European Journal of Pharmacology (v.645, #1-3)

Tenuigenin protects cultured hippocampal neurons against methylglyoxal-induced neurotoxicity by Yu-Jing Chen; Xiao-Bo Huang; Zong-Xin Li; Lin-Lin Yin; Wen-Qiang Chen; Lin Li (1-8).
Methylglyoxal is a metabolite of glucose. Since serum methylglyoxal level is increased in diabetic patients, methylglyoxal is implicated in diabetic complications such as cognitive impairment. This study aimed to evaluate the effects of tenuigenin, an active component of roots of Polygala tenuifolia Willdenow, on methylglyoxal-induced cell injury in a primary culture of rat hippocampal neurons. MTT and Hoechst 33342 staining, together with flow cytometric analysis using annexin-V and propidium (PI) label, indicated that tenuigenin pretreatment attenuated methylglyoxal -induced apoptotic cell death in primary cultured hippocampal neurons, showing a dose-dependent pattern. Furthermore, 2, 7-dichlorodihydrofluorescein diacetate was used to detect the level of intracellular reactive oxygen species. Tenuigenin decreased the elevated reactive oxygen species induced by methylglyoxal. In addition, tenuigenin inhibited activation of caspase-3 and reversed down-regulation of the ratio of Bcl-2/Bax, both of which were induced by methylglyoxal stimulation. The results suggest that tenuigenin displays antiapoptotic and antioxidative activity in hippocampal neurons due to scavenging of intracellular reactive oxygen species, regulating Bcl-2 family and suppressing caspase-3 activity induced by methylglyoxal, which might explain at least in part the beneficial effects of tenuigenin against degenerative disorders involving diabetic cognitive impairment.
Keywords: Tenuigenin; Methylglyoxal; Hippocampal neurons; Apoptosis; Oxidative stress;

Gossypol inhibits phosphorylation of Bcl-2 in human leukemia HL-60 cells by Li-heng Huang; Jia-qi Hu; Wei-qun Tao; Yuan-hong Li; Guan-ming Li; Pei-yi Xie; Xiao-shan Liu; Jikai Jiang (9-13).
Gossypol is an attractive therapeutic anti-tumor agent as an apoptosis inducer and is being evaluated in preclinical tests. However, the molecular mechanisms underlying apoptosis induction by gossypol in malignant cells have not been completely enunciated. Here we investigate the alterations of Bcl-2/Bcl-xL/Mcl-1 protein levels and Bcl-2 phosphorylation in gossypol-induced apoptosis in human leukemia HL-60 cells. We found that gossypol treatment inhibited cell growth and induced apoptosis in HL-60 cells. Bcl-2/Bcl-xL/Mcl-1 protein levels were slightly reduced and phosphorylation of Bcl-2 at threonine 56 (phospho T56) was not altered. However, phosphorylation of Bcl-2 at serine 70 (phospho S70) was strikingly down-regulated in gossypol-exposed cells. This reduction was found to be not only in both dose- and time-dependent fashion but also obviated by phorbol l2,13-dibutyrate (PDBu), an activator of protein kinase C (PKC). In addition, pre-treatment of PDBu partially prevented gossypol-induced apoptosis in HL-60 cells. Collectively, gossypol treatment can reduce phosphorylation of Bcl-2 at serine 70 in leukemia HL-60 cells and gossypol may be a promising therapeutical candidate for leukemia patients especially expressing phosphorylated Bcl-2 at Ser70.
Keywords: Gossypol; Bcl-2 phosphorylation; Leukemia;

Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway by Hua-Li Xu; Xiao-Feng Yu; Shao-Chun Qu; Rui Zhang; Xiang-Ru Qu; Yan-Ping Chen; Xing-Yuan Ma; Da-Yuan Sui (14-22).
Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities including anti-tumor. Here, for the first time, we studied the molecular mechanism of Juglone-induced apoptosis in human leukemia HL-60 cells. In the present study, HL-60 cells were incubated with Juglone at various concentrations. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry. Expression of Bcl-2 and Bax mRNA was determined by quantitative polymerase chain reaction (qPCR). The results showed that Juglone inhibits the growth of human leukemia HL-60 cells in dose- and time-dependent manner. Topical morphological changes of apoptotic body formation after Juglone treatment were observed by Hoechst 33342 staining. The percentages of Annexin V-FITC-positive/PI negative cells were 7.81%, 35.46%, 49.11% and 66.02% with the concentrations of Juglone (0, 0.5, 1.0 and 1.5 μg/ml). Juglone could induce the mitochondrial membrane potential (△ Ψm) loss, which preceded release of cytochrome c (Cyt c), Smac and apoptosis inducing factor (AIF) to cell cytoplasm. A marked increased of Bax mRNA and protein appeared with Juglone treatment, while an evidently decreased of Bcl-2 mRNA and protein appeared at the same time. These events paralleled with activation of caspase-9, -3 and PARP cleavage. And the apoptosis induced by Juglone was blocked by z-LEHD-fmk, a caspase-9 inhibitor. Those results of our studies demonstrated that Juglone-induced mitochondrial dysfunction in HL-60 cells trigger events responsible for mitochondrial-dependent apoptosis pathways and the elevated ratio of Bax/Bcl-2 was also probably involved in this effect.
Keywords: Juglone; Apoptosis; Mitochondrial membrane potential; Caspase; HL-60;

