European Journal of Pharmacology (v.628, #1-3)

Former studies in rats demonstrated that starvation or treatment with the hypolipidemic drug clofibrate causes a marked increase in the concentration of carnitine in the liver. The molecular mechanisms underlying these phenomena in rats, however, have been largely unknown. Since both, fasting and clofibrate treatment lead to an activation of peroxisome proliferator-activated receptor α (PPARα), the hypothesis has been raised that activation of this nuclear receptor could lead to an up-regulation of novel organic cation transporters (OCTN) which facilitate transport of carnitine and several other organic cations through membranes. Studies in rodents and pigs have indeed shown that treatment with PPARα agonists causes an up-regulation of OCTN2 in liver and other tissues such as muscle and small intestine. Additional experiments with PPARα-null and corresponding wild-type mice, which were either fasted or treated with the high-affinity PPARα agonist WY-14,643, revealed that transcriptional up-regulation of OCTN2 and OCTN3 is dependent on PPARα. An up-regulation of OCTN by PPARα activation could be regarded as a means to supply cells with sufficient carnitine required for transport of excessive amounts of fatty acids into the mitochondrion during fasting, and therefore plays an important role in the adaptive response of the metabolism to fasting. Due to the strong similarities in the gene response to PPARα agonists and the similar metabolic features and anatomic conditions between pigs and humans, it is likely that pharmacological PPARα agonists exert similar effects in humans.
Keywords: Novel organic cation transporter; Carnitine; Peroxisome proliferator-activated receptor-α;

In vitro pharmacological selectivity profile of oseltamivir prodrug (Tamiflu®) and active metabolite by Lothar Lindemann; Helmut Jacobsen; Diana Schuhbauer; Frederic Knoflach; Silvia Gatti; Joseph G. Wettstein; Hansruedi Loetscher; Tom Chu; Martin Ebeling; James C. Paulson; Eric Prinssen; Manfred Brockhaus (6-10).
Neuropsychiatric adverse events have been reported in influenza patients with and without exposure to oseltamivir (Tamiflu®), triggering speculation as to whether oseltamivir may be interacting with any human receptors and contributing to such neuropsychiatric events. In this study, the in vitro selectivity profile of oseltamivir prodrug and active metabolite was investigated. Both compounds lacked clinically relevant pharmacological activities on human, rodent and primate neuraminidases and on a panel of 155 other molecular targets, including those relevant for mood, cognition and behavior. Neuropsychiatric adverse events observed in influenza patients are likely a phenomenon caused by the infection rather than by oseltamivir.
Keywords: Influenza; Neuraminidase; Oseltamivir; Tamiflu; Selectivity; Neuropsychiatric adverse event;

Hypoxia-induced cell damage is reduced by mild hypothermia and postconditioning with catalase in-vitro: Application of an enzyme based oxygen deficiency system by Karina Zitta; Patrick Meybohm; Berthold Bein; Cornelia Rodde; Markus Steinfath; Jens Scholz; Martin Albrecht (11-18).
Mild hypothermia and pharmacological postconditioning are widespread therapeutical treatment options that positively influence the clinical outcome after tissue hypoxia. In the study presented, a two-enzyme based in-vitro oxygen deficiency model in combination with cultured HT-1080 fibrosarcoma cells was employed to mimic the in-vivo situation of hypoxia and to evaluate the influence of mild hypothermia and postconditioning with catalase on hypoxia-mediated cell damage. Using the in-vitro oxygen deficiency model, partial pressure of oxygen was rapidly reduced to levels below 5 mmHg in the culture media and cells responded with an increased expression of hypoxia inducible factor-1 on protein level. Hypoxia resulted in significant cell rounding and retraction of cytoplasmic cell extensions. Evaluation of cytotoxicity revealed a 3.5-fold increase in lactate dehydrogenase levels which was accompanied by 40-fold elevated levels of hydrogen peroxide. The hypoxia-induced increase of lactate dehydrogenase was 2.5-fold reduced in the hypothermia group, although morphological correlates of cytotoxicity were still visible. Hypothermia did not significantly influence hydrogen peroxide concentrations in the culture media. Pharmacological postconditioning with catalase however dose-dependently decreased hypoxia-induced lactate dehydrogenase release. This cytoprotective effect was accompanied by a dose-dependent, up to 50-fold reduction of hydrogen peroxide concentrations and retention of normal cell morphology. We suggest that the described in-vitro oxygen deficiency model is a convenient and simple culture system for the investigation of cellular and subcellular events associated with oxygen deficiency. Moreover, our in-vitro results imply that catalase postconditioning may be a promising approach to attenuate hypoxia-induced and hydrogen peroxide-mediated cell and tissue damage.
Keywords: Hypoxia; Postconditioning; Catalase; Mild hypothermia; Hydrogen peroxide; Reactive oxygen species;

The dopaminergic stabilizers pridopidine (ACR16) and (−)-OSU6162 display dopamine D2 receptor antagonism and fast receptor dissociation properties by Tino Dyhring; Elsebet Ø. Nielsen; Clas Sonesson; Fredrik Pettersson; Jonas Karlsson; Peder Svensson; Palle Christophersen; Nicholas Waters (19-26).
A new pharmacological class of CNS ligands with the unique ability to stimulate or suppress motor and behavioral symptoms depending on the prevailing dopaminergic tone has been suggested as “dopaminergic stabilizers”. The molecular mode-of-action of dopaminergic stabilizers is not yet fully understood, but they are assumed to act via normalization of dopaminergic signaling, through interactions with the dopamine D2 receptor. Here we have evaluated the dopaminergic stabilizers pridopidine (ACR16) and (−)-OSU6162, as well as the new compound N-{[(2S)-5-chloro-7-(methylsulfonyl)-2,3-dihydro-1,4-benzodioxin-2-yl]methyl}ethanamine (NS30678) in a series of cellular in vitro dopamine D2 receptor functional and binding assays. Neither ACR16, (−)-OSU6162, nor NS30678 displayed detectable dopamine D2 receptor-mediated intrinsic activity, whereas they concentration-dependently antagonized dopamine-induced responses with IC50 values of 12.9 μM, 5.8 μM, and 7.0 nM, respectively. In contrast to the high-affinity typical antipsychotics haloperidol and raclopride, the dopaminergic stabilizers ACR16 and (−)-OSU6162 both displayed fast dopamine D2 receptor dissociation properties, a feature that has previously been suggested as a contributing factor to antipsychotic atypicality and attributed mainly to low receptor affinity. However, the finding that NS30678, which is equipotent to haloperidol and raclopride, also displays fast receptor dissociation, suggests that the agonist-like structural motif of the dopaminergic stabilizers tested is a critical dissociation rate determinant. The results demonstrate that dopaminergic stabilizers exhibit fast competitive dopamine D2 receptor antagonism, possibly allowing for temporally variable and activity-dependent dopamine D2 receptor occupancy that may partly account for their unique stabilization of dopamine dependent behaviors in vivo.
Keywords: Dopaminergic stabilizer; Dopamine D2 receptor; Pridopidine; ACR16; Fast dissociation; Antipsychotic;

