular arrhythmias, creatine kinase-MB release, lactate accumulation and oxidative stress. Nicorandil (3 mg/kg) was more effective in improving the energy production and lowering the elevated myeloperoxidase activity. Both doses of pioglitazone (10 or 20 mg/kg) were equally effective in reducing lactate accumulation and completely counteracting the oxidative stress. Pioglitazone (10 mg/kg) was more effective in improving energy production and reducing ventricular arrhythmias, plasma creatine kinase-MB release and total nitrate/nitrite. It seems that selective mitochondrial KATP channel opening by lower doses of nicorandil and pioglitazone in the present study provided more cardioprotection against ventricular arrhythmias and biochemical changes induced by ischemia/reperfusion. Histological examination revealed also better improvement by the lower dose of nicorandil than that of pioglitazone.
Keywords: Arrhythmias; Ischemia; Nicorandil; Pioglitazone; Preconditioning; Reperfusion;

Ginsenoside Rg3 decelerates hERG K+ channel deactivation through Ser631 residue interaction by Sun-Hye Choi; Tae-Joon Shin; Sung-Hee Hwang; Byung-Hwan Lee; Jiyeon Kang; Hyeon-Joong Kim; Su-Hyun Jo; Han Choe; Seung-Yeol Nah (59-67).
The human ether-a-go-go-related gene (hERG) cardiac K+ channels are one of the representative pharmacological targets for development of drugs against cardiovascular diseases such as arrhythmia. Panax ginseng has been known to have cardio-protective effects. However, little is known about the molecular mechanisms of how ginsenosides, the active ingredients in Panax ginseng, interact with hERG K+ channel proteins. In the present study, we first examined the effects of various ginsenosides on hERG K+ channel activity by expressing human α subunits in Xenopus oocytes. Among them ginsenoside Rg3 (Rg3) most potently enhanced outward I hERG and peak I tail . Rg3 induced a large persistent deactivating-tail current (I deactivating-tail ) and profoundly decelerated deactivating current decay in both concentration- and voltage-dependent manners. The EC50 for steady-state I hERG , peak I tail , and persistent I deactivating-tail was 0.41 ± 0.05, 0.61 ± 0.11, and 0.36 ± 0.04 μM, respectively. Rg3 actions were blocked by bepridil, a hERG K+ channel antagonist. Site-directed mutation of S631, which is located at the channel pore entryway, to S631C in hERG K+ channel abolished Rg3 actions on hERG K+ channels. These results indicate that S631 residue of hERG K+ channel plays an important role in Rg3-mediated induction of a persistent I deactivating-tail and in a deceleration of hERG K+ channel deactivation.
Keywords: Panax ginseng; Ginsenoside Rg3; hERG K+ channel; Interaction site; Human heart;

Acute lung injury or acute respiratory distress syndrome is a serious clinical problem with high mortality. Oxidative stress was found to play a major role in mediating lung injury and antioxidants have been shown to be effective in attenuating acute lung injury. In this study, we determine the effects of tempol, a membrane-permeable radical scavenger, in lipopolysaccharide (LPS)-induced acute lung injury and the underlying mechanism. Acute lung injury was induced by intraperitoneal injection of LPS (1 mg/kg) and mice were treated with tempol 30 min before injection of LPS. One hour later, bronchoalveolar lavage fluid was collected and subjected to estimation of total and differential cell counts as well as the proinflammatory cytokines; tumor necrosis factor-alpha(TNF-α), interleukin-1beta(IL-1β) and interferon-gamma (IFN-γ). Lung tissue damage was confirmed by histopathological changes and by immunohistochemical analysis of myeloperoxidase (MPO). Moreover, lipid peroxidation, reduced glutathione (GSH) and nitric oxide (NO) were investigated in the lung tissue. Pretreatment with tempol produced significant attenuation of LPS-induced lung injury as well as inhibition of LPS mediated increase in MPO immunostaining, MDA and NO levels in lung tissue. Elevated cytokines levels in both bronchoalveolar lavage fluid and lung tissue homogenates of acute lung injury mice were significantly decreased after administration of tempol. These findings confirmed significant protection by tempol against LPS-induced acute lung injury and that superoxide anion scavenging appears to be a potential target for new potential therapy in pulmonary disorders.
Keywords: Acute lung injury; Lipopolysaccharide; Tempol; Superoxide anion;