Promoting human embryonic stem cell (hESC)-derived-neural progenitor survival in the pro-apoptotic niche is pivotal for stem cell replacement therapy. The present study was designed to investigate the protective effect of hepatocyte growth factor (HGF) on hESC-derived neural progenitor injured by hydrogen peroxide (H2O2) exposure. Treatment of hESC-derived neural progenitor cells with HGF prior to H2O2 exposure conferred protective effect against oxidative stress-induced apoptosis. HGF treatment increased both phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation. However, selective inhibition of each pathway supported that the activation of PI3K/AKT, but not ERK1/2, provides survival advantage to the neural progenitor cells. Further investigation indicated that HGF pretreatment could attenuate the decrease of the expression of Bcl-2 protein induced by H2O2, whereas the level of Bax was not affected. Additionally, we observed that H2O2-induced decrease of mitochondrial transmembrane potential, release of cytochrome c and increase of caspase-3 activation were alleviated by HGF pretreatment. These effects of HGF could be reversed by inhibition of the PI3K/Akt and ERKs pathways, indicating PI3K/Akt and ERKs signaling might be involved in HGF-mediated regulation of mitochondrial apoptotic pathway mediated by H2O2. The neuroprotective effect of HGF might potentially be useful in stem cell-based therapies for neurodegenerative disorders.
Keywords: Hepatocyte growth factor; Neural progenitor; Hydrogen peroxide; Apoptosis; PI3K/Akt; Mitochondrial;

Hydroxyurea stimulates the release of ATP from rabbit erythrocytes through an increase in calcium and nitric oxide production by Madushi Raththagala; Welivitya Karunarathne; Matthew Kryziniak; John McCracken; Dana M. Spence (32-38).
Hydroxyurea, a proven therapy for sickle cell disease, is known to improve blood flow and reduce vaso-occlusive crises, although its exact mechanism of action is not clear. The objective of this study was to determine if hydroxyurea results in an increase of ATP release from the red blood cell (RBC) via the drug's ability to stimulate nitric oxide (NO) production in these cells. A system enabling the flow of RBCs through microbore tubing was used to investigate ATP release from the RBC. Incubation of rabbit RBCs (7% hct) with 50 μM hydroxyurea resulted in a significant increase in the release of ATP from these cells. This level of ATP release was not detected in the absence of flow. Studies also showed that increments in hydroxyurea and NO (from spermine NONOate) resulted in an initial increase in ATP release, followed by a decrease in this release at higher concentrations of hydroxyurea and the NO donor. Incubation with L-NAME abolished the effect of the hydroxyurea, suggesting that NO production by the RBC was involved. Indeed, in the presence of 50 μM hydroxyurea, the amount of total Ca2+ measured (by atomic absorption spectroscopy) in a 7% solution of RBCs increased from 363 ± 47 ng/ml and 530 ± 52 ng/ml. Finally, EPR studies suggest that an increase in nitrosylated Hb in the RBC is only measured for those studies involving hydroxyurea and a Ca2+-containing buffer.
Keywords: Nitric oxide; Hydroxyurea; ATP [adenosine triphosphate]; eNOS [endothelial nitric oxide synthase]; Red cell; Sickle cell;

Cytoprotective effects of the volatile anesthetic sevoflurane are highly dependent on timing and duration of sevoflurane conditioning: Findings from a human, in-vitro hypoxia model by Karina Zitta; Patrick Meybohm; Berthold Bein; Henning Ohnesorge; Markus Steinfath; Jens Scholz; Martin Albrecht (39-46).
Using animal models, volatile anesthetics have been recognized for their neuroprotective effects. Nevertheless, there is still disagreement about the optimal duration and timing of conditioning with the volatile anesthetic sevoflurane in the human system. In the study presented, we employed a human neuronal cell culture model to investigate the effects of hypoxia and to evaluate potential cytoprotective properties of different sevoflurane conditioning strategies. Sevoflurane was applied to human IMR-32 cells in which hypoxic conditions were induced for 2 h using our recently described two-enzyme model (Zitta et al., Eur. J. Pharmacol., 2010). Cellular effects of hypoxia and sevoflurane conditioning were evaluated by lactate dehydrogenase (LDH) measurements, brightfield microscopy, ELISAs, cytometric bead arrays, Westernblotting and RT-PCR. Hypoxia increased the release of LDH into the culture medium after 24 h (normoxia: 0.15 ± 0.02 a.u; hypoxia: 0.69 ± 0.08 a.u, P < 0.001) and expression of hypoxia associated genes HIF-1alpha, VEGF, catalase. Cytoprotective effects were observed in cultures that received sevoflurane for 30 min before hypoxia (preconditioning: 0.41 ± 0.07 a.u., P < 0.01) and for 30 min during the hypoxic period (intraconditioning: 0.20 ± 0.01 a.u., P < 0.001). Application of sevoflurane after the hypoxic insult did not lead to cytoprotection (postconditioning: 0.73 ± 0.12 a.u., P > 0.05). Conditioning with sevoflurane for a total of 3 h before, during and after hypoxia, however, resulted in an enhanced release of LDH (periconditioning: 0.97 ± 0.10 a.u., P < 0.01) and additional cell damage. Hypoxia and sevoflurane intraconditioning were associated with changes in erk1/2 phosphorylation (T202/Y204) and HIF-1alpha protein levels, whereas phosphorylation of akt (S473) was not significantly altered. Our results suggest short pre- and intraconditioning with sevoflurane as most potent strategies to reduce hypoxia induced neuronal cell damage.
Keywords: Hypoxia; Pharmacological conditioning; Volatile anesthetic; Sevoflurane; Cell culture;

Cyclophosphamide, an antineoplastic drug effective against a wide variety of cancers is cytotoxic to normal cells also. Ascorbic acid (vitamin C) at higher concentrations possesses cytotoxic effects and it can also enhance the cytotoxicity of 5-fluorouracil in a dose-dependent manner in mouse lymphoma. In the present study, effect of cyclophosphamide treatment alone and in combination with ascorbic acid in vivo on the ultrastructure and some biochemical changes in Dalton's lymphoma tumor cells were investigated. Cyclophosphamide treatment causes disappearance of cell membrane processes, thickening and reduction in the number of mitochondrial cristae as well as the manifestation of rounded shape of mitochondria. The combination treatment with ascorbic acid plus cyclophosphamide caused further changes in tumor cells showing disintegration in the cell surface membrane, disruption in the nuclear membrane and roundish mitochondria with reduction and disruption in the mitochondrial cristae. The observed ascorbic acid plus cyclophosphamide-mediated decrease in reduced glutathione (GSH) in tumor cells may play an important role in the antitumor activity of cyclophosphamide by weakening cellular antioxidant-mediated defense mechanism, thereby increasing tumor cell's susceptibility to cell death. The cyclophosphamide-mediated decrease in lactate dehydrogenase activity in tumor cells and simultaneous increase in ascites supernatant may possibly indicate alteration in the membrane permeability of tumor cells for lactate dehydrogenase as well as tumor cell injury. Further investigation should determine detailed mechanism(s) involved in cyclophosphamide-induced ultrastructural and biochemical changes in tumor cells.
Keywords: Cyclophosphamide; Ascorbic acid; Dalton's lymphoma; Glutathione; Lactate dehydrogenase;

