European Journal of Pharmacology (v.589, #1-3)

Silybin is a flavonoid with antioxidant and free radical scavenging abilities. Silybin also acts as an iron chelator by binding Fe (III). The present study was undertaken to assess the biological effects of silybin on T leukemia cells in the presence or absence of iron and compare its effects with a well-known iron chelator; desferrioxamine. In these experiments, we studied the growth capacity of Jurkat while varying iron availability in the environment. Desferrioxamine significantly inhibited growth and proliferation of Jurkat cells, blocking treated cells in the G0/G1 phase and inducing apoptosis. In contrast, silybin showed a bimodal effect, inducing cell proliferation at lower concentrations whereas inhibition of DNA synthesis and significant cell death was observed at higher concentrations. Chelation of Fe totally abrogated antiproliferative, cytotoxic and apoptotic effects of desferrioxamine on Jurkat cells. Conversely, the silybin–Fe complex had no appreciable effect on its antiproliferative and cytotoxic activities. The cytotoxic effect of desferrioxamine was also prevented in iron-loaded Jurkat cells; however, the effect of silybin on the growth and viability of iron-loaded cells was similar to the effect of its iron complex on untreated Jurkat cells. Despite the Fe chelating activity of silybin that suggests its possible application in chelation therapy of chronic iron overload, the biological effects of silybin on Jurkat cells are different than those of desferrioxamine, probably due to antioxidant activity of silybin, which causes pro-oxidant effect via iron-catalyzed oxidation with the subsequent generation of reactive oxygen species.
Keywords: Flavonoid; Silybum marianum; Silybin; Desferrioxamine; Iron chelator; Jurkat cell line;

Aspirin induces lytic cytotoxicity in Epstein–Barr virus-positive cells by Su-Fang Liu; Hai Wang; Zi-Jian Li; Xi-Yun Deng; Hui Xiang; Yong-Guang Tao; Wei Li; Min Tang; Ya Cao (8-13).
Epstein–Barr virus (EBV) infection in tumor cells is generally restricted to the latent forms of viral infection. Switching the latent form of viral infection into the lytic form may induce tumor cell death. High levels of nuclear factor (NF)-κB can inhibit EBV lytic replication, and aspirin has the ability to inhibit NF-κB activity. The aims of the current study were to determine the effects of aspirin on inducing EBV lytic infection, and thus to reveal the possibility of targeting EBV-positive cancer cells by aspirin. Our results showed that aspirin depleted NF-κB (p65) in the nucleus and reactivated EBV into lytic replication. Cells exhibited decreased viability in a dose- and time-dependent manner when incubated with aspirin. When ganciclovir was used in combination with aspirin to treat EBV-positive B95.8 cells and Raji cells, the cytotoxic effect of aspirin was amplified. We demonstrated that aspirin reduced the viability of EBV-positive B lymphocytes due to its ability to induce EBV lytic replication.
Keywords: Aspirin; Epstein–Barr virus; Lytic replication; Nuclear factor kappa B;

β-adrenoceptor blockers protect against staurosporine-induced apoptosis in SH-SY5Y neuroblastoma cells by Maya Mikami; Farida Goubaeva; Joseph H. Song; H.T. Lee; Jay Yang (14-21).
The β-adrenoceptor blockers exhibit a well-characterized anti-apoptotic property in the heart and kidney while less is known about the effect of this class of drugs on neuronal apoptosis. We studied the effects of three β-adrenoceptor blockers propranolol (1-(isoproplyamino)-3-(naphthalene-1-yloxy)propan-2-ol), atenolol (2-[4-[2-hydroxy-3-(1-methylethylamino)propoxyl]phenyl]ehanamide), and ICI 118551 (1-[2,3-(dihydro-7-methyl-1H-iden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol), against staurosporine-induced apoptosis in SH-SY5Y human neuroblastoma cells. Staurosporine increased caspase 3-like activity, DNA fragmentation, PARP cleavage, and the number of TUNEL positive cells consistent with the induction of apoptosis. Propranolol and ICI 118551, but not atenolol, demonstrated a concentration-dependent inhibition of caspase 3-like activity. Propranolol and ICI 118551 directly inhibited the enzymatic activity of recombinant caspase 9 while atenolol did not; however, none of the β-adrenoceptor blockers that were examined directly blocked caspases 2 or 3 activity. In isolated mitochondria, propranolol and ICI 118551 inhibited staurosporine-induced cytochrome c release while atenolol did not. We conclude that propranolol and ICI 118551 protect SH-SY5Y cells against staurosporine-induced apoptosis through a dual action on the mitochondria and on caspase 9 in a cell type and an apoptotic paradigm where the conventional inhibitors of mitochondrial permeability transition such as cyclosporin A and bongkrekic acid demonstrate no protection.
Keywords: β-adrenoceptor blocker; Apoptosis; Staurosporine, SH-SY5Y; Caspase;

Effects of phytoestrogen genistein on cytogenetic biomarkers in postmenopausal women: 1 year randomized, placebo-controlled study by Marco Atteritano; Francesco Pernice; Susanna Mazzaferro; Stefania Mantuano; Alessia Frisina; Rosario D'Anna; Maria Letizia Cannata; Alessandra Bitto; Francesco Squadrito; Nicola Frisina; Michele Buemi (22-26).
To evaluate in a twelve-month, randomized placebo-controlled study whether pure administration of phytoestrogen genistein (54 mg/day) might reduce cytogenetic biomarkers in peripheral lymphocytes of postmenopausal women. A total of 57 postmenopausal women met the criteria and were randomly assigned to receive phytoestrogen genistein (n = 30) or placebo (n = 27). There was no significant difference in age, length of time since menopause or body mass index between the two groups. After one year, plasma genistein level was 0.14 ± 0.01 μmol/L in the control group and 0.72 ± 0.08 μmol/L in the genistein group (P < 0.0001). At baseline, sister chromatid exchange rate was 4.97 ± 2.17 in the control group and 4.96 ± 1.83 in the genistein group (P = 0.89). After one year, sister chromatid exchange rate was 4.96 ± 2.16 in the control group and 3.98 ± 1.14 in the genistein group (P < 0.05). High frequency cells count was 3% in the genistein group and 5% in the control group (P < 0.05) at the end of the study. Chromosomal aberration frequency was 5.55% in the control group at time 0 and 5.75% in the genistein group; after one year, the figures were 5.86% in the control group and 4.5% in the genistein group (P < 0.05). After one year, there was a negative relationship between sister chromatid exchange rate and plasma levels (r = − 0.43; P < 0.05) in the genistein group.Phytoestrogen genistein has been shown in postmenopausal women to be effective in the reduction of cytogenetic biomarkers. The protective effect on genoma damage appears to be a particularly promising tool in reducing the risk of cancer.
Keywords: Genistein; Cytogenetic biomarker; Postmenopausal woman; Osteopenic; Genomic damage;

Antitumor effects of Isatin on human neuroblastoma cell line (SH-SY5Y) and the related mechanism by Lin Hou; Chuanxia Ju; Jinyu Zhang; Jinlian Song; Yinlin Ge; Wang Yue (27-31).
The purpose of the study was to investigate the antitumor effects of Isatin and the related mechanism. Human neuroblastoma cells (SH-SY5Y) were exposed to Isatin at different concentrations for 48 h. Apoptotic features were demonstrated by means of nuclei staining with Hoechst 33258 and flow cytometry with Propidium Iodide (PI). Expressions of Bcl-2, Bax and Vascular endothelial growth factor (VEGF) mRNA were analyzed via RT-PCR. Expressions of Bcl-2, Bax proteins and phosphorylated extracellular signal regulated protein kinases (ERKs, p42/p44) were analyzed via Western blot. Activation of caspase-3 was assayed by flow cytometry with anti-active caspase-3-McAb-PE. VEGF protein was determined by ELISA kits. And the results showed that apoptosis of SH-SY5Y cells were induced by Isatin in a dose-dependent manner. Expressions of Bcl-2, VEGF mRNA and Bcl-2, VEGF proteins were down-regulated, while expressions of Bax mRNA and Bax protein were not changed obviously. Expression of phosphorylated ERKs decreased, but the level of activated caspase-3 increased after treatment of Isatin. These results suggest that Isatin promotes the apoptosis of neuroblastoma cells, therefore, it might be a potential candidate for the treatment of neuroblastoma.
Keywords: Isatin; Human neuroblastoma cell; Apoptosis; Bcl-2; Bax; Caspase-3; Vascular endothelial growth factor; Mechanism;

Raloxifene analogue LY117018 suppresses oxidative stress-induced endothelial cell apoptosis through activation of ERK1/2 signaling pathway by Jing Yu; Masato Eto; Koichi Kozaki; Masahiro Akishita; Tetsuro Okabe; Yasuyoshi Ouchi (32-36).
A selective estrogen receptor modulator, raloxifene, has been shown to reduce cardiovascular events in relatively high-risk postmenopausal women with osteoporosis. However, the mechanisms by which raloxifene exerts a pharmacological effect on cardiovascular organs have not been fully elucidated. The present study was designed to examine whether the raloxifene analogue, 6-hydroxy-2-(p-hydroxyphenyl)-benzo(b) thien-3-yl-p-(2-(pyrrolidinyl)ethoxy phenyl ketone (LY117018), could inhibit apoptosis and to clarify the signaling pathway in vascular endothelial cells. LY117018 significantly inhibited hydrogen peroxide-induced apoptosis in bovine carotid artery endothelial cells. The anti-apoptotic effect of LY117018 was abolished by an estrogen receptor antagonist, 7α,7β-(9[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl) estra-1,3,5(10)-triene-3,17-diol (ICI 182,780). Mitogen-activated protein kinases (MAPK), including p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase1/2 (ERK1/2), and Akt, have been shown to act as apoptotic or anti-apoptotic signals. Phosphorylation of p38, JNK, ERK1/2 and Akt was examined. LY117018 increased ERK1/2 phosphorylation but did not enhance the phosphorylation of p38, JNK, or Akt. The anti-apoptotic effect of LY117018 was prevented by treatment with 2-[2′-amino-3′-methoxyphenyl]-oxanaphthalen-4-one (PD98059), an upstream inhibitor of ERK1/2. LY117018 stimulated an increase in ERK1/2 phosphorylation, which was diminished by ICI 182,780. The activation of ERK/1/2 by LY117018 was not inhibited by the transcription inhibitor, actinomycin D. These results suggest that estrogen receptors and the ERK1/2 signaling pathway are involved in the anti-apoptotic action of LY117018 in vascular endothelial cells.
Keywords: Endothelial cell; Apoptosis; Selective estrogen receptor modulator; Raloxifene; ERK1/2;