Apoptosis of human vein endothelium cell caused by reactive oxygen species is implicated in the pathogenesis of cardiovascular diseases. Rutin, an active flavonoid compound, is well known to possess potent antioxidant properties against oxidative stress insults through undefined mechanism(s). In this study, we first investigated the possible protective effects of rutin against apoptosis of human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2) and the associated signaling pathways. Decreased viability and increased apoptosis were observed in the HUVECs incubated with 200 μM H2O2 for 12 h. By examining the effect of rutin on H2O2-induced apoptosis in HUVECs, we found that rutin pretreatment significantly attenuated H2O2-induced apoptosis in HUVECs. We next examined the signaling involved in rutin-mediated anti-apoptotic effects. It was found that rutin pretreatment attenuated excessive reactive oxygen species in HUVECs exposed to H2O2. Rutin also prevented the increased DNA fragment formation and glutathione (GSH) depletion and inhibited the collapse of mitochondrial membrane potentials (ΔΨm) that occurred in HUVECs exposed to H2O2, which protected HUVECs against oxidative damage and the further mitochondrial membrane integrity impairment, leading to apoptosis. In conclusion, the results suggested that rutin (50 µM) blocked apoptosis in HUVECs through decreasing reactive oxygen species, increasing GSH, restoring ΔΨm and thus protecting DNA damage. Our research indicated that rutin protected the intracellular GSH antioxidant system and prevented H2O2-induced apoptosis of HUVECs through regulating reactive oxygen species mediated mitochondrial dysfunction pathway.
Keywords: Rutin; Hydrogen peroxide; Human umbilical vein endothelial cell (HUVEC); Apoptosis; Reactive oxygen species; Mitochondrion;

Comparison between gentamycin and exon skipping treatments to restore ryanodine receptor subtype 2 functions in mdx mouse duodenum myocytes by Fabrice Dabertrand; Jean Mironneau; Morgana Henaff; Nathalie Macrez; Jean-Luc Morel (36-41).
In Duchenne muscular dystrophy, a stop-codon mutation in the dystrophin gene induces an impairment of skeletal and smooth muscles contraction. In duodenum from mdx mouse, the disease model, the decrease of contractility was linked with the decrease of calcium signals encoded by ryanodine receptor subtype 2. Aminoglycoside and antisense oligonucleotide strategies were investigated to restore calcium signalling in the mdx mouse. Mdx mice were treated by intraperitoneal injection of gentamycin or 2-O-methyl antisense ribonucleotide directed against exon 23 of dystrophin for 2 weeks. The efficiency of both therapeutic strategies was determined by the level of dystrophin protein expression. The physiological effects of both treatments on ryanodine receptor expression and function were followed by RT-PCR, western blot and calcium measurements. Fourteen days after injection of gentamycin or anti-dystrophin antisense, the expression of dystrophin was recovered in skeletal muscle from treated mdx mice. In duodenum cells, RT-PCR and western blot indicated that the expression of ryanodine receptor subtype 2 was similar in treated mice than in control mice in association with the recovery of caffeine-induced Ca2+ response. No significant difference was observed in the ryanodine subtype 3-dependent spontaneous Ca2+ oscillations in untreated and treated mice. Conclusions — these results may help to explain the efficiency of aminoglycoside and anti-dystrophin antisense treatments in smooth muscle. Both treatments could be an interesting therapeutic option to restore smooth muscle contraction in patients with Duchenne muscular dystrophy.
Keywords: Calcium signalling; Ryanodine receptor; (Mdx mice);

The stimulative effects of endogenous opioids on endothelial cell proliferation, migration and angiogenesis in vitro by Xu Dai; Hong-jin Song; Shi-gang Cui; Ting Wang; Qian Liu; Rui Wang (42-50).
The opioid peptides modulate extensive bioactivities, including pain, cardiovascular response, development and further responses. In the present study, the stimulative effects of endogenous opioid peptides on angiogenesis are evaluated in the proliferation, migration, adhesion and tube formation assays of the human umbilical vein endothelial cell (HUVEC) for the first time. Endomorphin-1, endomorphin-2 and deltorphin I at physiological concentrations could stimulate HUVECs proliferation, migration, adhesion and tube formation in a dose dependent manner; whereas, they exhibited the cytotoxic effects on HUVECs at the higher doses in these assays. Naloxone, the nonselective opioid receptor antagonist, did not influence angiogenesis when it was administrated on its own; but it could antagonize the stimulative effects of the opioid peptides on angiogenesis when it was administrated in combination with the opioid peptides. Taken altogether, the results suggested that endogenous opioid peptides (endomorphin-1 and -2 and deltorphin I) stimulated angiogenesis at the cellular level, and these effects were mediated by the opioid receptors. These data are significant for potential clinical implementation in future.
Keywords: Endomorphin-1; Endomorphin-2; Deltorphin I; Angiogenesis; HUVEC;

The effect of Telmisartan on collagen biosynthesis depends on the status of estrogen activation in breast cancer cells by Beata Kociecka; Arkadiusz Surazynski; Wojciech Miltyk; Jerzy Palka (51-56).
PPAR-γ and estrogen receptor belong to a family of nuclear hormone receptors that were shown to affect transcriptional activity of each other. The angiotensin II type 1 receptor antagonist Telmisartan is a well known PPAR-γ ligand. The effect of Telmisartan-induced PPAR-γ activation on collagen biosynthesis was studied in the estrogen-dependent (MCF-7 cells expressing α and β receptors) and estrogen-independent (MDA-MB 231, expressing only β receptor) cell lines. We have found that the presence of estrogen in growth medium (2 nM) augmented collagen biosynthesis in both cell lines. An addition to the growth medium of PPAR-γ agonist, Telmisartan, but not rosiglitazone or clofibrat, other PPAR-γ agonists, induced inhibition of collagen biosynthesis in MCF-7 cells, cultured in the presence of estrogen, while it had no effect on collagen biosynthesis in MDA-MB-231 cells. On the other hand, Telmisartan induced stimulation of collagen biosynthesis in MCF-7 cells cultured in the absence of estrogen (or in conditions of estrogen receptor removal by ICI 182-780-dependent degradation) and had no effect on similarly cultured MDA-MB-231 cells. The effect of Telmisartan on collagen biosynthesis was found specific for PPAR-γ and not for angiotensin II type 1 since Losartan (specific antagonist of angiotensin II type 1 receptor) in the presence of estradiol did not induce inhibition of this protein in MCF-7 cells. The mechanism of the inhibition was found at the level of NF-kB (known inhibitor of collagen gene expression) and MAPK signaling. PPAR-γ ligands stimulated expression of NF-kB, while they inhibited expression of p-38 but not ERK1/ERK2. The data suggest that the effect of Telmisartan on collagen biosynthesis in breast cancer cells depends on the status of estrogen receptor activation and the inhibitory effect of Telmisartan on the process requires functional alpha estrogen receptor.
Keywords: PPAR-γ; PPAR-γ agonist; Collagen; Estrogen; Breast cancer cell;

Exposure to antiepileptic drugs does not alter the functionality of P-glycoprotein in brain capillary endothelial and kidney cell lines by Kamila Ambroziak; Konstantin Kuteykin-Teplyakov; Carlos Luna-Tórtos; Marwan Al-Falah; Maren Fedrowitz; Wolfgang Löscher (57-66).
Several major antiepileptic drugs, including carbamazepine, phenytoin and phenobarbital, induce xenobiotic metabolizing enzymes via activation of nuclear receptors, including pregnane X receptor (NR1I2) and constitutive androstane receptor (NR1I3). Via activation of these xenobiotic sensors, antiepileptic drugs may also induce the expression of efflux transporters such as P-glycoprotein (Pgp) in different tissues, including intestine, liver, kidney and brain. Increased expression of Pgp in brain capillary endothelial cells, which form the blood–brain barrier, could limit the penetration of antiepileptic drugs into the brain and therefore decrease their therapeutic efficacy. As a consequence, it is important to know whether antiepileptic drugs alter the expression or functionality of Pgp in endothelial cells. In the present study, we studied the effects of exposure to phenobarbital, phenytoin and carbamazepine on Pgp expression and functionality in the rat brain endothelial cell line GPNT. For comparison with drug effects on endothelial cells, a dog kidney cell line (MDCK II) was used. Furthermore, several known Pgp inducers (dexamethasone, doxorubicin, and rifampicin) were included in the study. Functionality of Pgp was determined by uptake assays, using known Pgp substrates (digoxin and vinblastine) and transport inhibitors (tariquidar, MK571). In GPNT cells, exposure to dexamethasone increased Pgp functionality, while antiepileptic drug exposure at clinically relevant concentrations did not exert any significant induction of Pgp expression or function. Similarly, antiepileptic drug exposure did not affect Pgp in MDCK cells. The lack of antiepileptic drugs to induce Pgp in brain capillary endothelial cells and kidney cells is in contrast to their known effect on Pgp expression in hepatic and intestinal cells, substantiating tissue differences in the regulation of Pgp.
Keywords: Phenobarbital; Nuclear receptor; PXR; CAR; Pharmacoresistance; Epilepsy;