Hepatoma-derived growth factor (HDGF) is correlated with aggressive tumor behaviors such as invasion and angiogenesis. Nevertheless, the potential involvement of HDGF in cell resistance to natural plant phenols-based chemotherapy is still unclear. This study demonstrated that over-expression of HDGF could confer the resistance of human colorectal cancer (CRC) cells to nordihydroguaiaretic acid (NDGA) toxicity. Enforced expression of HDGF could inhibit NDGA-induced apoptosis through the mitochondrial pathway in CRC cells, and attenuate the inhibitory effect of NDGA on tumor growth. Therefore, our results suggest that HDGF represents a potential molecule associated with chemotherapy resistance, which may have major implications in improving chemotherapy of colorectal cancer.
Keywords: Colorectal carcinoma; Chemotherapy resistance; Hepatoma-derived growth factor; Nordihydroguairetic acid; Apoptosis;

In vitro binding characteristics of [3H]AZ11637326, a novel α7-selective neuronal nicotinic receptor agonist radioligand by John C. Gordon; Eifion Phillips; David A. Gurley; J. Richard Heys; Lois Ann Lazor; Herbert G. Barthlow; Mike A. Mallamaci; Rich A. Keith (63-69).
AZ11637326 (5′-(2-fluoro[3,4,5−3H3]phenyl)-spiro[1-azabicyclo [2.2.2]octane-3,2′(3′H)-furo[2,3-b]pyridine) is a potent partial agonist at the human α7 neuronal nicotinic receptor with sub-nanomolar affinity for the human and rat α7 [125I]α-bungarotoxin binding sites. In a search for novel agonist radioligands and imaging ligands for the α7 nicotinic receptor, [3H]AZ11637326 was synthesized and its in vitro membrane binding properties were characterized. [3H]AZ11637326 bound to hα7-HEK membranes with high specificity (> 95%), high affinity (230 pM) and a Bmax of 460 fmol/mg. The rank order of affinity of nicotinic standards determined with [3H]AZ11637326 strongly correlated with those determined using the classical α7 antagonist [125I]α-bungarotoxin, indicating that [3H]AZ11637326 bound to hα7-HEK membranes with an α7 nicotinic-like pharmacology. The Ki values for the standards were on average 2.3-fold lower affinity than determined using the prototypical α7 nicotinic antagonist [125I]α-bungarotoxin. Because [3H]AZ11637326 specific binding is rapid and reversible, the Ki values determined using this ligand are more accurate estimates of affinity than those determined using the kinetically sluggish [125I]α-bungarotoxin. [3H]AZ11637326 also bound to a high affinity (510 pM), nicotine-sensitive site on rat hippocampal membranes with an average Bmax of 55 fmol/mg. With rat hippocampal membranes, the nicotine-sensitive fraction of total binding was sub-optimal for a radioligand (~ 50%), yet the potencies and rank order of the Ki values for standards were consistent with an α7 nicotinic pharmacology. Overall, these studies indicate that [3H]AZ11637326 is a useful new in vitro probe of the α7 nicotinic receptor agonist site and support its potential utility for in vivo receptor occupancy studies.
Keywords: Nicotinic; Radioligand; Alpha 7;

Bioactive compounds found in many plant species have been used in Chinese, Unani, and Indian ayurvedic medicine. Accumulative evidences in both in vitro and in vivo studies using berberine demonstrated anti-cancer and anti-inflammatory properties in different cancer cells. In the present study, a putative compound from commercial sample was purified by chromatographic techniques. The structure of the pure compound was confirmed by spectroscopic studies. The purified berberine was tested against breast cancer (MCF-7) and normal human breast epithelial (MCF-12F) cells for 24, 48 and 72 h at various concentrations. Using MTT assay, berberine exhibited a significant cytotoxic effect on the MCF-7 cells (P  < 0.01) without affecting the breast normal epithelial cell growth at 25 μM concentration. Based on these results, MCF-7 cells were treated with 25 μM berberine for 48 and 72 h for further studies to illustrate induction of apoptosis through cell cycle distribution and DNA fragmentation with agarose gel electrophoresis. Western blotting with treated cells revealed that berberine induces apoptosis in MCF-7 cells through a mitochondria-dependent pathway by increasing levels of cytoplasmic cytochrome c, caspase-9 activity and cleavage of PARP while decreasing levels of Bcl-2. Furthermore, immunoblotting results demonstrated that p53 and p27 were up-regulated suggesting that barberine seems to play a pro-apoptotic role in cancer cells. In conclusion, berberine inhibits the proliferation of MCF-7 breast cancer cells through a mitochondria and caspase dependent apoptotic pathway. It is possible that berberine may serve as a potential naturally occurring compound for breast cancer therapy.
Keywords: Berberine; Identification; MCF-7 cells; Apoptosis; p53;

