European Journal of Pharmacology (v.622, #1-3)

Inhibition of H2O2-induced neuroblastoma cell cytotoxicity by a triazine derivative, AA3E2 by Hamed Shaykhalishahi; Razieh Yazdanparast; Hyung-Ho Ha; Young-Tae Chang (1-6).
Alzheimer's disease is the major cause of senile dementia with the hallmark of β-amyloid deposition in neurons. Although the main cause(s) of this deposition is not fully understood, however, the wealth of the present literature data supports the pivotal role of reactive oxygen and nitrogen species in both the initiation and progression of β-amyloid aggregation and deposition. In the present study, we were interested to evaluate the free-radical protecting effect of AA3E2, a triazine derivative with a β-amyloid-breaking activity, among SK-N-MC neuroblastoma cells exposed to hydrogen peroxide (H2O2) as an exogenous source of free radicals. Exposure of the cells to different doses of AA3E2 (1–16 µM) for 3 h followed by subsequent exposure to a single dose of H2O2 (mainly 150 µM) attenuated the extent of superoxide dismutase (SOD) and catalase (CAT) inhibition by H2O2, in a dose dependent manner. Furthermore, significant reduction was observed in the extent of cellular lactate dehydrogenase release, intracellular ROS and the extent of apoptosis among the cells pre-treated with AA3E2. Based on these data, an antioxidant mode of action is proposed for AA3E2 besides its previously β-amyloid-breaking activity.
Keywords: Alzheimer's disease; Antioxidant; Apoptosis; Hydrogen peroxide; SK-N-MC cell; Triazine compound;

Isoform specificity of cardiac glycosides binding to human Na+,K+-ATPase α1β1, α2β1 and α3β1 by Christian Hauck; Tatjana Potter; Michaela Bartz; Thorsten Wittwer; Thorsten Wahlers; Uwe Mehlhorn; Georgios Scheiner-Bobis; Alicia A. McDonough; Wilhelm Bloch; Robert H.G. Schwinger; Jochen Müller-Ehmsen (7-14).
Cardiac glycosides inhibit the Na+,K+-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na+,K+-ATPase are expressed: α1β1, α2β1 and α3β1. It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na+,K+-ATPase isoforms α1β1, α2β1 and α3β1 were expressed in yeast which has no endogenous Na+,K+-ATPase. Isoform specific affinities of digoxin, digitoxin, β-acetyldigoxin, methyldigoxin and ouabain were assessed in [³H]-ouabain binding assays in the absence or presence of K+ (each n  = 5). The subcellular localizations of the Na+,K+-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K+, methyldigoxin (α1  > α3  > α2) and ouabain (α1  = α3  > α2) showed distinct isoform specific affinities, while for digoxin, digitoxin and β-acetyldigoxin no differences were found. In the presence of K+, also digoxin (α2  = α3  > α1) and β-acetyldigoxin (α1  > α3) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only α1β1 and α2β1 are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na+,K+-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance.
Keywords: Heart failure; Digoxin; Digitoxin; β-Acetyldigoxin; Methyldigoxin;