The effects of jatrorrhizine on contractile responses of rat ileum by JianYe Yuan; JiYan Zhou; ZhiBi Hu; Guang Ji; JianQun Xie; DaZheng Wu (74-79).
This study was designed to evaluate the effect of jatrorrhizine on smooth muscle contractions isolated from rat ileum longitudinal muscles. Jatrorrhizine increased the amplitude of spontaneous contractions of ileum longitudinal muscles in concentration-dependent manner with an EC50 of 30.0 ± 8.4  μM. Preincubation of ileum strips with atropine (1  μM), 4-diphenyllacetoxy-N (2-chloriethyl)-piperidine (4-DAMP, 1  μM) or darifenacin (1  μM) significantly inhibited acetylcholine (0.1  μM)- and jatrorrhizine (100  μM)-induced ileum longitudinal muscle contractions, whereas they were not affected by AF-DX116 (1  μM) or hexamethonium (100  μM). Pretreatment with SB204070 (1  μM) rather than 3-tropanyl-indole-3-carboxyleat (tropisetron, 1  μM) significantly inhibited 5-HT (10  μM)-induced ileum longitudinal muscle contractions. In contrast, jatrorrhizine-induced ileum longitudinal muscle contractions were not inhibited by tropisetron or SB204070. Furthermore, jatrorrhizine-induced ileum longitudinal muscle contractions were strongly inhibited by nifedipine (1  μM), and also attenuated by removal of extracellular Ca2+, U73122 (1  μM), ruthenium red (50  μM) or 2-aminoethoxydiphenylborate (2-APB, 10  μM). Taken together, jatrorrhizine-elicited spontaneous contractions in rat ileum longitudinal muscles are mediated by activation of acetylcholine receptors, mostly the M3 receptor. Ca2+ influx through L-type Ca2+ channel is significantly contributed to jatrorrhizine-elicited spontaneous contractions, and Ca2+ release via IP3 and ryanodine pathways are also involved.
Keywords: Jatrorrhizine; Ileum; Muscarinic receptor; 5-hydroxytryptamine (5-HT) receptor; Ca2+-depentent signal pathway; Rat;

Chronic glucokinase activation reduces glycaemia and improves glucose tolerance in high-fat diet fed mice by Maria Sörhede Winzell; Matthew Coghlan; Brendan Leighton; Georgia Frangioudakis; David M. Smith; Leonard H. Storlien; Bo Ahrén (80-86).
Glucokinase (GK) plays a key role in maintaining glucose homeostasis by promoting insulin secretion from pancreatic beta cells and increasing hepatic glucose uptake. Here we investigate the effects of acute and chronic GK activation on glucose tolerance and insulin secretion in mice with diet-induced insulin resistance. In the acute study, a small molecule GK activator (GKA71) was administered to mice fed a high-fat diet for 8 weeks. In the long-term study, GKA71 was provided in the diet for 4 weeks to high-fat diet-fed mice. Glucose tolerance was measured after intravenous glucose administration, and insulin secretion was measured both in vivo and in vitro. Acute GK activation efficiently improved glucose tolerance in association with increased insulin secretion after intravenous glucose both in control and high-fat fed mice. Chronic GK activation significantly reduced basal plasma glucose and insulin, and improved glucose tolerance despite reduced insulin secretion after intravenous glucose, suggesting improved insulin sensitivity. Isolated islets from chronically GKA71-treated mice displayed augmented insulin secretion at 8.3 mmol/l glucose, without affecting glucose oxidation. High-fat diet fed mice had reduced glycogen and increased triglyceride in liver compared to control mice, and these parameters were not altered by long-term GK activation. We conclude that GK activation in high-fat diet-fed mice potently reduces glycaemia and improves glucose tolerance, with combined effect both to stimulate insulin secretion from islets and improve insulin sensitivity.
Keywords: Islet; Insulin secretion; Glucokinase; Glucose tolerance; Insulin resistance; High-fat diet;