Involvement of temporomandibular joint P2X3 and P2X2/3 receptors in carrageenan-induced inflammatory hyperalgesia in rats by Juliana Maia Teixeira; Maria Cláudia G. Oliveira; F.H. Nociti; J.T. Clemente-Napimoga; Adriana Pelegrini-da-Silva; Carlos Amílcar Parada; Cláudia Herrera Tambeli (79-85).
The aim of this study was to investigate the role of P2X3, P2X2/3 and P2X7 receptors in the development of TMJ hyperalgesia induced by carrageenan. We also investigated the expression of mRNA of P2X7 receptors in the trigeminal ganglia and the existence of functional P2X7 receptors in the rat's TMJ. The P2X1, P2X3 and P2X2/3 receptor antagonist TNP-ATP, but not the selective P2X7 receptor antagonist A-438079, significantly reduced carrageenan-induced TMJ inflammatory hyperalgesia. The qPCR assay showed that mRNA of P2X7 receptors are expressed in the trigeminal ganglia but this expression is not increased by the inflammation induced by carrageenan in the TMJ region. The P2X7 receptor agonist BzATP induced TMJ inflammatory hyperalgesia that was significantly reduced by pretreatment with dexamethasone. These results indicate that P2X3 and P2X2/3 but not P2X7 receptors are involved in carrageenan-induced TMJ inflammatory hyperalgesia. However, functional P2X7 receptors are expressed in the TMJ region. The activation of these receptors by BzATP sensitizes the primary afferent nociceptors in the TMJ through the previous release of inflammatory mediators. The findings of this study point out P2X3 and P2X2/3 receptors, but not P2X7 receptors, as potential targets for the development of new analgesic drugs to control TMJ inflammatory pain.
Keywords: ATP; P2X3 receptors; P2X2/3 receptors; P2X7 receptors; Inflammatory hyperalgesia; TMJ; Carrageenan;

Linalool blocks excitability in peripheral nerves and voltage-dependent Na+ current in dissociated dorsal root ganglia neurons by José Henrique Leal-Cardoso; Kerly Shamyra da Silva-Alves; Francisco Walber Ferreira-da-Silva; Tiago dos Santos-Nascimento; Humberto Cavalcante Joca; Flávio Henrique Pequeno de Macedo; Pedro Militão de Albuquerque-Neto; Pedro Jorge Caldas Magalhães; Saad Lahlou; Jader Santos Cruz; Roseli Barbosa (86-93).
Linalool is a terpene that occurs as a major constituent of essential oils of many plants of widespread distribution. It possesses several biological and pharmacological activities, including depressant effects on the central nervous system and olfactory receptors. The present study investigated whether linalool affects the excitability of peripheral components of the somatic sensory system. We used sciatic nerve and preparations of intact and dissociated neurons of dorsal root ganglion for extracellular, intracellular and patch-clamp recordings. Linalool concentration-dependently (0.3–2.0 mM) and reversibly blocked the excitability of the sciatic nerve. It inhibited peak-to-peak amplitude of the compound action potential (IC50 was 0.78 ± 0.04 mM). At 0.8 mM, it reversibly increased rheobase and chronaxy (from 3.2 ± 0.1 V and 52.4 ± 4.1 μs to 4.2 ± 0.3 V and 71.2 ± 5.5 μs (n = 5), respectively) and inhibited with greater pharmacological potency the amplitude of the compound action potential components corresponding to axons with slower velocity of conduction. In a similar concentration range (0.1–6 mM), linalool concentration-dependently and reversibly blocked the generation of action potentials of intact dorsal root ganglion neurons without alteration of resting membrane potential and input resistance, and inhibited the voltage-gated Na+ current of dissociated dorsal root ganglion neurons. In conclusion, we demonstrated that linalool acts on the somatic sensory system with local anesthetic properties, since it blocked the action potential by acting on voltage-dependent Na+ channels. This finding is important in showing the potential usefulness of linalool as a pharmacotherapeutic agent.
Keywords: Linalool; Compound action potential; Intracellular recording; Patch-clamp; Sciatic nerve; Dorsal root ganglia;

Effect of metamizol on morphine pharmacokinetics and pharmacodynamics after acute and subchronic administration in arthritic rats by Adriana Miriam Domínguez-Ramírez; Alma Rosa Cortés-Arroyo; Marcela Hurtado y de la Peña; José Raúl Medina López; Francisco Javier López-Muñoz (94-101).
The aim of the present study was to investigate whether metamizol alters the pharmacokinetics of morphine and to determine the relationship between morphine plasma levels and antinociceptive effect produced after co-administration of drugs under acute and subchronic treatments using the pain-induced functional impairment model in rat (PIFIR model). Administration of morphine + metamizol under acute treatment produced a significantly higher antinociceptive effect than that obtained with morphine alone (P <  0.05). This effect remained unaltered after subchronic treatments for 6 and 12 days. In addition, after the simultaneous administration of the drugs in a single dose, a pharmacokinetic interaction was found, which significantly (P <  0.001) increased maximum plasma concentration (C max), concentration at 4 h (C 4h), partial areas under the plasma concentration–time curve from zero to 4 h (AUC0–4) and from zero to 24 h (AUC0–24). Moreover, whereas plasma concentration of morphine markedly decreased up to 4 h (C 4h) after subchronic administration of the opioid, multiple dosing of the morphine + metamizol combination produced an accumulation of the drug in plasma (P <  0.001). The increase observed in morphine plasma levels after co-administration of metamizol may be explained by a possible enzymatic inhibition of the glucuronosyl–transferase system involved in the metabolism of morphine. This study reveals both a pharmacodynamic and a pharmacokinetic interaction between morphine and metamizol, leading to an increased antinociceptive effect and a delay in tolerance development.
Keywords: Morphine; Metamizol; Pharmacokinetics; Pharmacodynamics; Drug interactions;

It is well documented that cannabinoid receptor agonist WIN 55,212-2 had protective effect against cerebral ischemic injury. Our previous study indicated that WIN 55,212-2 pretreatment induced ischemic tolerance to focal cerebral ischemia in a dose-dependent manner. The aim of the present study was to investigate the time–effect relationship of the WIN 55,212-2 pretreatment and explore the role of phosphorylated extracellular signal-regulated kinase 1/2. Rats were pretreated with 1 mg/kg WIN 55,212-2 once a day for 1, 3 and 5 days. Twenty four hours after the end of pretreatment, focal cerebral ischemia was induced by the middle cerebral artery occlusion. Brain ischemic injury was evaluated by neurological function scores and infarction volumes. The effect of U0126, a potent and specific inhibitor of mitogen-activated protein kinase kinase, on WIN 55,212-2 pretreatment was also studied. Moreover, the expression of phosphorylated extracellular signal-regulated kinase 1/2 in the penumbra of ischemic side 4 h after reperfusion was investigated by immunohistochemistry and Western blotting. The results showed that WIN 55,212-2 pretreatment can protect the rat brain against transient focal cerebral ischemia injury, and its protective effect was enhanced gradually with increasing numbers of pretreatment, and was partially reversed by U0126. We further found that WIN 55,212-2 pretreatment up-regulated the levels of phosphorylated extracellular signal-regulated kinase 1/2. These findings suggest that the neuroprotective effect of WIN 55,212-2 pretreatment against focal cerebral ischemia is through the activation of extracellular signal-regulated kinases in rats.
Keywords: WIN 55,212-2; Pretreatment; Ischemia; Brain; Neuroprotection;