Effect of lipid raft disruption on TRPV1 receptor activation of trigeminal sensory neurons and transfected cell line by Éva Szőke; Rita Börzsei; Dániel Márton Tóth; Orsolya Lengl; Zsuzsanna Helyes; Zoltán Sándor; János Szolcsányi (67-74).
The transient receptor potential vanilloid 1 (TRPV1) is a noxious heat-sensitive, chemonociceptive cation channel which is expressed in primary sensory neurons of polymodal nociceptors. The present study is devoted to analyse the role of lipid raft constituents in calcium influx evoked by various TRPV1 agonists on sensory neurons and on rTRPV1-transfected CHO cell line. Depletion of cholesterol by methyl β-cyclodextrin (MCD, 1–10 mM) diminished the percent of the calcium uptake response of cultured trigeminal neurons to capsaicin (100 nM) or resiniferatoxin (RTX, 3 nM). In contrast, in TRPV1-transfected cells the inhibition was observed only when capsaicin or N-oleoyldopamine (OLDA, 10 µM) was applied, but not when RTX, anandamide (AEA, 10 µM) or pH 5.5 was used for gating. The magnitude of Ca2+-transients evoked by capsaicin (330 nM) was also inhibited in both cell types. Treatment of rTRPV1-expressing cells with sphinomyelinase inhibited the capsaicin-evoked 45Ca-uptake leaving the RTX-induced response unchanged. On the other hand, in trigeminal neurons the effect of both compounds was inhibited by sphingomyelinase treatment. Inhibition of ganglioside biosynthesis by d-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP, 10–20 µM) or myriocyn (5–50 nM) diminished similarly capsaicin- or RTX-evoked calcium uptake in both cultured trigeminal neurons and rTRPV1-expressing cells. The present study revealed that depletion of different constituents of lipid raft inhibited gating the TRPV1 cation channel by various vanilloid and non-vanilloid agents. Evidence for a supporting role of cholesterol, sphingomyelin and gangliosides were obtained both in native and TRPV1-transfected cells. Differential modulation of responses to capsaicin and RTX was often observed.
Keywords: Capsaicin; Resiniferatoxin; Methyl β-cyclodextrin; Sphingomyelinase; Ganglioside; N-oleoyldopamine;

Analysis of the antinociceptive interactions in two-drug combinations of gabapentin, oxcarbazepine and amitriptyline in streptozotocin-induced diabetic mice by Maja A. Tomić; Sonja M. Vučković; Radica M. Stepanović-Petrović; Ana M. Micov; Nenad D. Ugrešić; Milica Š. Prostran; Bogdan Bošković (75-82).
Antiepileptic and antidepressant drugs are the primary treatments for pain relief in diabetic neuropathy. Combination therapy is a valid approach in pain treatment, where a reduction of doses could reduce side effects and still achieve optimal analgesia. We examined the effects of two-drug combinations of gabapentin, oxcarbazepine, and amitriptyline on nociception in diabetic mice and aimed to determine the type of interaction between components. The nociceptive responses in normal and diabetic mice were assessed by the tail-flick test. The testing was performed before and three weeks after the diabetes induction with streptozotocin (150 mg/kg; i.p.), when the antinociceptive effects of gabapentin, oxcarbazepine, amitriptyline and their two-drug combinations were examined. Gabapentin (10–40 mg/kg; p.o.) and oxcarbazepine (20–80 mg/kg; p.o.) produced a significant, dose-dependent antinociception in diabetic mice while amitriptyline (5–60 mg/kg; p.o.) produced weak antinociceptive effect. In normal mice, neither of the drugs produced antinociception. Gabapentin and oxcarbazepine, co-administered in fixed-dose fractions of the ED50 to diabetic mice, induced significant, dose-dependent antinociception. Isobolographic analysis revealed synergistic interaction. Oxcarbazepine (10–60 mg/kg; p.o.) + amitriptyline (5 mg/kg; p.o.) and gabapentin (10–30 mg/kg; p.o.) + amitriptyline (5 mg/kg; p.o.) combinations significantly and dose-dependently reduced nociception in diabetic mice. Analysis of the log dose–response curves for oxcarbazepine or gabapentin in a presence of amitriptyline and oxcarbazepine or gabapentin applied alone, revealed a synergism in oxcarbazepine–amitriptyline and additivity in gabapentin–amitriptyline combination. These findings provide new information about the combination therapy of painful diabetic neuropathy and should be explored further in patients with diabetic neuropathy.
Keywords: Gabapentin; Oxcarbazepine; Amitriptyline; Streptozotocin; Diabetic neuropathy; Synergistic interaction;

A longitudinal study of 5-HT outflow during chronic fluoxetine treatment using a new technique of chronic microdialysis in a highly emotional mouse strain by Daniela Popa; Julie Cerdan; Christelle Repérant; Bruno P. Guiard; Jean-Philippe Guilloux; Denis J. David; Alain M. Gardier (83-90).
The onset of a therapeutic response to antidepressant treatment exhibits a delay of several weeks. The present study was designed to know whether extracellular serotonin (5-HT) levels need to be increased in territories of 5-HT innervation in order to obtain beneficial effects from a chronic treatment with a serotonin-selective reuptake inhibitor (SSRI). Thus, we performed a longitudinal study of a chronic fluoxetine treatment in a model of highly emotional mice (BALB/cJ). The function of the 5-HT system in the raphe nuclei and hippocampus, was assessed by using repeated in vivo microdialysis sessions in awake freely moving mice, then studying its relation with behavior, analyzed mainly with open field paradigm. One of the neural mechanisms underlying such delay has been proposed to be the functional status of 5-HT1A autoreceptors in raphe nuclei. Thus, we also assessed the degree of 5-HT1A autoreceptor desensitization by using a local infusion in the raphe of the antagonist, WAY 100635 via reverse microdialysis. We report that the anxiolytic-like effects of fluoxetine correlate in time and amplitude with 5-HT1A autoreceptor desensitization, but neither with the extracellular levels of 5-HT in the raphe nuclei, nor in the hippocampus. Our study suggests that the beneficial anxiolytic/antidepressant-like effects of chronic SSRI treatment indeed depend on 5-HT1A autoreceptor internalization, but do not require a sustained increase in extracellular 5-HT levels in a territory of 5-HT projection such as hippocampus.
Keywords: Serotonin-selective reuptake inhibitor SSRI; Depression; Anxiety; Repeated intracerebral microdialysis; Open field; Tail suspension test; Novelty suppressed feeding test; Awake mice;