Regulation of the maturation of osteoblasts and osteoclastogenesis by glutamate by Tzu-Hung Lin; Rong-Sen Yang; Chih-Hsin Tang; Ming-Yueh Wu; Wen-Mei Fu (37-44).
Glutamate, an important central excitatory neurotransmitter, is also secreted by osteoblasts and may be involved in the regulation of bone metabolism. Glutamate receptors for N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) are demonstrated in bone cells. Here we investigated the in vivo effects of glutamate by local injection of AMPA, NMDA, and their antagonists into tibia as well as their in vitro effects on the maturation of osteoblasts and formation of osteoclasts. AMPA receptor antagonist CNQX and NMDA receptor antagonist MK-801 significantly inhibited the maturation and mineralization of osteoblasts in high-glutamate α-MEM. On the other hand, AMPA and NMDA up-regulated the mineralized deposition and osteocalcin mRNA expression of primary osteoblasts cultured in glutamate-free DMEM. AMPA and NMDA induced the phosphorylation of extracellular signal-related kinases (ERK) in osteoblasts within 15 min. In addition, NMDA but not AMPA up-regulated the number of osteoclasts while MK801 antagonized this potentiating effect. To explore the action of glutamate agonists on bone formation in animal model, AMPA was locally injected into tibia and it was found that the bone volume in secondary spongiosa significantly increased and co-treatment of CNQX antagonized the enhancing effect of AMPA. These results suggest that glutamate may play a physiological role in regulating the maturation of osteoblasts and osteoclastogenesis. Activation of both AMPA and NMDA receptors regulates the maturation of osteoblasts. NMDA but not AMPA affects receptor for activation of NF-κB ligand (RANKL)-induced osteoclastogenesis.
Keywords: Glutamate; AMPA; NMDA; Osteoblast; Osteoclastogenesis; ERK;

C6 glioma cells were treated with clinically relevant concentrations of valproic acid (0.5 or 1.0 mM) for 1–7 days and RT-PCR used to examine expression of the melatonin MT1 receptor and selected epigenetic modulators. Valproic acid caused significant time-dependent changes in the mRNA expression of the melatonin MT1 receptor, histone deacetylase (HDAC) 1, 2 and 3, and methyl CpG binding protein 2 (MeCP2). A structurally distinct HDAC inhibitor, trichostatin A, also caused a significant concentration-dependent induction of melatonin MT1 receptor mRNA expression, suggesting involvement of an epigenetic mechanism. The ability of clinical concentrations of valproic acid to significantly alter melatonin MT1 receptor expression, suggests a role for this receptor in the diverse neuropharmacological and oncostatic effects of this agent.
Keywords: Histone acetylation; Epigenetic modulation; Trichostatin A; Combinatorial therapy;

Effects of the mGlu4 receptor allosteric modulator N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) were tested on closely related mGlu6 receptors. Modulation of native calcium currents in isolated sympathetic neurons from rat superior cervical ganglion by expressed mGlu4 and mGlu6 receptor was used to assay receptor activity. Glutamate concentration–response curves with and without PHCCC confirmed that the drug is an allosteric modulator of mGlu4 receptor, without direct agonist activity. Conversely, PHCCC directly activates the mGlu6 receptor and does not enhance activity of glutamate. Therefore, PHCCC is a direct mGlu6 receptor agonist, but lacks allosteric modulatory properties.
Keywords: PHCCC; Sympathetic neurons; Calcium channel;

Microglia are the prime effector cells involved in immune and inflammatory responses in the central nervous system (CNS). In pathological conditions, microglia are activated to restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various toxic proinflammatory molecules. Thus, negative regulators of microglial activation have been identified as potential therapeutic candidates in many neurological diseases. A number of selenium-containing compounds show antioxidant activity. In this study, we investigated 2-amino-1,3-selenazole derivatives with regard to anti-inflammatory activity or inhibitory effects on microglial activation. Among 26 derivatives of 2-amino-1,3-selenazole and bis-(2-amino-5-selenazoyl) ketones, we observed that 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1) and 5-chloroacetyl-2-morpholino-1,3-selenazole (CS2) strongly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) release from BV2 microglial cells. In rat primary cultured microglia, CS1 and CS2 significantly reduced LPS-induced production of NO, tumor necrosis factor (TNF)-α, and prostaglandin E2. Real-time reverse transcription-polymerase chain reaction analysis revealed that the pretreatment of primary microglial cells with CS1 and CS2 attenuated LPS-induced mRNA expression for inducible NO synthase, TNF-α, interleukin-1β, and cyclooxygenase-2. In addition, CS1 and CS2 suppressed LPS-induced activation of nuclear factor-κB and Akt. These results suggest that CS1 and CS2 may provide neuroprotection by suppressing the proinflammatory pathway in activated microglia.
Keywords: Akt; 2-amino-1,3-selenazole; Brain inflammation; Microglia; NF-κB; Nitric oxide; TNF-α;

Lamotrigine prevents MK801-induced alterations in early growth response factor-1 mRNA levels and immunoreactivity in the rat brain by Sang-Ha Park; Young Ho Seo; Bo-Hyun Moon; Song-hyen Choi; Seungwoo Kang; Kuem-Ju Lee; Sang-Hyun Choi; Min-Soo Lee; Boe-Gwun Chun; Kyung-Ho Shin (58-65).
MK801 (dizocilpine) induces selective neurotoxic effects in the retrosplenial cortex, ranging from neuronal vacuolization to irreversible neurodegeneration depending on the dose administered. Although lamotrigine prevents MK801-induced neuronal vacuolization in the retrosplenial cortex 4 h after injection, it is not clear whether lamotrigine attenuates the subsequent neurodegeneration that occurs 3–4 days later. Because early growth response factor-1 (egr-1) plays a key role in neurodegeneration and its expression is induced in the retrosplenial cortex following MK801 treatment, it is possible that lamotrigine may attenuate MK801-induced neurodegeneration via inhibition of egr-1 expression in the retrosplenial cortex. To address this issue, we treated rats with lamotrigine (10 or 20 mg/kg) followed by MK801 (2 mg/kg) and measured changes in the levels of egr-1 mRNA and immunoreactivity in the retrosplenial cortex and other brain regions 3 h later. We also evaluated the effects of these treatments on neurodegeneration 4 days following treatment using Fluoro-Jade B staining. MK801 treatment increased egr-1 mRNA and immunoreactivity in the restrosplenial, cingulate, entorhinal and piriform cortices, but decreased levels in hippocampal subfields. These MK801-induced changes in egr-1 expression were significantly inhibited by lamotrigine pretreatment. In addition, MK801-induced neurodegeneration in the retrosplenial cortex was partially blocked by lamotrigine pretreatment in a dose dependent manner. These results demonstrate that lamotrigine pretreatment prevents the MK801-induced upregulation of egr-1 expression in a region-selective manner, and suggest that this effect may contribute, in part, to the attenuation of MK801-induced neurodegeneration in the retrosplenial cortex.
Keywords: Early growth response factor-1; MK801; Lamotrigine; Retrosplenial cortex;

Blockade of sensory abnormalities and kinin B1 receptor expression by N-Acetyl-l-Cysteine and ramipril in a rat model of insulin resistance by Mahmoud Ali Ismael; Sébastien Talbot; Cynthia L. Carbonneau; Christian M. Beauséjour; Réjean Couture (66-72).
Glucose-fed rat is a model of insulin resistance that displays sensory polyneuropathy and hypertension. This study aimed at comparing the beneficial effects of N-Acetyl-l-Cysteine (NAC, antioxidant) and ramipril (angiotensin-1 converting enzyme inhibitor) on tactile and cold allodynia induced by chronic glucose feeding. Impact of these treatments was also assessed on hypertension, plasma glucose and insulin concentrations, insulin resistance and kinin B1 receptor expression.Male Wistar rats (50–75 g) were given 10% d-glucose in their drinking water for 11 weeks or tap water (controls). Glucose-fed rats were treated either with NAC (1 g/kg/day, gavage), ramipril (1 mg/kg/day in drinking water) or no drug during the last 5 weeks. Glucose feeding for 6 weeks induced a significant increase in systolic blood pressure and hyperglycaemia which was accompanied by tactile and cold allodynia. At 11 weeks, plasma insulin, insulin resistance (HOMA index), kinin B1 receptor mRNA in spinal cord and renal cortex and B1 receptor binding sites in spinal cord were enhanced in glucose-fed rats. NAC and ramipril caused a progressive to complete inhibition of tactile and cold allodynia from 6 to 11 weeks. High systolic blood pressure, hyperinsulinemia, insulin resistance and kinin B1 receptor expression were also normalized or attenuated in glucose-fed rats by either treatment. Results suggest that chronic treatment with an antioxidant or an ACE inhibitor provides similar beneficial effects on sensory polyneuropathy, hypertension and insulin resistance in glucose-fed rats. Both therapies were associated with a reduction of the expression of the pro-nociceptive kinin B1 receptor.
Keywords: ACE inhibitor; Allodynia; Diabetes; Hypertension; Insulin resistance; N-Acetyl-l-Cysteine; Oxidative stress; Pain neuropathy;

Deficiency in dopaminergic activity has been linked to a depressed state in pharmacological and clinical studies. Current pharmacological treatment for depression primarily involves modulation of serotonergic and noradrenergic systems but not dopaminergic neurotransmission. Available pharmacotherapy for depression has a number of drawbacks as a significant number of people are either refractory or develop tolerance to the antidepressant agents resulting in relapse. Furthermore, the slow onset of action of current therapies often poses a challenge for effective treatment. In our effort to develop novel molecules impacting all three above mentioned monoamine systems, we discovered structurally unique pyran derivatives with various profiles in inhibiting monoamine transporters. One of our lead molecules, D-161 exhibited triple monoamine transporter inhibitory activity with the highest affinity for norepinephrine transporter (NET) followed by its affinity for serotonin transporter (SERT) and dopamine transporter (DAT). D-161 exhibited potent activity in reducing immobility significantly in the rat forced swim test as well as in the mouse tail suspension test. Moreover, results from locomotor activity tests indicated that the reduction of immobility by D-161 was not due to motor activation as no significant motor activation was observed when the rats were subjected to the same doses of drug under the same conditions as in the forced swim test. These results suggest that the novel asymmetric pyran derivative D-161 with unique molecular structure exhibiting triple monoamine transporter inhibitory activity could possess potent antidepressant activity.
Keywords: Antidepressant; Dopamine; Serotonin; Norepinephrine; Monoamine transporter;