Distinct effects of carvone analogues on the isolated nerve of rats by Juan Carlos R. Gonçalves; Aron de Miranda H. Alves; Anna Erika V. de Araújo; Jader Santos Cruz; Demetrius Antônio Machado Araújo (108-112).
Carvone (p-mentha-6,8-dien-2-one) is a monoterpene ketone found as the main active component of various essential oils. It is obtained by distillation and occurs naturally as the enantiomers (+)- and (−)-carvone. Our group have shown that the in vivo antinociceptive activity of (−)-carvone is impaired with decreased nerve excitability. To better characterize the neuropharmacology of such a monoterpene, we investigated the profile of several carvone analogues to establish a structure–function relationship related to the compound action potential (CAP) inhibitory effect. We performed ex vivo assays to evaluate the effects of (+)- and (−)-carvone, carvacrol, (−)-carveol, and limonene on CAP characteristics using a modified single sucrose-gap method. Our results demonstrated that (−)-carvone was less potent (IC50  = 10.7 ± 0.07 mM) in reducing nerve excitability than its enantiomer, (+)-carvone (IC50  = 8.7 ± 0.1 mM), although they shared a similar mode of action, since their effects were partially extinguished by nerve washing and also by reduction of depolarization velocity, probably as a result of voltage-gated sodium channel blockades. In a structure–activity relationship study, we demonstrated that hydroxyl groups in the (−)-carveol and carvacrol molecules enhanced the CAP blocking-effect, while the absence of oxygen moiety in (+)-limonene resulted in the effect being almost abolished. Therefore, inhibition of CAP conduction in peripheral nerves by monoterpenes could expand our understanding concerning the pharmacology of such natural bioactive compounds. Moreover, activation or inhibition of nerve excitability with these tested monoterpenes can be achieved by altering their chemical structures, and this can lead to further implications for target-directed drug design.
Keywords: Carvone; Carveol; Carvacrol; Limonene; Single sucrose-gap; Compound action potential;

CaV2.1 is highly expressed in the nervous system and plays an essential role in the presynaptic modulation of neurotransmitter release machinery. Recently, the antiepileptic drug levetiracetam was reported to inhibit presynaptic CaV2.1 functions, reducing glutamate release in the hippocampus, although the precise physiological role of CaV2.1-regulated synaptic functions in cognitive performance at the system level remains unknown. This study examined whether CaV2.1 mediates hippocampus-dependent spatial short-term memory using the object location and Y-maze tests, and perirhinal cortex-dependent nonspatial short-term memory using the object recognition test, via a combined pharmacological and genetic approach. Heterozygous rolling Nagoya (rol/+) mice carrying the CaV2.1α1 mutation had normal spatial and nonspatial short-term memory. A 100 mg/kg dose of levetiracetam, which is ineffective in wild-type controls, blocked spatial short-term memory in rol/+ mice. At 5 mg/kg, the N-methyl-d-aspartate (NMDA) receptor blocker (+/−)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), which is ineffective in wild-type controls, also blocked the spatial short-term memory in rol/+ mice. Furthermore, a combination of subthreshold doses of levetiracetam (25 mg/kg) and CPP (2.5 mg/kg) triggered a spatial short-term memory deficit in rol/+ mice, but not in wild-type controls. Similar patterns of nonspatial short-term memory were observed in wild-type and rol/+ mice when injected with levetiracetam (0–300 mg/kg). These results indicate that CaV2.1-mediated NMDA receptor signaling is critical in hippocampus-dependent spatial short-term memory and differs in various regions. The combination subthreshold pharmacological and genetic approach presented here is easily performed and can be used to study functional signaling pathways in neuronal circuits.
Keywords: CPP; Levetiracetam; Object location test; Rolling Nagoya mouse strain; Subthreshold dose; Y maze test;

Activation of the Nociceptin/Orphanin FQ (NOP) receptor may have anti-abuse effects. The present study examined the consequence of NOP receptor activation on the rewarding effect of opiates and psychostimulants in the conditioned place preference task in rats. First, the motivational effect of the NOP receptor agonists Ro64-6198 (0.316–3.16 mg/kg i.p.) and Ro65-6570 (1–10 mg/kg i.p.) when administered alone, was assessed. Ro65-6570 was selected for further drug combination studies since, unlike Ro64-6198, it was devoid of an intrinsic motivational effect. Next, the minimal effective dose to induce reward for the opiates heroin (0.1–3.16 mg/kg i.p.), morphine (1–10 mg/kg i.p.), hydrocodone (0.316–10 mg/kg i.p.), tilidine (1–31.6 mg/kg i.p.), hydromorphone (0.1–10 mg/kg i.p.), and oxycodone (0.0316–10 mg/kg i.p.), as well as for the psychostimulants cocaine (3.16–31.6 mg/kg i.p.) and dexamphetamine (0.316–3.16 mg/kg i.p.) in combination with Ro65-6570 (0 or 3.16 mg/kg i.p.) was determined. All drugs produced conditioned place preference, and for opiates and cocaine, but not for dexamphetamine, the minimal effective dose was higher when combined with Ro65-6570 (3.16 mg/kg i.p.). Attenuation of the rewarding effect of tilidine (3.16 mg/kg i.p.) and oxycodone (1 mg/kg i.p.) by Ro65-6570 (3.16 mg/kg i.p.) could be reversed by pre-treatment with the NOP receptor antagonist J-113397 (4.64 mg/kg i.p.), suggesting that the attenuating effect of Ro65-6570 on opiates is due to activation of the NOP receptor. Taken together, the present study suggests that activation of NOP receptors effectively attenuates the rewarding effect of opiates, but may be less effective in reducing psychostimulant-induced reward.
Keywords: Drug dependence and abuse; Nociceptin; Opiate; Behavioural pharmacology; ORL1;