Emerging evidence recently indicates that the anterior cingulate cortex is critically involved in the central processing and modulation of noxious stimulus, although the neuroadaptation in the anterior cingulate cortex has not been well documented in the conditions of chronic pain. Meanwhile, the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex remains unclear. To address these issues, the present study was undertaken to explore the adaptation of excitatory glutamatergic transmission and mu opioid receptor-mediated modulation of glutamatergic transmission in the anterior cingulate cortex slices from the complete Freund′s adjuvant (CFA)-inflamed rats. The results demonstrated that glutamatergic paired-pulse facilitation was decreased in the anterior cingulate cortex neurons from the CFA-inflamed rats, indicating an enhanced presynaptic glutamate release. In addition, activation of mu opioid receptor significantly inhibited the glutamatergic excitatory postsynaptic currents (EPSCs) in the anterior cingulate cortex neurons, which was attained through the suppression of presynaptic glutamate release. Taken together, these findings provided the evidence for the functional adaptation of central glutamatergic transmission induced by peripheral inflammation, and elucidated the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex.
Keywords: Excitatory postsynaptic current; Opioid receptor; Anterior cingulate cortex; Complete Freund′s adjuvant;

Effects of MDMA (“ecstasy”) during adolescence on place conditioning and hippocampal neurogenesis by Briony J. Catlow; Kimberly A. Badanich; Ashley E. Sponaugle; Amanda R. Rowe; Shijie Song; Igor Rafalovich; Vasyl Sava; Cheryl L. Kirstein; Juan Sanchez-Ramos (96-103).
The use of 3,4,methylenedioxymethamphetamine (MDMA), the active agent in ecstasy, during adolescence is widespread yet the effects on adolescent behavior and brain development are unknown. The aim of the present study was 1) to evaluate effects of MDMA in adolescent rats using the conditioned place preference (CPP) paradigm to measure MDMA-induced reward and 2) assess effects of MDMA administration on cellular proliferation, survival and neurogenesis in the dentate gyrus of the hippocampus. During the adolescent period, MDMA CPP was measured in adolescents [postnatal day (PND) 28–39] by training rats to associate 1.25, 2.5, 5.0 mg/kg MDMA or saline administration with environmental cues. After CPP ended, bromodeoxyuridine (BrdU) was injected and rats were euthanized either 24 h (to evaluate cell proliferation) or 2 weeks (to assess neurogenesis) after the last MDMA injection. Adolescents expressed a CPP for 2.5 mg/kg MDMA. Repeated exposure to 5.0 mg/kg MDMA during adolescence increased cell proliferation, yet diminished neurogenesis, an effect that was replicated using flow cytometry. These findings suggest differential dose effects of adolescent MDMA exposure on reward related behaviors and hippocampal neurogenesis.
Keywords: Adolescent; MDMA; Conditioned place preference; Hippocampus; Proliferation; Neurogenesis; Flow cytometry; Development;

Impaired spatial working memory and decreased frontal cortex BDNF protein level in dopamine transporter knockout mice by BingJin Li; Yosefu Arime; F. Scott Hall; George R. Uhl; Ichiro Sora (104-107).
Brain-derived neurotrophic factor (BDNF), one of the key brain neurotrophins, has been implicated in neuronal plasticity and memory. Recent studies document the importance of BDNF for normal long-term memory functions. However, there are few studies of the roles of BDNF in short-term memory. Dopamine is likely to play important roles in BDNF gene expression in specific brain regions, including frontal cortical regions that are implicated in short-term working memory processes that include spontaneous alternation. We have thus tested spatial working memory in dopamine transporter knockout (DAT KO) and wild-type mice. Spontaneous alternation in the Y-maze, an index of short-term spatial working memory in mice, was significantly decreased in DAT KO mice compared to wild-type mice. BDNF protein was significantly decreased in frontal cortex, though not in striatum or hippocampus, of the DAT KO mice. The data support the hypothesis that impaired spatial working memory in DAT KO mice may be related to decreased frontal cortical BDNF in these animals, and document apparent roles for BDNF in a short-term memory process.
Keywords: Brain-derived neurotrophic factor (BDNF); Y-maze; Frontal cortex; (Dopamine transporter knockout (DAT KO) mouse);

Alzheimer's disease is a chronic neurodegenerative disorder marked by a progressive loss of memory and cognitive function. Stress level glucocorticoids are correlated with dementia progression in patients with Alzheimer's disease. In this study, twelve month old male mice were chronically treated for 21 days with stress-level dexamethasone (5 mg/kg). We investigated the pathological consequences of dexamethasone administration on learning and memory impairments, amyloid precursor protein processing and neuronal cell apoptosis in 12-month old male mice. Our results indicate that dexamethasone can induce learning and memory impairments, neuronal cell apoptosis, and mRNA levels of the amyloid precursor protein, β-secretase and caspase-3 are selectively increased after dexamethasone administration. Immunohistochemistry demonstrated that amyloid precursor protein, caspase-3 and cytochrome c in the cortex and CA1, CA3 regions of the hippocampus are significantly increased in 12-month old male mice. Furthermore, dexamethasone treatment induced cortex and hippocampus neuron apoptosis as well as increasing the activity of caspase-9 and caspase-3. These findings suggest that high levels of glucocorticoids, found in Alzheimer's disease, are not merely a consequence of the disease process but rather play a central role in the development and progression of Alzheimer's disease. Stress management or pharmacological reduction of glucocorticoids warrant additional consideration of the regimen used in Alzheimer's disease therapies.
Keywords: Memory and learning impairment; Dexamethasone; Amyloid precursor protein; Apoptosis; Alzheimer's disease;

Pinacidil-primed ATP-sensitive potassium channels mediate feedback control of mechanical power output in isolated myocardium of rats and guinea pigs by Diethart Schmid; Dawid L. Staudacher; Christian A. Plass; Hans G. Loew; Eva Fritz; Guenter Steurer; Peter Chiba; Thomas Moeslinger (116-127).
We tested the hypothesis, that ATP-sensitive potassium (KATP) channels limit cardiac energy demand by a feedback control of mean power output at increased cardiac rates. We analysed the interrelationships between rising energy demand of adult rat and guinea pig left ventricular papillary muscle and down-regulatory electromechanical effects mediated by KATP channels. Using the KATP-opener pinacidil the stimulation frequency was increased stepwise and the mechanical parameters and action potentials were recorded. Power output was derived from force–length area or force–time integral calculations, respectively. Simultaneously oxygen availability in the preparations was estimated by flavoprotein fluorescence measurements. ADP/ATP ratios were determined by HPLC. We found highly linear relationships between isotonic power output and the effects of pinacidil on isotonic shortening in both rat (r 2  = 0.993) and guinea pig muscles (r 2  = 0.997). These effects were solely observed for the descending limb of shortening–frequency relationships. In addition, a highly linear correlation between total force–time integral-derived power and pinacidil effects on action potential duration (APD50, r 2  = 0.92) was revealed. Power output became constant and frequency-independent in the presence of pinacidil at higher frequencies. In contrast, the KATP-blocker glibenclamide produced a lengthening of APD50 and increased force transiently at higher power levels. Pinacidil prevented core hypoxia and a change in ADP/ATP ratio during high frequency stimulation. We conclude, that pinacidil-primed cardiac KATP channels homeostatically control power output during periods of high energy demand. This effect is associated with a reduced development of hypoxic areas inside the heart muscle by adapting cardiac function to a limited energy supply.
Keywords: ATP (adenosine 5′-triphosphate)-sensitive potassium channels; Flavoprotein oxidation; Glibenclamide; Pinacidil; Mean power output; Papillary muscles;