In vivo modulation of dopaminergic nigrostriatal pathways by cytisine derivatives: Implications for Parkinson's Disease by J. Andrés Abin-Carriquiry; Gustavo Costa; Jessika Urbanavicius; Bruce K. Cassels; Marco Rebolledo-Fuentes; Susan Wonnacott; Federico Dajas (80-84).
Nicotinic acetylcholine receptor agonists are considered potential pharmacological agents for Parkinson's Disease treatment, due to their ability to improve experimental Parkinson symptomatology, reduce 3,4-dihydroxy-l-phenylalanine-induced dyskinesias and stop the neurodegenerative process at an experimental level. In the present work, the ability of the nicotinic agonist cytisine and two halogenated derivatives (3-bromocytisine and 5-bromocytisine) to induce striatal dopamine release was characterized in vivo by microdialysis. Cytisine, 5-bromocytisine and nicotine were much more efficacious than 3-bromocytisine in eliciting dopamine release in response to their local application through the microdialysis probe. Moreover, the agonists were intermittently administered before and after an intranigral injection of 6-hydroxydopamine (6-OHDA), and striatal dopamine tissue levels were assessed 8 days after the lesion. Both cytisine and its 5-bromo derivative (but not the 3-bromo derivative) significantly prevented the decrease of striatal dopamine tissue levels induced by 6-OHDA. These results suggest that the efficacy of nicotinic agonists to stimulate dopamine release in vivo through presynaptic nicotinic receptors could be related to their potential to induce striatal protection.
Keywords: Cytisine; Dopamine; Neuroprotection; Parkinson's Disease; Nicotinic acetylcholine receptor; 6-Hydroxydopamine;

The study of ischemic tolerance is critical in the development of strategies for the treatment of ischemic stroke. We used the oxygen and glucose deprivation (OGD) paradigm in cultured cortical neurons as an in vitro approach to elucidate the mechanism of protection conferred by glutamate preconditioning. Pretreatment of neurons with N-methyl-d-aspartate (NMDA) receptor antagonists prevented OGD-induced cell death whereas α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor and voltage-dependent Ca++ channel (VDCC) blockers were without effect. Neurons preconditioned with glutamate exhibited resistant to damage induced by OGD. The ischemic tolerance depended on the duration of preconditioning exposure and the interval between preconditioning exposure and test challenge. Protective efficacy was blocked by the NMDA or AMPA receptor antagonists but not by the VDCC blocker. Furthermore, neuroprotective effect was not seen if extracellular Ca++ was omitted or removed with EGTA. Pretreatment with staurosporin and 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93) but not 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY294002) or 1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio] butadiene (U0126) significantly reduced ischemic tolerance. Preconditioning increased phosphorylated levels of cAMP responsive element binding protein (CREB) and pretreatment with CRE-decoy oligonucleotide completely blocked preconditioning-induced increase in cell viability. Importantly, glutamate preconditioning increased Bcl-2 expression that was blocked by KN93, staurosporin and CRE-decoy oligonucleotide. These results suggest that preconditioning with glutamate conferred neuroprotection against subsequent OGD by inducing p-CREB-mediated Bcl-2 expression.
Keywords: Preconditioning; Neuronal death; Apoptosis; Ischemic tolerance; cAMP responsive element binding protein; CRE-decoy oligonucleotide;

Amphetamine causes dopamine depletion and cell death in the mouse olfactory bulb by Fidelis E. Atianjoh; Bruce Ladenheim; Irina N. Krasnova; Jean Lud Cadet (94-97).
Amphetamine is a neurotoxic psychostimulant that causes dopamine depletion and neuronal death in the rodent striatum. In the present study, we sought to determine if toxic doses of the drug can also induce pathological changes in the mouse olfactory bulb. We found that injections of amphetamine (10 mg/kg × 4, given 2 h apart) caused significant decreases in dopamine levels in that structure. This dose of the drug also induced substantial increases in the number of terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate (dNTP) nick end labeling (TUNEL)-positive cells in the olfactory bulb indicative of elevated DNA fragmentation. These results show that the toxic effects of amphetamine involve the olfactory bulb in addition to the striatum. These observations need to be taken into consideration when discussing the clinical course of amphetamine addiction.
Keywords: Neurotoxicity; Striatum; TUNEL staining; DNA fragmentation;

Minodronic acid, a third-generation bisphosphonate, antagonizes purinergic P2X2/3 receptor function and exerts an analgesic effect in pain models by Shuichiro Kakimoto; Yukinori Nagakura; Seiji Tamura; Tomonari Watabiki; Kumiko Shibasaki; Shohei Tanaka; Masamichi Mori; Masao Sasamata; Masamichi Okada (98-101).
The P2X2/3 receptor has an important role in the nociceptive transmission. Minodronic acid is a third third-generation bisphosphonate and a potent inhibitor of bone resorption. We found that minodronic acid inhibited α,β-methylene ATP-induced cation uptake with the potency higher than that of suramin in the P2X2/3 receptor receptor-expressing cells. Other bisphosphonates did not show such activity. Subcutaneously administered (10–50 mg/kg) minodronic acid significantly inhibited the α,β-methylene ATP-, acetic acid- and formalin-induced nociceptive behaviors in mice. These unique effects of minodronic acid would be beneficial for the treatment of accelerated bone turnover diseases accompanied by bone pain, including bone metastases.
Keywords: Bisphosphonate; Minodronic acid; P2X2/3 receptor; Analgesia; Pain;

To explore the therapeutic potential of imidazoline I1 receptor ligands in motor dysfunction related to the basal ganglia, rigidity was induced in mice by intraperitoneal administration of reserpine. The imidazoline I1 receptor agonists moxonidine and tizanidine reduced rigidity in a dose-dependent manner. Although rigidity was reduced by efaroxan (an imidazoline I1 receptor and α2-adrenoceptor antagonist) and idazoxan (an imidazoline I1 and I2 receptor and α2-adrenoceptor antagonist), SKF86466 and yohimbine, both of which are α2-adrenoceptor antagonists with no affinity for imidazoline receptors, also suppressed rigidity, suggesting that activation rather than blockade of imidazoline I1 receptors contributes to reduction of reserpine-induced muscle rigidity.
Keywords: Parkinson's disease; Rigidity; Electromyogram; Imidazoline receptor; Moxonidine; Tizanidine;

Gamma-butyrolactone (GBL) elevates striatal and prefrontal cortex dopamine levels; only the striatal dopamine levels are elevated by increased dopamine synthesis. If increased dopamine synthesis is necessary in order for dopamine levels to be affected by tyrosine availability, then GBL-induced prefrontal cortex dopamine levels should be tyrosine insensitive. Rats received either vehicle, tyrosine (50 or 200 mg/kg i.p.) or a tyrosine-depleting mixture prior to GBL 750 mg/kg i.p.. GBL-induced dopamine levels in prefrontal cortex were lowered by tyrosine depletion. GBL-induced striatal dopamine levels were not affected. Hence, increased dopamine synthesis may not be necessary in order for tyrosine availability to affect pharmacologically elevated prefrontal cortex dopamine levels.
Keywords: dopamine; gamma-butyrolactone; prefrontal cortex; striatum; tyrosine; tyrosine hydroxylase;

The aim of the present study was to investigate the effects of chronic administration of morphine on the expression of an endogenous opioid peptide in the spinal dorsal horn. Bovine adrenal medulla 22-like immunoreactivity (BAM22-IR) was found in the superficial layers of the spinal cord. Intrathecal (i.t.) administration of morphine (20 μg) for 6 days, but not 2 days, significantly reduced the expression of BAM22-IR whereas i.t. administration of saline for 2 and 6 days did not alter the expression of BAM22-IR. The present study suggests that reduction of BAM22-IR in the spinal cord is involved in the development of morphine tolerance.
Keywords: Intrathecal; Spinal dorsal horn; Chronic morphine; BAM22 (Bovine adrenal medulla 22);

Lacosamide, the new anticonvulsant, effectively reduces harmaline-induced tremors in rats by Thomas Stöhr; Delphine Lekieffre; Joachim Freitag (114-116).
The present study aimed at evaluating the efficacy of lacosamide (0.3 to 30 mg/kg), a new anticonvulsant drug, in a model of essential tremor in comparison to the reference compounds propranolol and primidone. We observed a high tremorlytic effect of lacosamide reducing the intensity of tremors following harmaline administration in a dose-dependent manner. The highest dose also modified the latency and intensity of tremor at onset. The effect of lacosamide was equal or even superior to propranolol and primidone indicating that lacosamide may be a new antitremorgenic drug that merits further clinical investigation.
Keywords: Essential tremor; Harmaline; Lacosamide; Antitremorgenic drug;

Δ9-tetrahydrocannabinol prolongs the immobility time in the mouse forced swim test: Involvement of cannabinoid CB1 receptor and serotonergic system by Nobuaki Egashira; Tomomi Matsuda; Emi Koushi; Fuminori Higashihara; Kenichi Mishima; Shozo Chidori; Nobuyoshi Hasebe; Katsunori Iwasaki; Ryoji Nishimura; Ryozo Oishi; Michihiro Fujiwara (117-121).
In the present study, we investigated the effect of Δ9-tetrahydrocannabinol (THC), the principal psychoactive component of marijuana, on immobility time during the forced swim test. THC (2 and 6 mg/kg, i.p.) significantly prolonged the immobility time. In addition, THC at the same doses did not significantly affect locomotor activity in the open-field test. The selective cannabinoid CB1 receptor antagonist rimonabant (3 mg/kg, i.p.) significantly reduced the enhancement of immobility by THC (6 mg/kg). Similarly, the selective serotonin (5-HT) reuptake inhibitor (SSRI) citalopram (10 mg/kg, i.p.) and 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.3 mg/kg, i.p.) significantly reduced this THC-induced effect. Moreover, the selective 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide dihydrochloride (WAY100635, 1 mg/kg, i.p.) and the postsynaptic 5-HT1A receptor antagonist MM-77 (0.1 mg/kg, i.p.) reversed this reduction effect of 8-OH-DPAT (0.3 mg/kg). In contrast, the selective 5-HT7 receptor antagonist (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulfonyl]phenol hydrochloride (SB269970) had no effect on this reduction effect of 8-OH-DPAT. WAY100635 (1 mg/kg) also reversed the reduction effect of citalopram (10 mg/kg). These findings suggest that the 5-HT1A receptors are involved in THC-induced enhancement of immobility.
Keywords: Cannabinoid; Δ9-tetrahydrocannabinol; Forced swim test; Cannabinoid CB1 receptor; Serotonin (5-hydroxytryptamine, 5-HT); 5-HT1A receptor1;