Metabolic regulation of coronary vascular tone: Role of hydrogen peroxide, purinergic components, and angiotensin by Takashi Kaneshiro; Shu-ichi Saitoh; Hirofumi Machii; Osamu Yamaguchi; Toshiyuki Ishibashi; Yukio Maruyama; Yasuchika Takeishi (127-134).
Metabolic regulation plays an important role in modifying coronary vascular tone. We hypothesized that hydrogen peroxide, purinergic components, and angiotensin, produced by cardiac myocytes control coronary vascular tone in proportion to metabolism. We measured changes in the diameter of isolated, pressurized coronary arterioles in response to supernatant from isolated cardiac myocytes in rats (stimulated for 20-, 60-, and 120-min at 400 bpm). Changes in the diameter of arterioles were determined under control conditions following treatment of arterioles with an adenosine receptor antagonist, 8-PSPT, a P2Y1 receptor antagonist, MRS-2179, or an angiotensin II receptor antagonist, olmesartan. A supernatant (500 μl to a 2 ml bath) from myocytes stimulated for 20-, 60- and 120-min caused graded vasodilation (14.1 ± 0.4, 20.2 ± 1.6, 53.8 ± 6.2%, P < 0.01 vs. non-stimulated, respectively). In 20-min stimulation, catalase with myocyte supernatants eliminated vasodilation. Following 60-min stimulation, catalase converted myocyte supernatant-induced vasodilation to a vasoconstriction (− 15.1 ± 1.0%), and this vasoconstriction was eliminated by olmesartan. Upon 120-min stimulation, catalase partially reduced the vasodilation by myocyte supernatants (37.2 ± 3.8%). The remaining vasodilation was converted to a vasoconstriction with 8-PSPT and MRS-2179, and this vasoconstriction was completely eliminated with olmesartan. Cardiac myocytes modulate vascular tone through the net effects of hydrogen peroxide, purinergic components (adenosine and ADP), and angiotensin in proportion to ischemia.
Keywords: Coronary circulation; Metabolic regulation; Hydrogen peroxide; ADP; Adenosine; Angiotensin;

Some cardiovascular therapeutics inhibit paraoxonase 1 (PON1) from human serum by Mehmet Mustafa İşgör; Şükrü Beydemir (135-142).
Harel et al. (2004) report that atherosclerosis is the underlying cause for 50% of the mortality in Western societies, and organophosphates in nature constitute an important risk as well as a terrorist threat for all living things. Since paraoxonase enzyme (PON) is a bioscavenger against both atherosclerosis and organophosphate toxicity, studies on paraoxonase enzyme (PON) occupy an important place in the scientific world. In this study, we purified PON1 enzyme from human serum by using a simple three-step purification method: ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. In addition, we investigated the effects of certain cardiovascular drugs on human serum paraoxonase enzyme activity. IC50 values and K i constants were calculated for digoxin, metoprolol tartrate, verapamil, diltiazem, amiodarone, dobutamine, and methylprednisolone, which show inhibitory effects. IC50 values were determined to be 0.012 μM, 0.621 μM, 0.672 μM, 1.462 μM, 3.255 μM, 4.495 μM and 47.803 μM, respectively, and K i constants were calculated to be 0.035 ± 0.01273 μM, 1.115 ± 0.27003 μM, 1.188 ± 0.11529 μM, 3.104 ± 1.00478 μM, 5.427 ± 1.34063 μM, 10.7 ± 3.14572 μM and 109 ± 17.47875 μM, respectively. A comparison of the IC50 and K i values of the drugs revealed that digoxin has the maximum inhibition rate. Furthermore, methylprednisolone and amiodarone were found to be competitive inhibitors, verapamil and dobutamine were uncompetitive inhibitors, while others inhibited the enzyme in noncompetitive manner.
Keywords: Paraoxonase; Cardiovascular disease; Drugs; Chromatography; Inhibition;

Sulfur dioxide upregulates the aortic nitric oxide pathway in rats by Junling Li; Ruijin Li; Ziqiang Meng (143-150).
Sulfur dioxide (SO2) is a common gaseous pollutant. It is also, however, endogenously generated from sulfur-containing amino acids. Recent studies have demonstrated that rat blood pressure can be lowered by SO2-exposure in vivo and that vasodilation caused by SO2 at low concentrations (< 450 μM) is endothelium-dependent in rat aorta. However, effects of SO2 on nitric oxide synthase (NOS) and nitric oxide (NO) production have not been previously studied in rat aorta. The objective of the present study is to assess the effects of acute (10 min) and prolonged (2 h) stimulation with different concentrations of SO2 on NO/cGMP pathway in isolated rat aorta. The results show that: (1) the acute and prolonged pretreatments with SO2 produced an inhibition of vasoconstrictions induced by norepinephrine. (2) SO2 potentiated activity of endothelial nitric oxide synthase (eNOS), but not of induced NOS (iNOS). (3) SO2 could increase expression of eNOS gene on the transcription and translation levels in rat aorta. (4) SO2 enhanced NO formation in aortic tissue. (5) The level of cGMP in rat aorta was increased by SO2 and no change of cAMP. These findings led to the conclusion: there were acute and prolonged effects of SO2 on the NO/cGMP signalling pathway; and SO2 could upregulate the eNOS–NO–cGMP pathway and at least partly by which the SO2 might cause vasodilation and inhibition to vasoconstriction.
Keywords: Sulfur dioxide; Vasodilation; Nitric oxide synthase; Nitric oxide; cGMP (3′,5′-cyclic guanosine monophosphate); Acute exposure; Prolonged exposure;