Estrogen rapidly attenuates cannabinoid-induced changes in energy homeostasis by Brian A. Kellert; Mike C. Nguyen; Cara Nguyen; Que H. Nguyen; Edward J. Wagner (15-24).
We examined whether estrogen negatively modulates cannabinoid-induced regulation of food intake, core body temperature and neurotransmission at proopiomelanocortin (POMC) synapses. Food intake was evaluated in ovariectomized female guinea pigs abdominally implanted with thermal DataLoggers and treated s.c. with the cannabinoid CB1/CB2 receptor agonist WIN 55,212-2, the CB1 receptor antagonist AM251 or their cremephor/ethanol/0.9% saline vehicle, and with estradiol benzoate (EB) or its sesame oil vehicle. Whole-cell patch clamp recordings were performed in slices through the arcuate nucleus. WIN 55,212-2 produced dose- and time-dependent increases in food intake. EB decreased food intake 8–24 h after administration, but rapidly and completely blocked the increase in consumption caused by WIN 55,212-2. EB also attenuated the WIN 55,212-2-induced decrease in core body temperature. The AM251-induced decrease in food intake was unaffected. The diminution of the WIN 55,212-2-induced increase in food intake caused by EB correlated with a marked attenuation of cannabinoid receptor-mediated decreases in glutamatergic miniature excitatory postsynaptic current frequency occurring within 10–15 min of steroid application. Furthermore, EB completely blocked the depolarizing shift in the inactivation curve for the A-type K+ current caused by WIN 55,212-2. The EB-mediated, physiologic antagonism of these presynaptic and postsynaptic actions elicited upon cannabinoid receptor activation was observed in arcuate neurons immunopositive for phenotypic markers of POMC neurons. These data reveal that estrogens negatively modulate cannabinoid-induced changes in appetite, body temperature and POMC neuronal activity. They also impart insight into the neuroanatomical substrates and effector systems upon which these counter-regulatory factors converge in the control of energy homeostasis.
Keywords: Estrogen; POMC; Appetite; Cannabinoids; Glutamate; K+ channels; Electrophysiology;

Neuroprotective effects of the new diterpene, CBNU06 against beta-amyloid-induced toxicity through the inhibition of NF-kappaB signaling pathway in PC12 cells by Hyo-Shin Kim; Ji-Youn Lim; Donggeun Sul; Bang Yeon Hwang; Tae-Jun Won; Kwang Woo Hwang; So-Young Park (25-31).
Alzheimer's disease is the most common form of dementia, causing progressive cognitive dysfunction, particularly memory loss. Recently, modulation of beta-amyloid (Aβ) toxicity, one of the major potential causes of Alzheimer's disease, has emerged as a possible therapeutic approach to control the onset of Alzheimer's disease. In this study, we investigated the neuroprotective effects and possible mechanisms by which 19-hydroxy-1α,6-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide (named as CBNU06), a new diterpene isolated from Isodon japonicus, acts against Aβ-induced toxicity in PC12 cells. Pretreatment with CBNU06 (20 μg/ml) prior to Aβ25 –35 (25 μM) significantly increased the viability of PC12 cells in a dose-dependent manner when examined by Hoechst staining, MTT assay and Trypan blue exclusion assay. This protective effect was accompanied by the decrease in translocation of NF-κB p50 and p65 from the cytoplasm to the nucleus, and followed by the decrease in cyclooxygenase-2 (COX-2) levels. In addition, pretreatment with CBNU06 significantly reversed the effect of Aβ on Bax and Bcl-2. Taken together, these results suggest that CBNU06 protected PC12 cells against Aβ-induced neurotoxicity through the inhibition of the NF-κB signaling pathways. Therefore, CBNU06 has the possible beneficial effects in Alzheimer's disease by attenuating Aβ-induced toxicity.
Keywords: CBNU06 (19-hydroxy-1α,6-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide); Beta-amyloid; NF-kappaB; Alzheimer's disease; PC12 cells;