Cannabinoid CB1 receptor antagonist rimonabant attenuates reinstatement of ketamine conditioned place preference in rats by Fangqiong Li; Qin Fang; Yu Liu; Mei Zhao; Dapeng Li; Jishi Wang; Lin Lu (122-126).
Recent evidence suggests that cannabinoid CB1 receptors may represent effective targets for therapeutic agents used to treat cocaine and heroin relapse. However, the role of cannabinoid CB1 receptors in the potential treatment for other drugs of abuse is still largely unknown. The present study was conducted to determine whether cannabinoid CB1 receptors play a similar role in relapse to ketamine abuse. To establish a ketamine reinstatement model in the conditioned place preference paradigm, rats were trained to develop place preference conditioned by ketamine, which was subsequently extinguished through daily exposure to the test chambers in the absence of ketamine. On the day following the last extinction session, four groups of rats were injected with ketamine (1, 5, 10 and 15 mg/kg, i.p.) to reinstate previously extinguished conditioned place preference. To investigate the effects of rimonabant, a cannabinoid CB1 receptor antagonist, on reinstatement of ketamine-induced place preference, different doses of rimonabant (0.1, 0.5 and 3 mg/kg, i.p) were injected 30 min prior to the ketamine (5 and 15 mg/kg, i.p.) priming injection in a separate group of rats. To determine whether rimonabant itself produces conditioned place preference or conditioned place aversion, rats were trained for conditioned place preference or place aversion with rimonabant (0, 0.1, 0.5, 3.0 mg/kg, i.p.). While ketamine priming injections reinstated extinguished place preference, rimonabant administration significantly attenuated the reinstatement of ketamine-induced place preference in a dose-dependent manner. Importantly, rimonabant itself did not produce conditioned place preference or place aversion. Since the reinstatement effects of ketamine administration were inhibited by rimonabant, these findings suggest that a cannabinoid CB1 receptor antagonist may be useful in preventing relapse to ketamine abuse.
Keywords: Cannabinoid CB1 receptor antagonist; Conditioned place preference; Ketamine; Rimonabant; Relapse; Reinstatement;

Inhibition of α4 integrin mediated adhesion was involved in the reduction of B16-F10 melanoma cells lung colonization in C57BL/6 mice treated with Gambogic acid by Jie Zhao; Qi Qi; Yong Yang; Hong-Yan Gu; Na Lu; Wei Liu; Wei Wang; Lei Qiang; Ling-Bo Zhang; Qi-Dong You; Qing-Long Guo (127-131).
Gambogic acid, the major active ingredient of gamboge, has been shown to exhibit anti-cancer activity both in vivo and in vitro. However, its potential activity in tumor metastasis remains unclear. In the present study, we found that Gambogic acid strongly inhibited the adhesion of highly metastatic mouse melanoma B16-F10 cells in vitro. Gambogic acid also exhibited significant anti-metastasis activity on the development of in vivo artificial metastases (i.e. following tail vein i.v. injection of the B16-F10 melanoma tumor cells in C57BL/6 mice). Flow cytometric analysis and Western blot showed that Gambogic acid inhibited the cell surface expression of α4 integrin, while exhibited negligible effects on the expression of α5 and β1 integrin. Thus we concluded for the first time that Gambogic acid could inhibit the adhesion and migration of B16-F10 cells in vitro and in vivo, in which down-regulation of α4 integrin expression was involved.
Keywords: Gambogic acid; Integrin; Adhesion; Metastasis; B16-F10 cell;

Memantine prevents the cognitive impairment induced by 3,4-methylenedioxymethamphetamine in rats by Jorge Camarasa; José M. Marimón; Teresa Rodrigo; Elena Escubedo; David Pubill (132-139).
Amphetamine abuse is an important risk factor for the development of cognitive impairment involving learning and memory. Since in previous studies we have demonstrated the effectiveness of alpha-7 nicotinic receptor antagonists in preventing the neurotoxicity induced by amphetamine derivatives, the present paper seeks to determine whether pre-treatment with memantine (MEM) (an antagonist of both nicotinic and NMDA receptors) counteracts the memory impairment induced by 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) administration in male Long Evans rats. In mice, MDMA and MEM induced a locomotor stimulant response but with a different profile. Moreover, MEM inhibited the rearing and thygmotaxis behaviour induced by MDMA. Non-spatial memory was tested in the object recognition test and the spatial learning and memory was tested in the Morris water maze. In our experimental conditions, rats receiving MEM pre-treatment recovered the ability to discriminate between the familiar and the novel object that had been abolished by MDMA treatment. Animals treated with MDMA showed impaired learning in the Morris water maze. Results of the probe trial demonstrated that MDMA-treated rats did not remember the location of the platform, but this memory impairment was also prevented by the MEM pre-treatment. Moreover, MEM alone improved the learning task. No differences were observed between the different groups as regards swim speed. In conclusion, MEM significantly improved the learning and memory impairment induced by MDMA and constitutes the first approach to the treatment of the long-term cognitive deficits found in ecstasy users.
Keywords: Memantine; MDMA; Ecstasy; Learning and memory; Object recognition; Morris water maze;

Small-conductance calcium-activated potassium channels (KCa2) generating the medium afterhyperpolarization seen after an action potential modulate the neuronal integration signal. The effects of two KCa2 channel blockers, apamin, specific to KCa2.2 and KCa2.3 channels, and lei-Dab7, which binds to KCa2.2 channels only, were compared to evaluate the involvement of KCa2 channel subunits in behavior, spatial learning and memory in rats. Intracerebroventricular (9–5 ng) injections of lei-dab7 decreased locomotor activity, food intake and body weight in rats deprived of food. A dose of 3 ng lei-Dab7 had no effect on these types of behavior. We therefore used this dose for attention and memory tasks. No modification to attention or memory was observed in a spatial radial-arm maze task with rats given 3 ng lei-Dab7, whereas apamin (0.3 ng) improved reference memory and accelerated changes of strategy from egocentric to allocentric. These findings suggest that KCa2.3 blockade improves memory in rats. Lei-Dab7 entirely outcompeted the binding of iodinated apamin to 64 brain structures (mean IC50: 34.5 nM), although IC50 values were highly variable. By contrast, overall IC50 values for apamin were close to mean values (11.3 pM). The very low affinity of the hippocampus and neocortex for lei-Dab7 may account for the absence of a behavioral effect of this compound. The variability of IC50 values suggests that KCa2 channel composition varies considerably as a function of the brain structure considered.
Keywords: Potassium channel; Hippocampus; Afterhyperpolarization; Spatial learning; Memory; Behavior; Calcium-activated potassium channel; Toxin; SK channel; Competition; Rodents;

The main clinically used antidepressant drugs are selective monoamine reuptake inhibitors, including selective serotonin reuptake inhibitors (citalopram, sertraline), selective dopamine reuptake inhibitor (nomifensine) and selective noradrenaline reuptake inhibitor (reboxetine), but they have various side effects. Because cannabinoid CB1 receptor antagonists (SR141716A, AM251) enhance monoamine release, they might be beneficial in the therapy of affective disorders. We hypothesized that the use of monoamine reuptake inhibitors in combination with cannabinoid CB1 receptor antagonists would allow a lower dose of monoamine reuptake inhibitors to be used in the therapy of depression, thereby reducing or eliminating the side effects. To test this hypothesis, we examined the combination of SR141716A or AM251 with citalopram, sertraline, nomifensine or reboxetine at subthreshold doses to see whether these combinations would show an additive effect in the forced swimming test and the tail suspension test with mice. Subthreshold doses of cannabinoid CB1 receptor antagonist and selective serotonin reuptake inhibitors, which separately had no effect on the immobility of mice in the tests, showed a clear effect when the drugs were administered at 40 and 30 min, respectively, before the tests, without any change of motor activity. Therefore, the use of subthreshold doses of these agents in combination might be useful to enhance mainly serotonergic neurotransmission, and to reduce or eliminate the side effects of citalopram and sertraline.
Keywords: Cannabinoid CB1 receptor antagonist; Selective serotonin reuptake inhibitor; Forced swimming test; Tail suspension test; Subthreshold dose;

5-HT2C receptor activation is a common mechanism on proerectile effects of apomorphine, oxytocin and melanotan-II in rats by Yasuharu Kimura; Yuki Naitou; Fumikazu Wanibuchi; Tokio Yamaguchi (157-162).
5-Hydroxytryptamine (5-HT), dopamine, oxytocin and melanocortin pathways are known to be involved in the induction of penile erections in rats. Although a dopamine–oxytocin–5-HT link in the central nervous system has been suggested to be important to the control of penile erections, the 5-HT receptor subtype that mediates dopamine–oxytocin–5-HT action and the relationship between the dopamine–oxytocin–5-HT pathway and melanocortin pathway have not been fully elucidated. In this study, in order to clarify these matters, we examined the effects of a selective 5-HT2B/5-HT2C receptors antagonist, 1-(1-methylindol-5-yl)-3-(3-pyridyl)urea (SB200646) and a selective 5-HT2C receptor antagonist, 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy) pyridin-3-yl carbamoyl] indoline (SB242084) on penile erections induced by a dopamine receptor agonist, 10, 11-dihydroxyaporphine (apomorphine), oxytocin, or a melanocortin receptor agonist, melanotan-II (MT-II) in rats. SB200646 at 10 mg/kg and SB242084 at 3 mg/kg, these doses which completely antagonize penile erections induced by 5-HT2C receptor agonists, m-chlorophenylpiperazine (mCPP) and (S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine (YM348), significantly inhibited penile erections elicited by apomorphine, oxytocin or MT-II. In addition, in order to clarify further the suggestion that the 5-HT pathway projecting from medulla oblongata to lumbosacral spinal site and lumbosacral 5-HT2C receptor are involved in the induction of penile erection, we also examined the proerectile effect of YM348 in spinal and a 5-HT depletor, p-chlorophenyl alanine (pCPA)-treated rats. YM348 induced intracavernous pressure increase in spinal and pCPA-treated rats as well as normal rats. These results suggest that 5-HT2C receptor in lumbosacral spinal sites mediates not only dopamine–oxytocin–5-HT action but melanocortin action on penile erections, and that the 5-HT pathway is located downstream from melanocortin pathway as well as the dopamine–oxytocin pathway.
Keywords: 5-HT2C receptor; Penile erection; Intracavernous pressure; Apomorphine (10, 11-dihydroxyaporphine); Oxytocin; MT-II (melanotan-II);

On the mechanism of antidepressant-like action of berberine chloride by Shrinivas K. Kulkarni; Ashish Dhir (163-172).
Berberine, an alkaloid isolated from Berberis aristata Linn. has been used in the Indian system of medicines as a stomachic, bitter tonic, antiamoebic and also in the treatment of oriental sores. Evidences have demonstrated that berberine possesses central nervous system activities, particularly the ability to inhibit monoamine oxidase-A, an enzyme involved in the degradation of norepinephrine and serotonin (5-HT). With this background, the present study was carried out to elucidate the antidepressant-like effect of berberine chloride in different behavioural paradigms of despair. Berberine (5, 10, 20 mg/kg, i.p.) inhibited the immobility period in mice in both forced swim and tail-suspension test, however, the effect was not dose-dependent. Berberine (5 and 10 mg/kg, i.p.) also reversed the reserpine-induced behavioral despair. Berberine (5 mg/kg, i.p.) enhanced the anti-immobility effect of subeffective doses of various typical but not atypical antidepressant drugs in forced swim test. Berberine (5 mg/kg, i.p.) following its acute administration in mice resulted in increased levels of norepinephrine (31%), serotonin (47%) and dopamine (31%) in the whole brain. Chronic administration of berberine (5 mg/kg, i.p.) for 15 days significantly increased the levels of norepinephrine (29%), serotonin (19%) as well as dopamine (52%) but at higher dose (10 mg/kg, i.p.), there was no change in the norepinephrine (12%) levels but a significant increase in the serotonin (53%) and dopamine (31%) levels was found. The antidepressant-like effect of berberine (5 mg/kg, i.p.) in forced swim test was prevented by pretreatment with l-arginine (750 mg/kg, i.p.) or sildenafil (5 mg/kg, i.p.). On the contrary, pretreatment of mice with 7-nitroindazole (7-NI) (25 mg/kg, i.p.) or methylene blue (10 mg/kg, i.p.) potentiated the effect of berberine (2 mg/kg, i.p.) in the forced swim test. Pretreatment of mice with (+)-pentazocine (2.5 mg/kg, i.p.), a high-affinity sigma1 receptor agonist, produced synergism with subeffective dose of berberine (2 mg/kg, i.p.). Pretreatment with various sigma receptor antagonists viz. progesterone (10 mg/kg, s.c.), rimcazole (5 mg/kg, i.p.) and N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047; 1 mg/kg, i.p.) reversed the anti-immobility effects of berberine (5 mg/kg, i.p.). Berberine at lower dose did not affect the locomotor activity and barbiturate-induced sleep time. It produced mild hypothermic action in rats and displayed analgesic effect in mice. Taken together, theses findings demonstrate that berberine exerted antidepressant-like effect in various behavioural paradigms of despair possibly by modulating brain biogenic amines (norepinephrine, serotonin and dopamine). Further, nitric oxide pathway and/or sigma receptors are involved in mediating its antidepressant-like activity in mouse forced swim test.
Keywords: Berberine chloride; Antidepressant; Nitric oxide; Sigma receptors; Monoamines;