Halichlorine is a novel L-type Ca2+ channel inhibitor isolated from the marine sponge Halichondria okadai Kadota by Yoshiki Tsubosaka; Takahisa Murata; Kazuya Kinoshita; Kaoru Yamada; Daisuke Uemura; Masatoshi Hori; Hiroshi Ozaki (128-131).
Halichlorine, isolated from a marine sponge Halichondria okadai Kadota, has a unique structure and its physiological activity is virtually unknown. In the present study, we investigated the direct effect of halichlorine on vascular contractility. In endothelium-denuded rat aorta, while the treatment of halichlorine (0.01–10 μM) did not induce vascular contraction, halichlorine (0.01–10 μM) dose-dependently inhibited both the steady-state precontractions induced by high K+ (65.4 mM) and phenylephrine (1 μM). The vasodilator effect of halichlorine (10 μM) on high K+ (65.4 mM)-induced contraction was more potent than that on phenylephrine (1 μM)-induced contraction (65.4 mM high K+: 72.7 ± 3.4%; 1 μM phenylephrine: 34.7 ± 2.3%). To investigate the mechanism underlying the suppressive effect of halichlorine on vascular contractility, we examined the effect of halichlorine on intracellular Ca2+ concentration in vascular smooth muscle with a fluorescent Ca2+ indicator, fura-2. Treatment of halichlorine (10 μM) significantly inhibited the sustained [Ca2+]i elevation induced by high K+ (65.4 mM) (45.3 ± 5.5%). Furthermore, current measurements by whole-cell mode patch-clamp recording in rat aortic smooth muscle cells (A7r5 cells) demonstrated that halichlorine (10 μM) decreased the current density of the L-type Ca2+ channel (peak Ca2+-channel current densities: − 2.09 ± 0.27 pA/pF for control; − 0.58 ± 0.07 pA/pF for halichlorine). These results suggest that halichlorine inhibits L-type Ca2+ channels in vascular smooth muscle cells, which inhibit intracellular Ca2+ influx, and then reduce vascular contractions.
Keywords: Halichroline; L-type Ca2+ channel; Vascular smooth muscle cell; Contraction;

Diabetes mellitus has been known to mitigate ischemic or pharmacologic preconditioning in ischemia–reperfusion injuries. Remifentanil is a widely used opioid in cardiac anesthesia that possesses a cardioprotective effect against ischemia–reperfusion. We evaluated whether diabetes affected remifentanil preconditioning induced cardioprotection in ischemia–reperfusion rat hearts in view of anti-apoptotic pathways of survival and Ca2+ homeostasis. Streptozotocin-induced, diabetic rats and age-matched wild-type Sprague–Dawley rats were subjected to a left anterior descending coronary artery occlusion for 30 min followed by 1 h of reperfusion. Each diabetic and wild-type rat was randomly assigned to the sham, ischemia–reperfusion only, or remifentanil preconditioning group. Myocardial infarct size, activities of ERK1/2, Bcl2, Bax and cytochrome c, and gene expression influencing Ca2+ homeostasis were assessed. Remifentanil preconditioning significantly reduced myocardial infarct size compared to ischemia–reperfusion only in wild-type rats but not in diabetic rats. Remifentanil preconditioning increased expression of ERK1/2 and anti-apoptotic protein Bcl-2 and decreased expression of pro-apoptotic proteins, Bax and cytochrome c, compared to ischemia–reperfusion only in wild-type rats. In diabetic rat hearts, however, remifentanil preconditioning failed to recover the phosphorylation state of ERK1/2 and to repress apoptotic signaling. In addition, diabetes minimized remifentanil induced modulation of abnormal changes in sarcoplasmic reticulum genes and proteins in ischemia–reperfusion rat hearts. In conclusion, diabetes mitigated remifentanil induced cardioprotection against ischemia–reperfusion, which might be associated with reduced recovery of the activities of proteins involved in anti-apoptotic pathways including ERK1/2 and the abnormal expression of sarcoplasmic reticulum genes as a result of ischemia–reperfusion in rat hearts.
Keywords: Diabetes mellitus; Remifentanil; Ischemia–reperfusion;

Vascular insulin resistance in prehypertensive rats: Role of PI3-kinase/Akt/eNOS signaling by Rong Li; Haifeng Zhang; Wenqing Wang; Xiaoming Wang; Yuxiao Huang; Chen Huang; Feng Gao (140-147).
It is well known that systemic insulin resistance is closely associated with the metabolic syndrome including type 2 diabetes and hypertension. However, it remains unclear whether vascular insulin resistance acts as an early etiologic factor for the development of hypertension. Male spontaneously hypertensive rats (SHRs) aged 5 weeks (young) and 15 weeks (adult) were studied and vascular insulin resistance was assessed as the function of isolated aortic vasodilatory response to insulin in vitro. Compared with Wistar–Kyoto (WKY) rats, adult SHRs exhibited significant hypertension with significantly decreased aortic vasodilatation to insulin, whereas young SHRs had normal blood pressure but exhibited similar vascular insulin resistance. Both young and adult SHRs showed significant downregulated expression of PI3-kinase and decreased insulin-stimulated phosphorylations of Akt and eNOS in vascular tissues. Treatment with rosiglitazone (RSG), an insulin sensitizer, for 2 weeks increased vascular PPARγ expression and restored PI3-kinase/Akt/eNOS-mediated signaling pathway only in young SHRs. More importantly, this treatment improved aortic vasodilatory response to insulin in young but not in adult SHRs. In summary, vascular insulin resistance, characterized by the impairment of PI3-kinase/Akt/eNOS-mediated signaling in vascular endothelium, may play important roles in endothelial dysfunction and subsequent development of hypertension in normotensive young SHRs.
Keywords: Insulin resistance; Hypertension; Vasorelaxation; PI3-kinase; Akt;

The present study investigated the mediated effect of protein kinase C (PKC) in arginine vasopressin (AVP)-induced restoration of vascular responsiveness and calcium sensitization following hemorrhagic shock. Using both isolated superior mesenteric artery from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cell (VSMC), we investigated the roles of PKC-α, δ and ε isoforms in AVP-induced restoration of vascular reactivity and calcium sensitivity. Meanwhile, effects of their specific inhibitors on the activity of myosin light chain phosphatase (MLCP), myosin light chain kinase (MLCK), and the phosphorylation of myosin light chain (MLC20) in VSMC were observed. The results indicated that AVP improved the reactivity of superior mesenteric artery and VSMC to norepinephrine and calcium following hemorrhagic shock and hypoxia. PKC-α inhibitor and PKC-ε inhibitory peptide antagonized these effects of AVP, while PKC-δ inhibitor only partially antagonized these effects of AVP. AVP up-regulated the expression of PKC-α and ε in the particulate fractions of hypoxia-treated VSMC with the decrease of the activity of MLCP and the increase of the phosphorylation of MLC20. These effects of AVP were inhibited by PKC-α inhibitor and PKC-ε inhibitory peptide, but not by the PKC-δ inhibitor. The results suggested that PKC plays an important role in AVP-induced restoration of vascular reactivity and calcium sensitivity following hemorrhagic shock. PKC-α and ε may be the main isoforms involved in this process and play effect via MLC20 phosphorylation dependent mechanism, while PKC-δ may be partially involved in AVP action by other mechanisms.
Keywords: Hemorrhagic shock; Arginine vasopressin; Protein kinase C isoforms; Vascular reactivity; Calcium sensitivity;