The protective effect of 17β-estradiol postconditioning against hypoxia/reoxygenation injury in human gastric epithelial cells by Mei-Jing Liu; Su-Juan Fei; Wei-Li Qiao; Dong-Shu Du; Yong-Mei Zhang; Yu Li; Jian-Fu Zhang (151-157).
The purpose of this study was to investigate the effects and mechanisms of 17β-estradiol pharmacological postconditioning on gastric epithelial cells hypoxia/reoxygenation injury by using an in vitro model of human gastric epithelial cells. The model of hypoxia/reoxygenation was established with human gastric epithelial cell line. The gastric epithelial cell viability was detected by 3-(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays. Gastric epithelial cellular apoptosis was determined by Hoechst 33258 fluorochrome staining and flow cytometric analysis. Contents of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured by Colorimetry analysis. The protein expression of Bcl-2 and Bax in different groups was determined by Western blot analyses and immunocytochemistry assay. 17β-estradiol (10−8, 10−7 and 10−6  mol/l) inhibited hypoxia/reoxygenation injury and 17β-estradiol (10−6  mol/l) obviously attenuated hypoxia/reoxygenation injury 3 h hypoxia followed by 4 h reoxygenation. 17β-estradiol promoted gastric epithelial cell viability and inhibited the gastric epithelial cell apoptosis, and meanwhile, decreased the MDA content and increased SOD activity. The level of Bcl-2 protein was restored to the normal level by 17β-estradiol pharmacological postconditioning. In contrast, the Bax protein level was markedly reduced by 17β-estradiol pharmacological postconditioning. These effects of 17β-estradiol were inhibited by pretreatment with fulvestrant. These data suggested that 17β-estradiol seems involved in regulation of gastric hypoxia/reoxygenation injury and gastroprotection, and its protective effects were strongly related to estrogen receptor.
Keywords: 17β-estradiol; Human gastric epithelial cells; Hypoxia/reoxygenation; Pharmacological postconditioning; Apoptosis; Bcl-2; Bax;

Mechanisms of relaxant activity of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in rat tracheal smooth muscle by Haroldo A. Toque; Fabíola Z.T. Mónica; Rafael P. Morganti; Gilberto De Nucci; Edson Antunes (158-164).
The soluble guanylyl cyclase is expressed in airway smooth muscle, and agents that stimulate this enzyme activity cause airway smooth muscle relaxation and bronchodilation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in airway smooth muscle. Therefore, this study aimed to investigate the mechanisms underlying the relaxations of rat tracheal smooth muscle induced by BAY 41-2272. Tracheal rings were mounted in 10-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed carbachol-precontracted tracheal rings (pEC50  = 6.68 ± 0.14). Prior incubation with the NO synthesis inhibitor l-NAME (100 μM) or the soluble guanylyl cyclase inhibitor ODQ (10 μM) caused significant rightward shifts in the concentration–response curves to BAY 41-2272. Sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and completely inhibited by ODQ. In addition, BAY 41-2272 shifted to the right the tracheal contractile responses to either carbachol (0.01–1 μM) or electrical field stimulation (EFS, 1–32 Hz). BAY 41-2272 (1 μM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2, and such effect was not modified by pretreatment with ODQ. In addition, BAY 41-2272 (up to 1 μM) significantly increased the cGMP levels, and that was abolished by ODQ. Our results indicate that BAY 41-2272 causes cGMP-dependent rat tracheal smooth muscle relaxations in a synergistic fashion with exogenous NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca2+ entry blockade.
Keywords: Soluble guanylyl cyclase; Nitric oxide; Cyclic GMP; Tracheal smooth muscle;

Pro-healing potential of hemin: An inducer of heme oxygenase-1 by Azad A. Ahanger; Shahid Prawez; Marie Dennis Marcus Leo; Kandaswamy Kathirvel; Dinesh Kumar; Surendra K. Tandan; Jitendra K. Malik (165-170).
Hemin induces heme oxygenase (HO), an enzyme which degrades heme in a rate-limiting manner and has an important role in cellular protection against oxidative stress and apoptosis. This HO inducer may be of potential therapeutic value in wound healing and inflammation. To identify the beneficial activity of HO vis a vis wound healing, hemin was used as inducer of HO in rats using a full-thickness cutaneous wound model. Hemin treatment increased cellular proliferation and collagen synthesis as evidenced by increase in wound contraction and hydroxyproline and glucosamine contents. mRNA expression of cytokines endorsed fast healing as was indicated by inhibition of pro-inflammatory cytokines such as ICAM-1 and TNF-α and up-regulation of anti-inflammatory cytokine IL-10.
Keywords: Heme oxygenase; Hemin; Wound healing;

Comparison of the efficacy of tacrolimus and cyclosporine A in a murine model of dinitrofluorobenzene-induced atopic dermatitis by Hirotaka Yamashita; Tomokazu Ito; Hideo Kato; Shusei Asai; Hiroyuki Tanaka; Hiroichi Nagai; Naoki Inagaki (171-176).
Tacrolimus (FK506) and cyclosporine A (Cys A) are immunosuppressive drugs used in the treatment of inflammatory diseases and for preventing rejection of allogeneic transplants. Tacrolimus forms a complex with FK506 binding protein (FKBP), and Cys A forms a complex with cyclophilin. These tacrolimus–FKBP and Cys A–cyclophilin complexes interact with calcineurin (CaN), thereby suppressing activation of T cells. In contrast, steroidal anti-inflammatory drugs suppress the immune system mainly via inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the activating protein-1 (AP-1) pathway. Previously, we reported that tacrolimus, but not dexamethasone, reduced scratching behavior in a murine model of atopic dermatitis. To elucidate the mechanism involved in the inhibition of scratching behavior, we used a mouse model of allergic dermatitis to compare the characteristics of tacrolimus and Cys A treatment. We found that Cys A suppressed scratching behavior induced by application of 2,4-dinitrofluorobenzene, as did tacrolimus. In addition, both drugs attenuated increases in vascular permeability and scratching behavior induced by passive cutaneous anaphylaxis. These results indicate that inhibition of the CaN pathway plays an important role in tacrolimus- and Cys A-induced inhibition of scratching behavior in mice. Furthermore, we observed that CaN inhibitors suppressed mast cell-dependent allergic reaction.
Keywords: Tacrolimus; Cyclosporine A; Calcineurin inhibitor; 2,4-Dinitrofluorobenzene; Scratching behavior;