Enhanced effect of neuropeptide Y on food intake caused by blockade of the V1A vasopressin receptor by Toshinori Aoyagi; Shinji Kusakawa; Atsushi Sanbe; Masami Hiroyama; Yoko Fujiwara; Junji Yamauchi; Akito Tanoue (32-36).
Food intake is regulated by various factors such as neuropeptide Y. Neuropeptide Y potently induces an increase in food intake, and simultaneously stimulates arginine-vasopressin (AVP) secretion in the brain. Recently, we reported that V1A vasopressin receptor-deficient (V1AR−/−) mice exhibited altered daily food intake accompanied with hyperglycemia and hyperleptinemia. Here, we further study the involvement of the AVP/V1A receptor in the appetite regulation of neuropeptide Y with V1AR−/− mice and antagonists for the AVP receptor. The intra-cerebral-ventricle administration of neuropeptide Y induced greater food consumption in V1AR−/− mice than wild-type (WT) mice, whereas an anorexigenic effect of leptin was not different between the two groups. This finding suggests that the orexigenic effect of neuropeptide Y was enhanced in V1AR−/− mice, leading to the increased food intake in response to the neuropeptide Y stimulation. In addition, the neuropeptide Y-induced orexigenic effect was enhanced by co-administration of OPC-21268, an antagonist for the V1A vasopressin receptor, into the cerebral ventricle in WT mice, whereas the neuropeptide Y-induced orexigenic effect was not affected by co-administration of SSR-149415, an antagonist for the V1B vasopressin receptor. These results indicate that AVP could suppress the neuropeptide Y-induced orexigenic effect via the V1A vasopressin receptor, and that blockade or inhibition of the AVP/V1A receptor signal resulted in the enhanced neuropeptide Y-induced orexigenic effect. Thus, we show that the AVP/V1A receptor is involved in appetite regulation as an anorexigenic factor for the neuropeptide Y-induced orexigenic effect.
Keywords: Arginine-vasopressin; V1A receptor; Neuropeptide Y; Feeding behavior;

Reduced spatial learning in mice treated with NVP-AAM077 through down-regulating neurogenesis by Mei Hu; Yong-Jun Sun; Qi-Gang Zhou; Yves P. Auberson; Ling Chen; Yao Hu; Chun-Xia Luo; Jia-Yi Wu; Dong-Ya Zhu; Li-Xin Li (37-44).
NR2A-containing N-methyl-d-aspartate (NMDA) receptors have important roles in influencing the long-term potentiation and spatial memory. Here using Morris water maze, we found that inhibition of NR2A-containing NMDA receptors by [(R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2, 3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) hindered the formation of spatial memory. An increasing number of reports suggest that adult hippocampal neurogenesis is involved in hippocampal-mediated learning. To explore the possible mechanisms understanding the reduced spatial memory by NVP-AAM077, we investigated the effects of NVP-AAM077 on neurogenesis. We found that NVP-AAM077 inhibited progenitor cells proliferation in the subventricular zone and dentate gyrus and reduced the survival of newborn cells in the dentate gyrus in the adult mice. In null mutant mice lacking neuronal nitric oxide synthase (nNOS) gene (nNOS−/−), the effects of NVP-AAM077 on neurogenesis disappeared. In addition, NVP-AAM077 increased nNOS enzymatic activity. Our findings suggest that NVP-AAM077 reduced spatial learning through down-regulating neurogenesis in the adult hippocampus.
Keywords: NVP-AAM077; NR2A; Spatial memory; Neurogenesis; nNOS;

PPARδ ligand L-165041 ameliorates Western diet-induced hepatic lipid accumulation and inflammation in LDLR−/− mice by Hyun-Joung Lim; Jin-Hee Park; Seahyoung Lee; Hye-Eun Choi; Kuy-Sook Lee; Hyun-Young Park (45-51).
Although peroxisome proliferator-activated receptor delta (PPARδ) has been implicated in energy metabolism and lipid oxidation process, detailed roles of PPARδ in lipid homeostasis under pathologic conditions still remain controversial. Thus, we investigated the effect of PPARδ ligand L-165041 on Western diet-induced fatty liver using low-density lipoprotein receptor-deficient (LDLR−/−) mice. LDLR−/− mice received either L-165041 (5 mg/kg/day) or vehicle (0.1 N NaOH) with Western diet for 16 weeks. According to our data, L-165041 drastically reduced lipid accumulation in the liver, decreasing total hepatic cholesterol and triglyceride content compared to the vehicle group. Gene expression analysis demonstrated that L-165041 lowered hepatic expression of PPARγ, apolipoprotein B, interleukin 1 beta (IL-1β), and interleukin-6. In contrast, L-165041 increased hepatic expressions of PPARδ, lipoprotein lipase (LPL), and ATP-binding cassette transporter G1 (ABCG1). Our data suggest that L-165041 might be effective in preventing Western diet-induced hepatic steatosis by regulating genes involved in lipid metabolism and the inflammatory response.
Keywords: Peroxisome proliferator-activated receptor delta; LDLR−/− mice; Steatosis; Inflammation;