Catalpol, an iridoid glucoside found in the root of Rehmannia glutinosa Libosch, has been demonstrated to reduce apoptosis in neuronal cell lines. Recent data suggests that catalpol also exerts anti-apoptotic effects on other cell types. The aim of the present study was to investigate whether catalpol protects against hydrogen peroxide (H2O2) induced apoptosis in human umbilical vein endothelial cells (HUVECs). Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling, Annexin V-fluorescein isothiocyanate binding assay and by assessment of caspase-3 activity. The level of intracellular reactive oxygen species was quantified by 2′, 7′-dichlorofluorescein diacetate assay. Expression of Akt, Bad, Bcl-2 and Bax mRNA and protein was determined by real-time semiquantitative reverse transcription-polymerase chain reaction and Western blotting. Apoptosis in HUVECs was associated with increased Bax, decreased Bcl-2 activity and inactivated phosphorylation of Akt and Bad after 24 h of H2O2 exposure. Pre-treatment of HUVECs with catalpol significantly reduced H2O2-induced intracellular reactive oxygen species release. Catalpol not only increased the expression of Bcl-2, while decreasing Bax expression, but also induced Akt activation and Bad phosphorylation, and ultimately reduced H2O2-induced apoptosis. The protective effects of catalpol were partially inhibited by the phosphatidylinositol 3-kinase (PI3K) antagonist wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Taken together, these results suggest that pre-treatment of HUVECs with catalpol can block H2O2-induced apoptosis, and that the underlying mechanism involves reactive oxygen species scavenging, activation of the PI3K/Akt–Bad signaling pathway and increased Bcl-2 and decreased Bax expression.
Keywords: Catalpol; Hydrogen peroxide; Apoptosis; HUVEC;

Chronic heart failure patients with high collagen type I degradation marker levels benefit more with ACE-inhibitor therapy by Sofia V. Chatzikyriakou; Dimitrios N. Tziakas; Georgios K. Chalikias; Dimitrios Stakos; Adina Thomaidi; Konstantina Mitrousi; Harisios Boudoulas (164-170).
Not all patients respond to angiotensin converting enzyme (ACE)-inhibitor equally. Genetic or other phenotypic variations might be useful in predicting the therapeutic efficacy of these drugs. With the present study we assessed the prognostic impact of ACE-inhibitor in chronic heart failure patients with different degrees of collagen metabolism as assessed by serum levels of a collagen type I degradation marker (CITP). One hundred ninety-six (126 male, 69 ± 10 years) chronic heart failure patients were studied prospectively for 12 months regarding survival. Serum concentrations of CITP were measured at study entry. Chronic heart failure patients were divided into groups according to whether (n  = 114) or not (n  = 82) they received ACE-inhibitor as well as to their CITP levels. Survival (52.2%) was significantly lower in ACE-inhibitor naive patients with high CITP levels compared to ACE-inhibitor naive patients with low CITP levels (83.3%, P  = 0.003), to ACE-inhibitor users with low CITP levels (80%, P  = 0.006) and to ACE-inhibitor users with high CITP levels (70.4%, P  = 0.015). ACE-inhibitor related improvement in mortality was most predominant in chronic heart failure patients with high CITP levels. CITP levels possibly reflecting an activated status of the renin–angiotensin–aldosterone system, may be of clinical relevance since they identify a subgroup of patients that is more susceptible to treatment with an ACE-inhibitor.
Keywords: Angiotensin converting enzyme-inhibitor; Collagen metabolism; Prognosis; Chronic heart failure;

Anti-inflammatory effects of formoterol and ipratropium bromide against acute cadmium-induced pulmonary inflammation in rats by Wenhui Zhang; Laurence Fievez; Esteban Cheu; Fabrice Bureau; Weifang Rong; Fan Zhang; Yong Zhang; Charles Advenier; Pascal Gustin (171-178).
In this study, the anti-inflammatory properties of formoterol and ipratropium bromide, alone or in combination, were investigated in a rat model of acute pulmonary inflammation induced by cadmium inhalation. Airway resistance and inflammatory responses, including matrix metalloproteinease-2 (MMP-2) and matrix metalloproteinease-9 (MMP-9) activities, were evaluated. Compared to values obtained in rats exposed to cadmium, pretreatment by bronchodilators administered alone significantly prevented the cadmium-induced increase of airway resistance. Formoterol elicited a significant decrease in total cell number, neutrophil and macrophage counts in bronchoalveolar lavage fluid, whereas ipratropium bromide reduced neutrophil numbers. The two compounds administered alone significantly attenuated the lung lesions associated with parenchyma inflammatory cell influx and congestion observed in the cadmium group. The increased MMP-9 activity was significantly attenuated. Although only formoterol induced a decrease protein concentration in bronchoalveolar lavage fluid, both compounds inhibited the pulmonary edema by reducing wet-to-dry weight ratio which returned to values similar to those recorded in the sham group. All the effects of formoterol on the cadmium-induced inflammatory responses were reversed by propranolol. Similar anti-inflammatory effects were obtained in rats pretreated with ilomastat which showed a significant reduction on inflammatory cell infiltration and MMP-9 activity in bronchoalveolar lavage fluid. Neither synergistic nor additive effects were obtained when the two bronchodilators were administered in combination. In conclusion, formoterol and ipratropium bromide partially protect the lungs against the inflammation by reducing neutrophilic infiltration. This protective effect is associated with reduced MMP-9 activity known to play an important pro-inflammatory role in acute inflammatory process.
Keywords: Pulmonary inflammation; Formoterol; Ipratropium bromide; Cadmium; Matrix metalloproteinase;

Mechanisms of action of hydrogen sulfide in relaxation of mouse distal colonic smooth muscle by Ingeborg Dhaese; Inge Van Colen; Romain A. Lefebvre (179-186).
Hydrogen sulfide (H2S) has been suggested as a gaseous neuromodulator in mammals. The aim of this study was to examine the influence of H2S on contractility in mouse distal colon. The effect of sodium hydrogen sulfide (NaHS; H2S donor) on prostaglandin F (PGF)-contracted circular muscle strips of mouse distal colon was investigated. In addition, tension and cytosolic calcium concentration ([Ca2+]cyt) in the mouse distal colon strips were measured simultaneously in the presence of NaHS. NaHS caused concentration-dependent relaxation of the pre-contracted mouse distal colon strips. The NaHS-induced relaxation was not influenced by the K+ channels blockers glibenclamide, apamin, charybdotoxin, barium chloride and 4-aminopyridine. The relaxation by NaHS was also not influenced by the nitric oxide inhibitor L-NAME, by the soluble guanylate cyclase respectively adenylate cyclase inhibitors ODQ and SQ 22536, by the nerve blockers capsazepine, ω-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The initiation of the NaHS-induced relaxation was accompanied by an increase in [Ca2+]cyt, but once the relaxation was maximal and sustained, no change in [Ca2+]cyt was measured. This calcium desensitization is not related to the best known calcium desensitizing mechanism as the myosin light chain phosphatase (MLCP) inhibitor calyculin-A and the Rho-kinase inhibitor Y-27632 had no influence. We conclude that NaHS caused concentration-dependent relaxations in mouse distal colon not involving the major known K+ channels and without a change in [Ca2+]cyt. This calcium desensitization is not related to inhibition of Rho-kinase or activation of MLCP.
Keywords: Calcium; Cystathionine β-synthase; Cystathionine γ-lyase; Hydrogen sulfide; Mouse distal colon; Potassium channel;