Suppression of hepatic oxidative events and regulation of eNOS expression in the liver by naringenin in fructose-administered rats by Sriramajayam Kannappan; Nallasamy Palanisamy; Carani Venkatraman Anuradha (177-184).
Previous studies show that naringenin promotes insulin sensitivity in fructose-fed rats. This study investigates whether naringenin prevents oxidative events and apoptotic changes triggered in the rat liver by a high fructose diet. Male Wistar rats of body weight 150–180 g were fed either diet containing starch (60% carbohydrate) or fructose (60% fructose diet). From the 16th day of feeding, rats in each dietary group were divided into two, and treated or not with naringenin (50 mg/kg b.w/day). After 60 days, oxidative and nitrosative damage and endothelial nitric oxide synthase (eNOS) expression and hepatocyte apoptosis were determined. To evaluate whether nitric oxide (NO) plays a role in naringenin action, insulin sensitivity indices, fasting plasma glucose and insulin were assessed in response to co-administration of l-nitro-arginine methyl ester (L-NAME), a NOS inhibitor. Fructose feeding caused oxidative damage to proteins and lipids and resulted in reduced antioxidant status, eNOS expression and nitrite level. Increased formation of 4-hydroxy nonenal (4-HNE), 2, 4-dinitrophenol (2, 4-DNP) and 3-nitrotyrosine (3-NT)-modified proteins and the presence of apoptotic nuclei were observed in the liver. Treatment with naringenin attenuated all these parameters to levels not significantly different from control. Treatment with naringenin improved insulin sensitivity. However, L-NAME plus naringenin administration abolished the insulin-sensitizing effects of naringenin in fructose-fed rats. Reduced oxidative events with simultaneous increase in NO bioavailability may be involved in the insulin-sensitizing and cytoprotective effects of naringenin in fructose-fed rats.
Keywords: Naringenin; Nitroxidative stress; Nitric oxide; Flavonoid; Antioxidant; L-NAME;

Effects of the novel Foxo1 inhibitor AS1708727 on plasma glucose and triglyceride levels in diabetic db/db mice by Hirotsugu Tanaka; Takeyuki Nagashima; Akiyoshi Shimaya; Yasuharu Urano; Teruhiko Shimokawa; Masayuki Shibasaki (185-191).
Recent evidence suggests that the forkhead transcription factor Foxo1 plays an important role in the regulation of glucose and triglyceride metabolism at the gene transcription level for glucose-6 phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and apolipoprotein C-III (apoC-III). Here, we report on the pharmacological effects of the novel Foxo1 inhibitor AS1708727, which we identified by compound screening. Chronic treatment of diabetic db/db mice with AS1708727 for four days significantly reduced blood glucose and triglyceride levels with decrease of gene expression levels of hepatic G6Pase, PEPCK, and apoC-III. No reports have yet examined the influence of Foxo1 inhibitors on these pharmacological effects. In this study, we newly identified a Foxo1 inhibitor compound capable of exerting both an anti-hypertriglyceridemic and anti-hyperglycemic effect. These effects were dependent on maintaining a stable blood concentration of AS1708727 and achieving a high rate of compound transition to the liver. We also investigated the action mechanism of AS1708727 on gluconeogenesis in vitro and in vivo. The compound inhibited gene expression of key gluconeogenic molecules and suppressed gluconeogenesis in Fao hepatocyte cells in vitro. Further, in the pyruvate challenge study using db/db mice in vivo, AS1708727 suppressed increases in blood glucose level by inhibiting gluconeogenic gene expression. These results indicate that the novel Foxo1 inhibitor AS1708727 may exert anti-diabetic and anti-hypertriglyceridemic effects by improving blood glucose and triglyceride metabolism at the gene expression level, and may represent a new class of drugs useful for treating type 2 diabetes mellitus and hypertriglyceridemaia.
Keywords: Foxo1; Type 2 diabetes mellitus; Gluconeogenesis; Hypertriglyceridemia; db/db mouse;

Gatifloxacin-induced histamine release and hyperglycemia in rats by Yasuyoshi Ishiwata; Masato Yasuhara (192-197).
Gatifloxacin, a fluoroquinolone antimicrobial agent, has been reported to cause both hypoglycemia and hyperglycemia in diabetic and non-diabetic patients. The purpose of the present study was to investigate the mechanism of gatifloxacin-induced hyperglycemia in normal and diabetic rats. Rats received a single intravenous injection of gatifloxacin and samples of their arterial blood were collected periodically. Diabetic rats were produced by the injection of streptozotocin and nicotinamide. In normal rats, the concentration of serum glucose decreased after the injection of gatifloxacin at 50 mg/kg, while it increased with gatifloxacin at 100 mg/kg. The concentrations of serum epinephrine and histamine increased after the injection of gatifloxacin at 100 mg/kg. The increases in serum glucose and epinephrine concentrations were reduced by pretreatment with diphenhydramine at 1 mg/kg. In diabetic rats, the concentration of serum glucose actually increased after the injection of gatifloxacin at 50 mg/kg, concomitant with increases in the serum epinephrine and histamine concentrations. The concentration of serum immunoreactive insulin slightly increased after the injection of gatifloxacin at 50 mg/kg. In addition, repeated oral administration of gatifloxacin to rats at 300 mg/kg twice a day for 7 days did not change glucose tolerance. In conclusion, gatifloxacin-induced release of histamine can contribute to an increase in the serum epinephrine concentration and hyperglycemia in normal rats. In diabetic rats, lower doses of gatifloxacin can induce hyperglycemia owing to the low level of insulin secretion that they exhibit compared with normal animals.
Keywords: Gatifloxacin; Hyperglycemia; Epinephrine; Histamine; Insulin; Diabetes mellitus;