Anxiety has been implicated in a number of psychiatric disorders, such as generalized anxiety disorder, depression, panic attacks, phobias, obsessive–compulsive disorders and posttraumatic stress disorders. In this study, we investigated the impact of high anxiety on the levels of intracellular reactive oxygen species in lymphocytes, granulocytes and monocytes from the peripheral blood of mice by using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe. The behavioural light/dark choice test was used to distinguish highly anxious from less anxious mice. Our results showed that a high anxiety level significantly increased the generation of reactive oxygen species in the peripheral blood lymphocytes, granulocytes and monocytes.
Keywords: Reactive oxygen species; Lymphocyte; Monocyte; Granulocyte; Light/dark test; Anxiety;

Rosiglitazone attenuates the cognitive deficits induced by high fat diet feeding in rats by Asif R. Pathan; Anil Bhanudas Gaikwad; Bhoomi Viswanad; Poduri Ramarao (176-179).
The present study was designed to test the hypothesis that insulin resistance plays a role in high fat diet feeding induced cognitive deficits. Rats consuming the high fat diet exhibited characteristic features of insulin resistance viz. mild hyperglycemia, hypertriglyceridemia, hypercholesterolemia, and hyperinsulinemia. Further, these rats showed a severe deficit in learning and memory. In contrast, rosiglitazone at the dose of 5 mg/kg, p.o. for 7 days prior to biochemical and behavioral testing significantly lowered the plasma glucose, triglycerides, cholesterol, and insulin levels. These animals also performed better on Morris water maze task, suggesting improved spatial memory. Our data demonstrate that the insulin sensitizers can overcome the cognitive deficits arising from high fat diet feeding, which may be in part mediated through the development of peripheral insulin resistance.
Keywords: High fat diet; Learning and memory; Insulin resistance; Rosiglitazone;

The effect of sildenafil on the altered thoracic aorta smooth muscle responses in rat pre-eclampsia model by Nergiz Hacer Turgut; Tijen Kaya Temiz; Ihsan Bagcivan; Bulent Turgut; Sefa Gulturk; Baris Karadas (180-187).
The pathophysiology of pre-eclampsia is still unknown thus effective primary prevention is not possible at the stage. The present study was conducted to research the smooth muscle responses in the pre-eclampsia model with suramin treated rats and the effect of phosphodiesterase-5 (PDE5) inhibitor on these responses.Rats of three groups; control, suramin and suramin + sildenafil were given intraperitoneal injections of saline, suramin or sildenafil citrate. Suramin injections caused increased blood pressure, protein in urine and caused fetal growth retardation. The use of sildenafil citrate straightened significantly both blood pressure and average fetus weight, but did not reach to control values. At the end of pregnancy, thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction responses, sodium nitroprusside and papaverine relaxation responses were similar in three groups. Contraction responses of phenylephrine, increased significantly in suramin group. Relaxation responses of acethylcholine and bradykinin decreased in suramin group. The use of sildenafil citrate partially straightened both relaxation and contraction responses, but did not reach to control values. In all groups in the presence of l-nitromonomethylarginine (l-NAME), 1H-(1, 2, 4) oxadiazole (4, 3-a) guinoxalin-1-one (ODQ) and indomethacin decreased the relaxation responses of acetylcholine and bradykinin. The cyclic guanosine monophosphate (cGMP) content of thoracic aorta tissue was determined by radioimmunoassay technique. The content of cGMP in suramin group decreased and use of sildenafil citrate increased the cGMP content but did not reach to control values. We conclude that in pre-eclampsia, the increase of contraction responses, the decrease of relaxation responses and the decrease of cGMP content can depend on insufficiency about synthesis or release of relaxant factors which was released from the vessel endothelium. The results in this study show that in pre-eclampsia; PDE5 inhibitors enhance endothelial function and may be used for protection. Further studies are needed to clear the efficiency and safety of PDE5 inhibitors.
Keywords: Pre-eclampsia; Endothelial dysfunction; Phosphodiesterase-5 inhibitor; (Rat);

Angiotensin II is known to potentiate vasoconstriction induced by electrical field stimulation (EFS), but the underlying mechanisms for this potentiation are not fully understood. This study was designed to investigate the role of superoxide anion in the potentiation effects of angiotensin II. Contraction of rat mesenteric arterial segments was induced by perivascular nerve stimulation with EFS, and superoxide production was measured with lucigenin-enhanced chemiluminescence. Extracellular signal-regulated kinase (ERK) phosphorylation was determined in cultured smooth muscle cells with Western blot. Angiotensin II concentration dependently potentiated the contraction of rat mesenteric arteries to EFS, which is frequency-dependent. This potentiation was blunted by an angiotensin AT1 receptor antagonist (2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylic acid, CV-11974), NAD(P)H oxidase inhibitor (apocynin), superoxide dismutase (SOD) and its mimetic tiron, but not affected by angiotensin AT2 receptor antagonist and inhibitors of xanthine oxidase, cytochrome P450, and cyclooxygenase. Angiotensin II increased superoxide production by mesenteric arteries, which was blunted by angiotensin AT1 receptor antagonist CV-11974, and NAD(P)H oxidase inhibitor apocynin. Superoxide generating compound pyrogallol mimicked the effects of angiotensin II. Tyrosine kinase inhibitor (tyrphostin A25) and mitogen-activated protein kinase (MAPK)/ERK inhibitors (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene (U 0126)) inhibited angiotensin II- and pyrogallol-induced potentiation of EFS-induced contraction, while inactive forms of these inhibitors did not show any inhibitory effects. In cultured smooth muscle cells from mesenteric arteries, angiotensin II and superoxide similarly induced ERK phosphorylation. These results showed that superoxide mediated angiotensin II-induced potentiation of contractile response to EFS and tyrosine kinase-MAPK/ERK activation was involved.
Keywords: Angiotensin II; Contraction; Electrical field stimulation; MAPK/ ERK phosphorylation; Mesenteric artery; NAD(P)H oxidase; Reactive oxygen species; Superoxide;

Comparison of pharmacodynamics between carvedilol and metoprolol in rats with isoproterenol-induced cardiac hypertrophy: Effects of carvedilol enantiomers by Kazuhiko Hanada; Kazuhiko Asari; Masako Saito; Jun-ichi Kawana; Mitsuo Mita; Hiroyasu Ogata (194-200).
A recent clinical study has shown that carvedilol has a significantly more favorable effect than metoprolol on survival rate in patients with heart failure. This may be due to actions of carvedilol such as β2-adrenoceptor blockade, α-adrenergic receptor blockade and other properties such as anti-oxidant effects that are not yet fully understood. We compared the effects of racemic carvedilol, metoprolol and carvedilol enantiomers on cardiac hypertrophy at similar heart rate in rats with isoproterenol-induced cardiac hypertrophy. Continuous administration of isoproterenol for 2 weeks produced heart failure, which is characterized by an increased heart rate, cardiac hypertrophy and downregulation of β-adrenoceptors. The doses of racemic carvedilol and metoprolol were adjusted to obtain a similar heart rate in rats with isoproterenol-induced cardiac hypertrophy. The reduction of left ventricular weight and improvement of cAMP production induced by carvedilol were superior to those induced by metoprolol. Although heart rate, blood pressure and cAMP production were not affected by R-carvedilol, left ventricular weight was significantly reduced as a result of α-adrenoceptor blockade. The improvement of cAMP production by S-carvedilol was significantly higher than that induced by coadministration of R-carvedilol and metoprolol, suggesting that β2-adrenoceptor blockade partly contributed to the improvement of signal transduction in rats with isoproterenol-induced cardiac hypertrophy. This study has demonstrated that the effects of carvedilol on cAMP production and cardiac hypertrophy in rats with isoproterenol-induced cardiac hypertrophy are superior to those induced by metoprolol at a similar heart rate.
Keywords: Carvedilol enantiomer; Isoproterenol-induced cardiac hypertrophy; Adenylate cyclase activity; Metoprolol;

We investigated the pharmacological profile of the vasoconstrictive response to clonidine in the isolated rabbit ear vein, and compared the characteristics of clonidine with those of noradrenaline and moxonidine. The maximal vasoconstrictive responses to clonidine and moxonidine in the rabbit ear vein were 35.94 ±11.18% and 88.78 ± 11.54% of the maximum response to noradrenaline, respectively. Prazosin 0.1 μM inhibited the vasoconstriction induced by lower concentrations of noradrenaline, and the concentration-dependent response curve for noradrenaline was significantly shifted to the right by 1 μM prazosin. Yohimbine (0.1 and 0.5 μM) only decreased the vasoconstrictive response to lower concentrations of noradrenaline, but did not affect the response to higher concentrations. Vasoconstrictive responses to lower but not higher concentrations of clonidine and moxonidine were inhibited by 0.1 μM yohimbine. In contrast, the same concentration of yohimbine significantly potentiated the maximal response to a high concentration of clonidine by 24.06%. In isolated rabbit ear vein pretreated with 0.1 μM yohimbine, prazosin competitively inhibited the concentration–response curve for clonidine with a pA2 value of 8.05 ± 0.06. We conclude that clonidine acts mainly on α2-adrenoceptors to produce vasoconstriction in the rabbit ear vein; however, in the preparation pretreated with yohimbine, the clonidine-induced vasoconstriction is mediated via α1-adrenoceptors and its maximal vasoconstriction is significantly potentiated.
Keywords: Clonidine; Yohimbine; α-Adrenoceptor; Vasoconstriction; Ear vein; (Rabbit);

Hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats by Stephen F. Rodrigues; Maria A. de Oliveira; Rosangela A. dos Santos; Antonio G. Soares; Rita de Cássia Tostes; Maria Helena C. Carvalho; Zuleica B. Fortes (206-214).
In addition to reducing blood pressure, hydralazine can reduce the production of inflammatory cytokines and reduce the expression of leukocyte adhesion molecules. Differences in leukocyte behavior and leukocyte adhesion molecule expression in spontaneously hypertensive rats (SHR) compared to normotensive rats have been reported. However, whether hydralazine can reduce leukocyte migration in vivo in hypertension and in normotension remains unknown. To address this question, male SHR and Wistar rats were treated for 15 days with hydralazine at a dose of ~ 3.5 mg/kg or ~ 14 mg/kg in their drinking water. The numbers of rollers and adherent and migrated cells were determined by direct vital microscopy, and blood pressure was assessed by tail plethysmography. In addition, following treatment with the higher dose, immunohistochemistry was used to measure the expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cells, while flow cytometry was used to evaluate the expression of leukocyte CD18 and L-selectin. Hydralazine reduced leukocyte adherence and migration in SHR either at the higher, that reduced blood pressure levels, or lower dose, which did not reduce it. Reduced ICAM-1 expression might be involved in the reduced migration observed in SHR. In Wistar rats, only at the higher dose hydralazine reduced blood pressure levels and leukocyte migration. Reduced P-selectin expression might be involved. We therefore conclude that hydralazine reduces leukocyte migration by different mechanisms in SHR and Wistar rats, specifically by reducing ICAM-1 expression in the former and P-selectin expression in the latter.
Keywords: SHR; Hydralazine; Leukocyte migration; ICAM-1; P-selectin; (Wistar rat);

Different effect of acute treatment with rosiglitazone on rat myocardial ischemia/reperfusion injury by administration method by Masahiro Abe; Yoshiharu Takiguchi; Satoshi Ichimaru; Shinichiro Kaji; Koichiro Tsuchiya; Koichiro Wada (215-219).
The present study was undertaken to examine the effect of rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-γ agonist, using different administration methods, on rat myocardial infarct size induced by 30 min of ischemia followed by 4 h of reperfusion. The infarct size was significantly reduced by the continuous infusion of rosiglitazone (0.5 mg/kg/h) from 30 min before occlusion for 2 h. On the other hand, limitation of the infarct size was shown by a bolus injection of 0.75 mg/kg at 5 min before reperfusion, but not by a bolus injection of 1 mg at 30 min before occlusion. The protective effect of rosiglitazone by the bolus injection before occlusion was obtained when an antioxidant, N-acetylcysteine, was concomitantly administered. The cardioprotection by rosiglitazone was associated with the inhibition of increased myeloperoxidase activity, tumor necrosis factor-α content and phosphorylation of inhibitor κB in the myocardium. The present study demonstrated that the protective effect of rosiglitazone on myocardial ischemia/reperfusion injury occurred most likely by inhibition of the nuclear factor-κB pathway through PPAR-γ activation. However, acute treatment with rosiglitazone is harmful if its concentration is high during ischemia.
Keywords: Rosiglitazone; Myocardial ischemia/reperfusion injury; Peroxisome proliferator-activated receptor-γ; Continuous infusion; Bolus injection;

Characterization of angiotensin II antagonism displayed by Ib, a novel nonpeptide angiotensin AT1 receptor antagonist by Jinhui Wu; Qiujuan Wang; Jiyuan Guo; Zheyi Hu; Zhiyong Yin; Jinyi Xu; Xiaoming Wu (220-224).
The pharmacologic profile of Ib, 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a novel nonpeptide angiotensin AT1 receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in rats. Ib inhibited [125I] angiotensin II binding to AT1 receptors in rat liver membranes (K i  = 2.5 ± 0.5 nM) and did not interact with AT2 receptors in bovine cerebellar membranes. In functional studies with rat and rabbit aorta, Ib inhibited the contractile response to angiotensin II (pD 2′ value: 7.43 and 7.29, respectively) with a significant reduction in the maximum. In pithed rats, Ib inhibited the angiotensin II induced pressor response in a dose-related manner. After intravenous administration, Ib produced a dose-dependent antihypertensive effects in spontaneously hypertensive rats and renal hypertensive rats. These results suggest that Ib is a potent angiotensin AT1 selective receptor antagonist with a mode of insurmountable antagonism.
Keywords: Ib; Angiotensin II; AT1 receptor; Insurmountable antagonism; Hypertension;

Lack of the serotonin transporter does not prevent mineralocorticoid hypertension in rat and mouse by Wei Ni; Huawei Zhou; Jessica Diaz; Dennis L. Murphy; Joseph R. Haywood; Stephanie W. Watts (225-227).
We hypothesized that lack of a functional serotonin transporter (SERT) would increase basal blood pressure and enhance the development of deoxycorticosterone acetate (DOCA)-salt hypertension compared to wild type (WT) controls. Mean arterial blood pressure was measured in WT and SERT knockout (KO) mice and rat models through radiotelemetry. Basal blood pressures were not different between respective WT and KO. Moreover, blood pressure elevated similarly (∼ 50 mm Hg) in all strains given DOCA and salt. Thus, the lack of functional SERT did not prevent development of DOCA-salt induced hypertension or modify basal blood pressure significantly.
Keywords: 5-HT; Serotonin transporter; Systemic hypertension;

The effects of interleukin-6 on the contraction and relaxation responses of the cavernous smooth muscle from rats by Soon Chul Myung; June Hyun Han; Kee Keun Song; Gun Hyun Kang; Shin Young Lee; Tae Hyoung Kim; Moo Yeol Lee; Hyun Woo Kim; Sae-Chul Kim (228-232).
The purpose of this study is to elucidate the effect of IL-6 on the vasomotor reactivity of the corpus cavernosum of the rats. The strips were either left untreated or treated with 1 ng/ml of IL-6 for 60 min. By increasing concentrations of phenylephrine, acetylcholine, or sodium nitroprusside, we assessed concentration–contraction or relaxation responses. The IL-6-treated strips were incubated for 30 min with or without l-NAME (NW-nitro-l-arginine methyl ester), l-arginine, indomethacin, BQ-123 (an endothelin receptor A inhibitor), or SQ 29,548 (a thromboxane A2 [TXA2] receptor blocker), and the effects on phenylephrine-induced contraction or acetylcholine-induced relaxation of phenylephrine-induced contraction were measured. The contractile responses to phenylephrine were significantly enhanced in the IL-6-treated strips, compared with the IL-6-nontreated strips, and the relaxation responses to acetylcholine were significantly inhibited in the IL-6-treated group compared with the IL-6-nontreated group. But after endothelial denudation, there was no difference between the IL-6-treated strips and the IL-6-nontreated strips on the contraction–relaxation responses to phenylephrine or acetylcholine. The relaxation responses to sodium nitroprusside were not inhibited in both groups. l-NAME completely inhibited the relaxation response to acetylcholine in the IL-6-treated strips, as well as the IL-6-nontreated strips. Indomethacin and SQ 29,548 significantly inhibited the increased contractile responses to phenylephrine in the IL-6-treated strips. But BQ 123 rarely affected the same responses. l-arginine reversed the inhibited relaxation responses to acetylcholine in the IL-6-treated strips. Therefore, IL-6 inhibits endothelium-dependent, NO-mediated relaxation and also enhances α1-adrenergic receptor-mediated contraction via an endothelium-dependent TXA2-mediated mechanism in the corpus cavernosum of the rat.
Keywords: Interleukin-6; Nitric oxide; Thromboxane A2;

Protective effect of quercetin and α-tocopherol on experimental reflux oesophagitis in rats was investigated. Rats received quercetin, (100 mg/kg), α-tocopherol (16 mg/kg), omeprazole (30 mg/kg) given at 1 h prior to surgery. Quercetin and α-tocopherol significantly inhibited the oesophagitis index to 1.33 ± 0.12 (P  < 0.001) and 1.83 ± 0.14 (P  < 0.001) respectively, as compare to control group 3.5 ± 0.21. Further, acid and pepsin out put of gastric contents were significantly decreased in treated groups. Indeed, quercetin significantly inhibited the lipid peroxidation (from 0.69 ± 0.05 to 0.43 ± 0.04 nmol of malonyldialdehyde (MDA)/mg protein) (P  < 0.001) and increased in levels of catalase to 29.5 ± 2.7 units of catalase activity/mg protein and superoxide dismutase (SOD) to 92.4 ± 10.5 units/mg protein (P  < 0.001). The α-tocopherol and omperazole showed significant inhibition in lipid peroxidation (0.34 ± 0.02 and 0.38 ± 0.01) (P  < 0.01) and enhanced the activities of catalase (34.3 ± 3.6 and 31.5 ± 3.4) (P  < 0.01) and SOD (87.3 ± 9.2 and 76.60 ± 6.9) activity. Quercetin and α-tocopherol treated group significantly increased the glutathione level to 36.5 ± 2.78 (P  < 0.01) and 32.1 ± 2.34 (P  < 0.05) respectively. However, it altered the elevated levels of sialic acid and hexose contents in oesophageal tissue. Indeed, quercetin significantly decreased the elevated plasma histamine content (P  < 0.05). Quercetin and α-tocopherol significantly attenuated the elevated level of collagen in oesophageal tissue as of the omeprazole. The results suggest that antioxidants could attenuate the severity of reflux oesophagitis and prevent the oesophageal mucosal damage and validate its therapeutic use in gastroesophageal reflux disease.
Keywords: Quercetin; α-tocopherol; Antioxidant; Oesophagitis; Gastroesophageal reflux disease;

Protective effects of antithrombin on puromycin aminonucleoside nephrosis in rats by Junji Yamashita; Kenji Nakajima; Yoichi Ohno; Yoshiaki Kaneshiro; Takato Matsuo; Hitoshi Tanaka; Kenji Kaneko (239-244).
We investigated the effects of antithrombin, a plasma inhibitor of coagulation factors, in rats with puromycin aminonucleoside-induced nephrosis, which is an experimental model of human nephrotic syndrome. Antithrombin (50 or 500IU/kg/i.v.) was administered to rats once a day for 10 days immediately after the injection of puromycin aminonucleoside (50mg/kg/i.v.). Treatment with antithrombin attenuated the puromycin aminonucleoside-induced hematological abnormalities. Puromycin aminonucleoside-induced renal dysfunction and hyperlipidemia were also suppressed. Histopathological examination revealed severe renal damage such as proteinaceous casts in tubuli and tubular expansion in the kidney of control rats, while an improvement of the damage was seen in antithrombin-treated rats. In addition, antithrombin treatment markedly suppressed puromycin aminonucleoside-induced apoptosis of renal tubular epithelial cells. Furthermore, puromycin aminonucleoside-induced increases in renal cytokine content were also decreased. These findings suggest that thrombin plays an important role in the pathogenesis of puromycin aminonucleoside-induced nephrotic syndrome. Treatment with antithrombin may be clinically effective in patients with nephrotic syndrome.
Keywords: Antithrombin; Nephrotic syndrome; Puromycin aminonucleoside;