Anti-inflammatory action of mollugin and its synthetic derivatives in HT-29 human colonic epithelial cells is mediated through inhibition of NF-κB activation by Kyoung-Jin Kim; Jong Suk Lee; Mi-Kyoung Kwak; Han Gon Choi; Chul Soon Yong; Jung-Ae Kim; Yong Rok Lee; Won Seok Lyoo; Young-Joon Park (52-57).
Mollugin is the active compound of Rubia cordifolia, which has been used as a traditional Chinese medicine for the treatment of various inflammatory diseases including arthritis and uteritis. In the present study, we investigated for the first time the inhibitory effects and the mechanisms of action of mollugin (M1) and its synthetic derivatives (M2–M4) on tumor necrosis factor (TNF)-α-induced inflammatory responses in HT-29 human colon epithelial cells. Treatment with M1 and its derivatives M2–M4 significantly inhibited TNF-α-induced attachment of U937 monocytic cells to HT-29 cells, which mimics the initial phase of colon inflammation. TNF-α-induced mRNA induction of the chemokines, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8, and the intercellular cell adhesion molecule (ICAM)-1, which are involved in adhesion between leukocytes and epithelial cells, was suppressed by M1–M4, and M1 was the most efficacious. In addition, M1–M4 significantly suppressed TNF-α-induced NF-κB transcriptional activity. Such NF-κB inhibitory activity of M1–M4 (20 µM) correlated with their ability to suppress TNF-α-induced chemokine expression and U937 monocytic cell adhesion to HT-29 colonic epithelial cells. Treatment of HT-29 cells with M1 and PDTC, a NF-κB inhibitor, synergistically suppressed both TNF-α-induced NF-κB activation and monocytic cell adhesion to HT-29 cells. These results suggest that M1–M4 inhibit TNF-α-induced expression of inflammatory molecules via NF-κB, and that M1, a potent NF-κB inhibitor, may be a valuable new drug candidate for the treatment of colon inflammation.
Keywords: Mollugin; Chemokine; Intercellular adhesion molecule (ICAM)-1; NF-κB; Intestinal inflammation;

Anti-inflammatory effects of phosphatidylcholine in neutrophil leukocyte-dependent acute arthritis in rats by Petra Hartmann; Andrea Szabó; Gábor Erős; Dóra Gurabi; Gyöngyi Horváth; István Németh; Miklós Ghyczy; Mihály Boros (58-64).
We investigated the effects of exogenous phosphatidylcholine (PC) and non-steroidal diclofenac supplementation on polymorphonuclear cell influx in carrageenan-induced arthritis in rats. The microcirculatory consequences were evaluated by a novel method developed for direct intravital microscopic observation of the synovial membrane. Arthritis was induced by injection of a mixture of 2% λ-carrageenan and 4% kaolin into the knee joints and the animals were treated orally with PC (150 mg/kg twice daily), sodium diclofenac (0.5 mg/kg twice daily) or saline vehicle. Intravital videomicroscopy was used to investigate the leukocyte-endothelial interactions directly in the synovial membrane at 6 h after the challenge. The inflammation–induced thermal and mechanical secondary hyperalgesic reactions were assessed at 24 h, and the knee volume changes at 48 h after the insult. The development of arthritis was accompanied by a significant increase in the number of adherent leukocytes in the synovial postcapillary venules, but this increase was reduced significantly (by ∼ 40%) by PC, and slightly (by 22%) by diclofenac treatment. The perivascular infiltration of the neutrophil leukocytes and the intercellular adhesion molecule-1 (ICAM-1) expressions were reduced only by PC treatment. The significant decrease (45%) in the thermal nociceptive latency, the 3-fold increase in the mechanical touch sensitivity and the knee cross-sectional area (which was increased by 35% by the arthritis induction) were significantly ameliorated by both treatments. The present study demonstrated the anti-inflammatory effects of PC in experimental arthritis. The therapeutic potential may be linked to the reduction of neutrophil leukocyte-mediated microcirculatory inflammatory reactions.
Keywords: Carrageenan; Synovium; Diclofenac; ICAM-1; Microcirculation intravital videomicroscopy;