Phospholipase C-independent effects of 3M3FBS in murine colon by Laura Dwyer; Hyun Jin Kim; Byoung Ho Koh; Sang Don Koh (187-194).
The muscarinic receptor subtype M3 is coupled to Gq/11 proteins. Muscarinic receptor agonists such as carbachol stimulate these receptors that result in activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and Ins(1,4,5)P 3 . This pathway leads to excitation and smooth muscle contraction. In this study the PLC agonist, 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benezenesulfonamide (m-3M3FBS), was used to investigate whether direct PLC activation mimics carbachol-induced excitation.We examined the effects of m-3M3FBS and 2, 4, 6-trimethyl-N-(ortho-3-trifluoromethyl-phenyl)-benzenesulfonamide (o-3M3FBS), on murine colonic smooth muscle tissue and cells by performing conventional microelectrode recordings, isometric force measurements and patch clamp experiments.Application of m-3M3FBS decreased spontaneous contractility in murine colonic smooth muscle without affecting the resting membrane potential. Patch clamp studies revealed that delayed rectifier K+ channels were reversibly inhibited by m-3M3FBS and o-3M3FBS. The PLC inhibitor, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), did not prevent this inhibition by m-3M3FBS. Both m-3M3FBS and o-3M3FBS decreased two components of delayed rectifier K+ currents in the presence of tetraethylammonium chloride or 4-aminopyridine. Ca2+ currents were significantly suppressed by m-3M3FBS and o-3M3FBS with a simultaneous increase in intracellular Ca2+. Pretreatment with U73122 did not prevent the decrease in Ca2+ currents upon m-3M3FBS application.In conclusion, both m-3M3FBS and o-3M3FBS inhibit inward and outward currents via mechanisms independent of PLC acting in an antagonistic manner. In contrast, both compounds also caused an increase in [Ca2+]i in an agonistic manner. Therefore caution must be employed when interpreting their effects at the tissue and cellular level.
Keywords: Smooth muscle; Contractility; Membrane potential; Delayed rectifier K+ channel; L-type Ca2+ current;

Pranlukast prevents cysteinyl leukotriene-induced emesis in the least shrew (Cryptotis parva) by Seetha Chebolu; Yaozhi Wang; Andrew P. Ray; Nissar A. Darmani (195-201).
Many chemotherapeutic agents activate multiple signaling systems, including potentially emetogenic arachidonic acid metabolites. Of these messengers, the emetic role of the leukotriene family has been neglected. The aims of this study were to test the emetic potential of key leukotrienes (LTA4, LTB4, LTF4, and the cysteinyl leukotrienes LTC4, LTD4 and LTE4), and to investigate whether the leukotriene CysLT1 receptor antagonist pranlukast or mixed leukotriene CysLT1/2 receptor antagonist Bay u9773 can prevent the LTC4-induced emesis. Least shrews were injected with varying doses of one of the six tested leukotrienes and vomiting parameters were measured for 30 min. LTC4 and LTD4 were most efficacious, and significantly increased both the frequency and percentage of animals vomiting at doses from 0.1 and 0.05 mg/kg, respectively. The other tested leukotrienes were either weakly emetic or ineffective at doses up to 4 mg/kg. The relative emetogenic activities of the cysteinyl leukotrienes (LTC4  = LTD4  > LTE4) suggest that leukotriene CysLT2 receptors have a key role in emesis. However, pranlukast dose-dependently, and at 10 mg/kg completely, blocked LTC4-induced vomiting, implicating a leukotriene CysLT1 receptor-mediated emetic effect. Bay u9773 dose-dependently reduced the percentage of animals vomiting, but did not significantly reduce vomiting frequency. Fos immunoreactivity, measured subsequent to LTC4-induced vomiting to define its putative anatomical substrates, was significantly increased in the enteric nervous system and medullary dorsal vagal complex following LTC4 (P  < 0.05) versus vehicle injections. This study is the first to show that some leukotrienes induce emesis, possibly involving both central and peripheral leukotriene CysLT1 and/or leukotriene CysLT2 receptors.
Keywords: Leukotriene A4; Leukotriene B4; Fos; Dorsal vagal complex; Enteric nervous system;

Contractile responses induced by physalaemin, an analogue of substance P, in the rat esophagus by Takahiko Shiina; Takeshi Shima; Haruko Hirayama; Hirofumi Kuramoto; Tadashi Takewaki; Yasutake Shimizu (202-206).
We examined the effects of physalaemin, an agonist of tachykinin receptors, on mechanical responses in the rat esophagus to clarify possible regulatory roles of tachykinins in esophageal motility. Exogenous application of physalaemin caused tonic contractions in rat esophageal segments when tension was recorded in the longitudinal direction but not when tension was recorded in the circular direction. The physalaemin-evoked contractions were blocked by pretreatment with nifedipine, a blocker of L-type calcium channels in both striated and smooth muscle cells. However, tetrodotoxin, a blocker of voltage-dependent sodium channels in striated muscle cells and neurons, did not affect the physalaemin-induced contractions. These results indicate that physalaemin might induce contractile responses in longitudinal smooth muscle of the muscularis mucosa via direct actions on muscle cells but not on neurons. Although pretreatment with a tachykinin NK1 receptor antagonist, N-acetyl-l-tryptophan 3,5-bis (trifluoromethyl) benzyl ester (L-732,138), did not significantly affect the physalaemin-evoked contractions in rat esophageal segments, a tachykinin NK2 receptor antagonist, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl] benzamide (SR48968), and a tachykinin NK3 receptor antagonist, (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide (SR142801), significantly inhibited the physalaemin-evoked contractions. These results suggest that tachykinins can activate longitudinal contraction of smooth muscle in the muscularis mucosa, mediated via tachykinin NK2 and NK3 receptors on muscle cells, in the rat esophagus.
Keywords: Esophagus; Muscularis mucosa; Physalaemin; Smooth muscle; Tachykinin; (Rat);

Ca2+ inflow responsible for neurotransmitter release at most peripheral junctions is mainly mediated by activation of CaV2.2 and CaV2.1 channels. The aim of the present study was to characterize the voltage-gated Ca2+ channels (VGCCs) responsible for the non-adrenergic non-cholinergic (NANC) relaxation and vasoactive intestinal polypeptide (VIP)-like immunoreactivity release in the rat gastric fundus. Precontracted longitudinal muscle strips of the rat gastric fundus were subjected to electrical field stimulation (EFS) under NANC conditions to evoke the relaxation and VIP-like immunoreactivity release. Nifedipine (1 µM) completely relaxed the preparations, so that its effects on EFS-induced NANC relaxations could not be investigated. ω-Conotoxin GVIA (0.3–100 nM) concentration-dependently reduced the amplitude of low frequency and the area under the curve (AUC) of high-frequency EFS-evoked relaxations (maximal reductions: approximately 55% and 42% of controls, respectively). The ω-conotoxin GVIA-resistant component of relaxation was not affected by ω-agatoxin IVA (300 nM), ω-conotoxin MVIIC (100 nM), SNX-482 (100 nM) or flunarizine (1 µM). ω-Conotoxin GVIA (30 nM), ω-agatoxin IVA (30 nM) and ω-conotoxin MVIIC (100 nM) reduced high-frequency EFS-evoked VIP-like immunoreactivity release by approximately 70%, 27% and 35% of controls, respectively. ω-Conotoxin GVIA (30 nM) plus ω-conotoxin MVIIC (100 nM) almost abolished the EFS-induced VIP-like immunoreactivity outflow. In the rat gastric fundus, the activation of CaV2.2 and P-type of CaV2.1 channels is responsible for the EFS-induced VIP-like immunoreactivity release. In contrast, CaV1 channels, novel VGCCs and/or molecular variants of VGCCs cloned to date may mediate a substantial component of the NANC relaxation.
Keywords: NANC (non-adrenergic non-cholinergic) relaxation; Rat gastric fundus; VGCCs (voltage-gated calcium channels); VIP (vasoactive intestinal polypeptide) release; Conotoxin; Agatoxin;