Ablation of primary afferent neurons by neonatal capsaicin treatment reduces the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury in cirrhotic rats by Paula R.S. Camara; Gerson J.N. Ferraz; Carla F. Franco-Penteado; Lourenco Sbragia-Neto; Luciana R. Meirelles; Simone A. Teixeira; Marcelo N. Muscara; Licio A. Velloso; Edson Antunes; Jose G.P. Ferraz (245-250).
Primary sensory afferent neurons modulate the hyperdynamic circulation in cirrhotic rats with portal hypertension. The stomach of cirrhotic rats is prone to damage induced by ethanol, a phenomenon associated with reduced gastric hyperemic response to acid-back diffusion. The aim of this study was to examine the impact of ablation of capsaicin-sensitive neurons and the tachykinin NK1 receptor antagonist A5330 on the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury and its effects on gastric cyclooxygenase (COX) and nitric oxide synthase (NOS) mRNA expression. Capsaicin was administered to neonatal, male, Wistar rats and the animals were allowed to grow. Cirrhosis was then induced by bile duct ligation in adult rats while controls had sham operation. Ethanol-induced gastric damage was assessed using ex vivo gastric chamber experiments. Gastric blood flow was measured as well as COX/NOS mRNA expression. Topical application of ethanol produced significant gastric damage in cirrhotic rats compared to controls, which was reversed in capsaicin- and A5330-treated animals. Mean arterial and portal pressure was normalized in capsaicin-treated cirrhotic rats. Capsaicin and A5330 administration restored gastric blood flow responses to topical application of ethanol followed by acid in cirrhotic rats. Differential COX and NOS mRNA expression was noted in bile duct ligated rats relative to controls. Capsaicin treatment significantly modified gastric eNOS/iNOS/COX-2 mRNA expression in cirrhotic rats. Capsaicin-sensitive neurons modulate the susceptibility of the portal hypertensive gastric mucosa to injury induced by ethanol via tachykinin NK1 receptors and signalling of prostaglandin and NO production/release.
Keywords: Cirrhosis; Portal hypertensive gastropathy; Portal hypertension; Capsaicin; Tachykinin;

Involvement of soluble guanylate cyclase α1 and α2, and SKCa channels in NANC relaxation of mouse distal colon by Ingeborg Dhaese; Gwen Vanneste; Patrick Sips; Emmanuel Buys; Peter Brouckaert; Romain A. Lefebvre (251-259).
In distal colon, both nitric oxide (NO) and ATP are involved in non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission. The role of the soluble guanylate cyclase (sGC) isoforms α1β1 and α2β1, and of the small conductance Ca2+-dependent K+ channels (SKCa channels) in the relaxation of distal colon by exogenous NO and by NANC nerve stimulation was investigated, comparing wild type (WT) and sGCα1 knockout (KO) mice. In WT strips, the relaxation induced by electrical field stimulation (EFS) at 1 Hz but not at 2–8 Hz was significantly reduced by the NO-synthase inhibitor l-NAME or the sGC inhibitor ODQ. In sGCα1 KO strips, the EFS-induced relaxation at 1 Hz was significantly reduced and no longer influenced by l-NAME or ODQ. The SKCa channel blocker apamin alone had no inhibitory effect on EFS-induced relaxation, but combined with ODQ or l-NAME, apamin inhibited the relaxation induced by EFS at 2–8 Hz in WT strips and at 8 Hz in sGCα1 KO strips. Relaxation by exogenous NO was significantly attenuated in sGCα1 KO strips, but could still be reduced further by ODQ. Basal cGMP levels were lower in sGCα1 KO strips but NO still significantly increased cGMP levels versus basal. In conclusion, in the absence of sGCα1β1, exogenous NO is able to partially act through sGCα2β1. NO, acting via sGCα1β1, is the principal neurotransmitter in EFS-evoked responses at 1 Hz. At higher stimulation frequencies, NO, acting at sGCα1β1 and/or sGCα2β1, functions together with another transmitter, probably ATP acting via SKCa channels, with some degree of redundancy.
Keywords: Soluble guanylate cyclase; Nitric oxide; SKCa channels (Ca2+-dependent K+ channels); Distal colon; sGCα1 knockout (soluble guanylate cyclase α1 knockout);

Validation of a conscious rat model for the discovery of novel agents that inhibit gastric acid secretion by Colin A. Campbell; Pamela J. Gaskin; Jill Darton; Peter Chiu; Kevin Lee; Peter G. McLean (260-263).
Identification of novel drug molecules requires the extensive evaluation in vitro and in vivo. Following in vitro evaluation it is necessary to efficiently screen numerous novel molecules in vivo using relatively simple methodology that requires small numbers of animals, is rapid to perform, and provides results that can definitely discriminate potential candidates for further investigation. Herein, we describe the results of three standard compounds (omeprazole, a proton pump inhibitor; cimetidine, an histamine H2 receptor antagonist; and AR-H047108, a potassium competitive acid blocker) in the rat aspiration model (under both basal and stimulated conditions), and compared the effects with those in the pyloric ligation model with a view to comparing the results in terms of sensitivity, robustness and simplicity of the methodology.In the aspiration model, drug or vehicle was administered orally 1 h prior to administration of pentagastrin or dimaprit. Ten minutes later 0.9% NaCl was administered orally and immediately recovered by aspiration. In the pyloric ligation model, drugs or vehicle were administered orally 2 h before ligation in a volume of 10 ml/kg. For each model, the volume of each sample was measured and the acidity was determined. In the aspiration model under basal acid secretion or following stimulation with pentagastrin omeprazole, cimetidine and AR-H047108 produced dose related inhibition of acidity. Omeprazole and cimetidine inhibited acid secretion following stimulation with dimaprit. In the pyloric ligation model omeprazole, cimetidine and AR-H047108 inhibited acid secretion.The profile of each of 3 inhibitors of acid secretion exhibited similar effects irrespective of the degree of stimulation (dimaprit, pentagastrin or pyloric ligation). Thus, based on these robust effects and ease of methodology we would recommend the use of the rat aspiration model with pentagastrin stimulation of gastric acid secretion as the primary in vivo methodology to screen novel inhibitors of acid secretion.
Keywords: Gastric acid secretion; Pyloric ligation; Pentagastrin; Aspiration; (Rat);

Spironolactone exhibits direct renoprotective effects and inhibits renal renin–angiotensin–aldosterone system in diabetic rats by Masateru Taira; Hiroe Toba; Masafumi Murakami; Ikumi Iga; Ryoko Serizawa; Shoko Murata; Miyuki Kobara; Tetsuo Nakata (264-271).
Aldosterone itself has been reported to participate in mediating renal injury, and it was confirmed that the aldosterone synthase CYP11B2 gene, protein, and aldosterone production are locally present in the kidney. To test the hypothesis that a mineralocorticoid receptor antagonist might ameliorate diabetic nephropathy and the inhibition of renal CYP11B2 expression might be associated with these renoprotective effects, spironolactone (50 mg/kg/day) was administered by gavage to uninephrectomized diabetic rats for 3 weeks. Streptozotocin (55 mg/kg, i.v.) significantly increased urinary protein excretion and collagen deposition in glomerular and tubulointerstitial areas in the kidney, which were attenuated by spironolactone treatment. RT-PCR and Western blot analysis revealed that the expression of mRNA for collagen I/IV, transforming growth factor-β, NADPH oxidase and mineralocorticoid receptor and the mineralocorticoid receptor protein in the kidney was enhanced in the uninephrectomized diabetic rat kidney and that the overexpression of these molecules was suppressed by spironolactone. Renal angiotensin converting enzyme was activated and overexpressed in diabetic rats, and spironolactone inhibited these changes. We demonstrated that spironolactone prevented the streptozotocin-induced increase in the renal CYP11B2 mRNA content. Controlling blood glucose level with insulin also attenuated the renal expression of mRNA for CYP11B2. On the other hand, the treatment of spironolactone in the present study did not affect blood glucose level or blood pressure in uninephrectomized streptozotocin-induced diabetic rats. These results suggest that spironolactone exerted renoprotective effects in uninephrectomized streptozotocin-induced diabetic rats and inhibited local renin–angiotensin–aldosterone system, such as the ACE expression and the hyperglycemia-induced overexpression of CYP11B2, in the kidney.
Keywords: Aldosterone; Diabetic nephropathy; Mineralocorticoid receptor antagonist; CYP11B2;

Trehalose: A biophysics approach to modulate the inflammatory response during endotoxic shock by Letteria Minutoli; Domenica Altavilla; Alessandra Bitto; Francesca Polito; Ersilia Bellocco; Giuseppina Laganà; Tiziana Fiumara; Salvatore Magazù; Federica Migliardo; Francesco Saverio Venuti; Francesco Squadrito (272-280).
We evaluated the effects of trehalose against endotoxic shock, a condition in which the loss of bio-membrane integrity plays a pivotal role. In addition we performed a biophysics experiment by Quasi Elastic Neutron Scattering (QENS) study, to investigate whether the membrane stability effect of trehalose might be correlated with its high capability to switch-off the water diffusive dynamics and, hence, the kinetic mechanisms of interaction. Endotoxic shock was induced in male rats by a single injection of Salmonella enteritidis lipopolysaccharide (LPS; 20 mg/kg/i.p.). Thirty minutes before and 2 h after LPS injection, the animals were randomized to receive vehicle (1 ml/kg/i.p. 0.9%NaCl), sucrose (1 g/kg/i.p.) or trehalose (1 g/kg/i.p.). Mean arterial blood pressure, Nuclear Factor-κß (NF-κB) binding activity, Iκ-Bα and Toll-like receptor-4 (TLR-4) activation were evaluated in both liver and lung. Plasmatic tumor necrosis factor-α (TNF-α), Interleukin-1 (IL-1), Interleukin-6 (IL-6) and malondialdehyde (MDA) were also investigated. We studied liver injury by means of blood alanine aminotransferase activity (ALT); inducible nitric oxide synthase (iNOS) expression, myeloperoxidase (MPO) activity and tissue edema evaluation. Lung injury was investigated by means of tissue monocyte chemoattractant protein-1 (MCP-1) levels, MPO activity, iNOS expression and edema formation. Trehalose reduced hypotension, NF-κB binding activity, IκBα protein loss and TLR-4 activation. In addition trehalose reduced TNF-α, IL-1, IL-6 and MDA levels. Trehalose also blunted liver and lung injury. QENS measurements showed also that trehalose possesses a high “switching off” capability. Sucrose did not modify endotoxic shock-induced sequelae. Trehalose blocked the inflammatory cascade triggered by endotoxin shock, stabilizing the bio-membranes and switching off the water diffusive dynamics.
Keywords: Trehalose; Disaccharide; Endotoxin shock; NF-κB; TLR-4; Bio-membrane stabilizing effect;