The present study was aimed to investigate the possible pancreatic tissue protective nature of D-Pinitol, a cyclitol present in soybean, against free radical-mediated oxidative stress in streptozotocin-induced diabetic rats by assaying the activity of pancreatic enzymatic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) and the levels of plasma non-enzymatic antioxidants such as vitamin E, vitamin C, ceruloplasmin and reduced glutathione (GSH). To assess the extent of oxidative stress, the levels of lipid peroxidation (LPO) and hydroperoxides in both plasma and pancreatic tissues were also measured. A significant increase in the levels of both lipid peroxides and hydroperoxides with a concomitant decrease in antioxidant status was observed in the diabetic rats when compared to control rats. Oral administration of D-Pinitol (50 mg/kg b.w./day for 30 days), a major cyclitol present in soybean, ameliorates the free radical-mediated alterations to near normalcy. The pancreatic tissue protective nature of D-Pinitol was further evidenced by histological observations. The results were statistically comparable with glyclazide, a standard hypoglycemic drug. Thus, the results of the present study suggest that D-Pinitol protects the pancreatic tissue from free radical-mediated oxidative stress in addition to its antidiabetic property.
Keywords: D-Pinitol; Diabetes mellitus; Streptozotocin; Lipid peroxidation; Oxidative stress;

Effects of the dipeptidyl peptidase-IV inhibitor ASP8497 on glucose tolerance in animal models of secondary failure by Yuka Someya; Ryosuke Nakano; Atsuo Tahara; Toshiyuki Takasu; Asako Takeuchi; Itsuro Nagase; Akiko Matsuyama-Yokono; Masahiro Hayakawa; Masao Sasamata; Keiji Miyata; Yasuo Uchiyama (71-77).
Sulfonylureas promote insulin secretion and potently lower blood glucose levels, however, they induce hypoglycemia and undergo a reduction in efficacy when administered long-term (secondary failure). The dipeptidyl peptidase (DPP)-IV inhibitor ASP8497, (2S,4S)-4-fluoro-1-({[4-methyl-1-(methylsulfonyl)piperidin-4-yl]amino}acetyl)pyrrolidine-2-carbonitrile monofumarate, inhibits the degradation of glucagon-like peptide-1 (GLP-1), an incretin hormone, and promotes insulin secretion in a glucose-dependent manner. ASP8497 is therefore less likely to induce hypoglycemia and less likely to show reduced efficacy even after repeated administration. Here, to determine whether or not ASP8497 improves glucose tolerance in Zucker fatty rats, we examined the effects of ASP8497 and gliclazide, a sulfonylurea, on glucose tolerance after repeated administration. We also developed an animal model of secondary failure using streptozotocin–nicotinamide-induced diabetic mice. Results: ASP8497 (3 mg/kg) improved glucose intolerance in Zucker fatty rat without any attenuation (blood glucose AUC: P  = 0.034 vs. vehicle) while gliclazide (10 mg/kg) did not (P  = 0.916 vs. vehicle). Furthermore, ASP8497 (3, 10 mg/kg) was found to effect glucose tolerance dose-dependently (3 mg/kg: P  = 0.230, 10 mg/kg: P  = 0.003 vs. glibenclamide (10 mg/kg)) by enhancing insulin secretion in mice inadequately controlled with glibenclamide. Our results suggest that ASP8497 may be effective even in patients with secondary failure who are unable to maintain satisfactory glycemic control using sulfonylureas.
Keywords: ASP8497; Dipeptidyl peptidase-IV; Glucagon-like peptide-1; Diabetes; Sulfonylurea; Secondary failure;