Anti-inflammatory drugs ameliorate opposite enzymatic changes in ileal 5-hydroxytryptamine metabolism in the delayed phase after cisplatin administration to rats by Chuanxia Ju; Naoya Hamaue; Takuji Machida; Yanxia Liu; Kenji Iizuka; Yue Wang; Masaru Minami; Masahiko Hirafuji (281-287).
The effects of anti-inflammatory drugs on ileal 5-hydroxytryptamine (5-HT) metabolic dynamics at 72 h after a single administration of cisplatin were investigated in rats. Cisplatin 5 mg/kg i.p. caused pathological changes, with an inflammatory response occurring 72 h after its administration. The inflammatory response was associated with the induction of cyclooxygenase-2, but not cyclooxygenase-1, in the ileal mucosa at 72 h after the cisplatin administration. Daily treatment with meloxicam 3 mg/kg s.c. ameliorated the cisplatin-induced mucosal damage, whereas dexamethasone 1 mg/kg s.c. did not. Cisplatin administration also caused a significant increase in cyclooxygenase-2 mRNA expression at 72 h after administration, which was blunted by dexamethasone, but not by meloxicam. Cisplatin increased the content of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), but had no effect on 5-HT turnover (5-HIAA/5-HT ratio). Meloxicam and dexamethasone did not significantly decrease 5-HT and 5-HIAA content. Cisplatin significantly decreased monoamine oxidase activity but increased tryptophan hydroxylase (TPH) activity and TPH1 mRNA expression in ileal tissue. Meloxicam and dexamethasone significantly restored the decreased monoamine oxidase activity and inhibited the cisplatin-induced increase in tryptophan hydroxylase activity toward the control levels. These drugs also decreased the cisplatin-induced increase in TPH1 mRNA expression. Neither cisplatin nor the anti-inflammatory drugs had significant effect on mRNA expression of the serotonin re-uptake transporter. These results suggest that the inflammatory response associated with cyclooxygenase-2 induction is involved in the opposite change in ileal tryptophan hydroxylase and monoamine oxidase activities in the delayed phase after single administration of cisplatin to rats.
Keywords: Cisplatin; 5-hydroxytryptamine (5-HT); Tryptophan hydroxylase; Monoamine oxidase; Cyclooxygenase-2; Anti-inflammatory drugs;

Differential effects of triptolide and tetrandrine on activation of COX-2, NF-κB, and AP-1 and virus production in dengue virus-infected human lung cells by Jun-Ting Liou; Zih-Yan Chen; Ling-Jun Ho; Shih-Ping Yang; Deh-Ming Chang; Chun-Chin Liang; Jenn-Haung Lai (288-298).
Most virus infections induce cycloxygenase-2 (COX-2) expression and subsequent prostaglandin E2 (PGE2) production in cells, an inflammatory response that might be detrimental to virus replication and pathogenesis. This response in dengue virus infection remains to be elucidated. Triptolide and tetrandrine, compounds derived from two commonly used Chinese herbs, both demonstrate anti-inflammatory and immunosuppressive effects partly through modulation of COX-2 expression and, hence, may have antiviral effects. In this study, we examined, firstly, the immune response to dengue virus infection with respect to COX-2 expression and PGE2 production in human lung cells (A549), liver cells (HepG2) and dendritic cells. Secondly, we assessed the potential antiviral effects of triptolide and tetrandrine on dengue virus infection vis-à-vis expression of COX-2, PGE2, transcription factors, as well as virus production. We found that dengue virus infection enhanced COX-2 expression and PGE2 production in A549 cells, similarly to the response in dendritic cells, but not in HepG2 cells. In dengue virus-infected A549 cells, nuclear factor κB (NF-κB) and activator protein 1 (AP-1) were also activated, and both were dose-dependently inhibited by triptolide (0.5–4 ng/ml). Tetrandrine (1–10 μM) had no similar immunosuppressive effects and, moreover, at higher concentrations, enhanced NF-κB and AP-1 activity, COX-2 expression and PGE2 production. However, unexpectedly, tetrandrine, but not triptolide, dose-dependently suppressed dengue virus production in A549 cells, independent of PGE2 level. Our findings imply that triptolide and tetrandrine may attenuate dengue virus infection in human lung cells, but through distinct pathways.
Keywords: Triptolide; Tetrandrine; Dengue virus; COX-2; PGE2; NF-κB; AP-1; A549 cell;

Protective effect of JBP485 on concanavalin A-induced hepatocyte toxicity in primary cultured rat hepatocytes by Jingjing Wu; Changyuan Wang; Qi Liu; Tao Yang; Qinghao Zhang; Jinyong Peng; Ying Gao; Huijun Sun; Taiichi Kaku; Kexin Liu (299-305).
Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide isolated from Laennec, and Laennec is a hydrolyzate of human placenta. Evidence has indicated that JBP485 exhibits potent anti-hepatitis activity. In this study, we investigated the protective effect and possible mechanisms of action of JBP485 in Concanavalin A (Con A)-induced hepatotoxicity in vitro. Two in vitro models were established. Model I: primary cultured female rat hepatocytes were only incubated with Con A (50 μg/ml); model II: co-culture system of hepatocytes and autologous splenic lymphocytes, both were stimulated with Con A (20 μg/ml). JBP485 (25 μM) was pre-incubated with the two models. Our results showed that JBP485 reduced cellular aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α) leakage following the application of Con A in both of the models. Potential protective mechanisms were elucidated by measuring DNA fragmentations, immunocytochemistry and RT-PCR. We showed that DNA fragmentations in hepatocytes were attenuated in the JBP485 pre-incubated groups, and at the same time, immunocytochemistry and RT-PCR indicated that expression levels of caspase-3 protein and mRNA in the JBP485 treated groups were decreased compared with those in the untreated groups. Moreover, intercellular adhesion molecule-1 (ICAM-1) was also down-regulated by this dipeptide. The results indicate that JBP485 exhibits hepatoprotective effect through inhibition of hepatocyte apoptosis and ICAM-1 expression.
Keywords: JBP485; Laennec; Concanavalin A; Apoptosis; ICAM-1; Primary cultured rat hepatocyte;

Pharmacokinetic, pharmacodynamic, and efficacy profiles of alogliptin, a novel inhibitor of dipeptidyl peptidase-4, in rats, dogs, and monkeys by Bumsup Lee; Lihong Shi; Daniel B. Kassel; Tomoko Asakawa; Koji Takeuchi; Ronald J. Christopher (306-314).
The aim of the present research was to characterize the pharmacokinetic, pharmacodynamic, and efficacy profiles of alogliptin, a novel quinazolinone-based dipeptidyl peptidase-4 (DPP-4) inhibitor. Alogliptin potently inhibited human DPP-4 in vitro (mean IC50, ~ 6.9 nM) and exhibited > 10,000-fold selectivity for DPP-4 over the closely related serine proteases DPP-2, DPP-8, DPP-9, fibroblast activation protein/seprase, prolyl endopeptidase, and tryptase (IC50 > 100,000 nM). Absolute oral bioavailability of alogliptin in rats, dogs, and monkeys was 45%, 86%, and 72% to 88%, respectively. After a single oral dose of alogliptin, plasma DPP-4 inhibition was observed within 15 min and maximum inhibition was > 90% in rats, dogs, and monkeys; inhibition was sustained for 12 h in rats (43%) and dogs (65%) and 24 h in monkeys (> 80%). From E max modeling, 50% inhibition of DPP-4 activity was observed at a mean alogliptin plasma concentration (EC50) of 3.4 to 5.6 ng/ml (10.0 to 16.5 nM) in rats, dogs, and monkeys. In Zucker fa/fa rats, a single dose of alogliptin (0.3, 1, 3, and 10 mg/kg) inhibited plasma DPP-4 (91% to 100% at 2 h and 20% to 66% at 24 h), increased plasma GLP-1 (2- to 3-fold increase in AUC0–20 min) and increased early-phase insulin secretion (1.5- to 2.6-fold increase in AUC0–20 min) and reduced blood glucose excursion (31%–67% decrease in AUC0–90 min) after oral glucose challenge. Alogliptin (30 and 100 mg/kg) had no effect on fasting plasma glucose in normoglycemic rats. In summary, these data suggest that alogliptin is a potent and highly selective DPP-4 inhibitor with demonstrated efficacy in Zucker fa/fa rats and potential for once-daily dosing in humans.
Keywords: Type 2 diabetes mellitus; Alogliptin; Dipeptidyl peptidase-4; Incretin; Glucagon-like peptide-1;

Choline, CDP-choline or phosphocholine increases plasma glucagon in rats: Involvement of the peripheral autonomic nervous system by Mehmet Cansev; Yesim Ozarda Ilcol; Mustafa Sertac Yilmaz; Emre Hamurtekin; Ismail H. Ulus (315-322).
The present study was designed to test the effects of choline, cytidine-5′-diphosphocholine (CDP-choline) and phosphocholine on plasma glucagon concentrations in rats. Intraperitoneal (i.p.) injection of 200–600 μmol/kg of choline, CDP-choline or phosphocholine produced a dose-dependent increase in plasma glucagon and choline concentrations. Pretreatment with hexamethonium (15 mg/kg; i.p.), a peripherally-acting ganglionic nicotinic acetylcholine receptor antagonist, entirely blocked the increases in plasma glucagon by 600 μmol/kg of choline, CDP-choline or phosphocholine. The increases in plasma glucagon by these choline compounds was reduced significantly (P  < 0.01) by about 25% by pretreatment with atropine methylnitrate (2 mg/kg), a peripherally-acting muscarinic acetylcholine receptor antagonist. Blockade of central acetylcholine receptors did not alter the increase in plasma glucagon induced by i.p. choline (600 μmol/kg). While α2-adrenoceptor blockade or bilateral adrenalectomy attenuated the increase in plasma glucagon evoked by choline compounds, blockade of α1- or β-adrenoceptors or chemical sympathectomy failed to alter this increase. Intracerebroventricular (i.c.v.) choline (1.5 μmol) administration also increased plasma glucagon; the effect was blocked by central pretreatment with a neuronal type nicotinic acetylcholine receptor antagonist, mecamylamine (50 μg; i.c.v.) or the neuronal choline uptake inhibitor, hemicholinium-3 (20 μg; i.c.v.). These data show that choline, CDP-choline or phosphocholine increases plasma glucagon concentrations by increasing peripheral nicotinic and muscarinic cholinergic neurotransmissions. Central choline also increases plasma glucagon by augmenting central nicotinic cholinergic neurotransmission by acting presynaptically. Stimulation of adrenal medullary catecholamine release and subsequent activation of α2-adrenoceptors are mainly involved in the increase in plasma glucagon induced by choline, CDP-choline or phosphocholine.
Keywords: Choline; CDP-choline; Phosphocholine; Glucagon; Catecholamine; Adrenoceptor;