The absorption sites of mizoribine (an imidazole nucleoside) and ribavirin (a purine nucleoside) in the small intestine were evaluated in rats. The intestinal absorption of mizoribine is known to be mediated by rat concentrative nucleoside transporter (CNT)1 and CNT2. In contrast, the absorption mechanism of ribavirin in rats is not yet fully understood. Thus, the intestinal absorption of ribavirin was characterized firstly. In in-situ jejunum loop studies, the absorption percentage of ribavirin at a dose of 25 mg/kg was significantly lower than those after 1 mg/kg and 5 mg/kg doses. Coadministration of adenosine, inosine and mizoribine, but not thymidine and gemcitabine, significantly suppressed the intestinal absorption of ribavirin, indicating that ribavirin absorption is mediated by CNT2 in rats. In in-situ loop studies, mizoribine and ribavirin were absorbed to the same extents both in the proximal and distal small intestine. In vivo study was carried out using mizoribine, in which the gastric emptying rates altered by a subcutaneous injection of metoclopramide or scopolamine butylbromide exerted no significant effects on the values of peak plasma level (Cmax), area under the plasma concentration–time profile from 0 to 6 h (AUC0–6), and urinary excretion percentage of mizoribine given orally, though the time to reach Cmax (Tmax) of mizoribine was altered by each treatment. In conclusion, mizoribine and ribavirin were found to be absorbed efficiently to the same extents from the whole small intestine. Also, the altered gastric emptying rates exerted no significant effects on the oral bioavailabilities of mizoribine and ribavirin.
Keywords: Absorption site; Bioavailability; Concentrative nucleoside transporter; CNT1; CNT2; Gastric emptying rate; Mizoribine; Ribavirin;

Inhibition of protein kinase Cδ reduces tristetraprolin expression by destabilizing its mRNA in activated macrophages by Tiina Leppänen; Ulla Jalonen; Riku Korhonen; Raimo K. Tuominen; Eeva Moilanen (220-225).
Tristetraprolin (TTP) binds to AU-rich elements within the mRNAs of several inflammatory genes and causes destabilization of the target mRNAs. The protein kinase C (PKC) pathway represents a major signalling system in inflammation and PKCδ is one of the key isoenzymes in the regulation of inflammatory processes. In the present study, we investigated the role of PKCδ in the regulation of the expression of tristetraprolin in activated macrophages by using the PKCδ inhibitor, rottlerin, and by downregulating PKCδ expression by using PKCδ siRNA. TTP protein and mRNA expression were measured by Western blotting and quantitative RT-PCR, respectively. TTP and TNFα mRNA decays were studied by the actinomycin D assay. In addition, we measured nuclear translocation of transcription factors believed to be important for TTP transcription, i.e. NF-κB, AP-2, SP1 and EGR1. Downregulation of PKCδ by siRNA decreased significantly TTP expression in activated macrophages. Rottlerin also decreased TTP expression in wild type cells but not in cells in which PKCδ had been downregulated by siRNA. Rottlerin decreased TTP mRNA half-life as measured by actinomycin D assay but it did not affect the nuclear translocation of transcription factors NF-κB, Sp1, AP-2 or EGR1 (which have been shown to be involved in TTP transcription). In addition, rottlerin reduced the decay of TNFα mRNA, which is an important target of TTP. The results suggest that PKCδ up-regulates the expression of TTP by stabilizing its mRNA which may serve as a feed-back loop to regulate the inflammatory response.
Keywords: Macrophage; mRNA decay; Protein kinase Cδ; Rottlerin; TNFα (tumour necrosis factor-α); TTP (tristetraprolin);

Miltefosine is currently marketed for treatment of skin metastasis of breast cancer and leishmaniasis. The mechanism of action is not fully understood, however, miltefosine is considered to be a prototype lipid raft modulator. The compound was shown to inhibit anti-IgE induced histamine release from human skin mast cells. After topical treatment it reduced skin reaction in allergic human volunteers undergoing a skin prick test. The aim of this study was to test whether miltefosine could also modify T-cell signalling and whether the drug may be useful for the treatment of atopic dermatitis. Miltefosine (20 µM) inhibited T-cell proliferation by > 50% in the mixed leukocyte test. In the toluene diisocyanate induced ear swelling test, miltefosine, administered topically as 2 and 6% solution or orally, attenuated ear swelling reaching 70% of the effect of dexamethasone at 100 mg/kg p.o. (P  < 0.01). The ear tissue content of the cytokines IL1ß, IL4 and IL6 was also reduced reaching 56% or 52% reduction of IL1ß (P  < 0.01) after 2% topical or 100 mg/kg p.o. Miltefosine significantly attenuated the allergic sensitization in the model of ovalbumin induced delayed - type hypersensitivity in mice. In a model of toluene diisocyanate induced scratching a significant (P  = 0.0047) reduction of scratching from 47 to 6 bouts was achieved with 100 mg/kg p.o. The data indicate that miltefosine modulates T-cell function in models for Th1 and Th2 related activity. This profile opens up the possibility for the treatment of T-cell related allergic diseases with a novel class of lipid raft modulator drugs such as miltefosine.
Keywords: Atopic dermatitis; Delayed - type hypersensitivity; Immune modulator; T-cell; Plasmalogen-phospholipid; Pruritus; Toluene diisocyanate; Arachidonic acid;

Cryptomerione induces Th1 cell polarization via influencing IL-10 production by cholera toxin-primed dendritic cells by Masao Takei; Akemi Umeyama; Je-Jung Lee; Noboru Shoji; Toshihiro Hashimoto (233-239).
Dendritic cells play an important role in the initiation and regulation of immune response. Dendritic cells have a key influence in the differentiation of naïve T cells into Th1, Th2 or Th17 effector cells. Cryptomerione is terpene isolated from the heartwood of Cryptomeria japonica. In this study, we investigated the effects of Cryptomerione on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Human monocytes were exposed to either Cryptomerione alone, or in combination with lipopolysaccaride (LPS) or cholera toxin (CT) and thereafter co-cultured with naïve T cells. We found no enhanced CD1a, CD80, CD83, CD86 and HLA-DR expression on Cryptomerione-primed dendritic cells. However, Cryptomerione augmented T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction to CT-primed dendritic cells and influenced the production of interleukin (IL)-10 and IL-12p70 by CT-primed dendritic cells, but not LPS-primed dendritic cells. Cryptomerione also inhibited Th2 cell polarization induced by CT-primed dendritic cells, but enhanced IFN-γ secretion by naïve T cells co-cultured with CT-primed dendritic cells. Cytokine production by CT-primed dendritic cells alone, or in combination with Cryptomerione was also influenced following treatment with anti-IL-10 mAb and anti-OX40L mAb. Thus, the potential mechanisms underlying the enhancement of Th1 polarization in CT-primed dendritic cells induced by Cryptomerione appeared to depend on IL-10 secretion and OX40L. These results suggest that Cryptomerione modulates human dendritic cells function in a fashion that favors Th1/Th2 cell polarization.
Keywords: Dendritic cells; Cryptomerione; Cholera toxin; Th1 response;

Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H92-E97 region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the peptide corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function.
Keywords: Proteinase-activated receptor; Adherent peritoneal cell; Phagocytosis; Spreading; S100A9; (Mouse);

A synthetic curcuminoid derivative inhibits nitric oxide and proinflammatory cytokine synthesis by Chau Ling Tham; Choi Yi Liew; Kok Wai Lam; Azam-Shah Mohamad; Min Kyu Kim; Yoke Kqueen Cheah; Zainul-Amirudin Zakaria; Mohd-Roslan Sulaiman; Nordin H. Lajis; Daud A. Israf (247-254).
Curcumin is a highly pleiotropic molecule with significant regulatory effects upon inflammation and inflammatory related diseases. However curcumin has one major important limitation in which it has poor bioavailability. Design of synthetic structural derivatives of curcumin is but one approach that has been used to overcome its poor bioavailability while retaining, or further enhancing, its drug-like effects. We have synthesized a series of curcumin analogues and describe the effects of 2,6-bis-4-(hydroxyl-3-methoxy-benzylidine)-cyclohexanone or BHMC upon nitric oxide and cytokine synthesis in cellular models of inflammation. BHMC showed a significant dose–response inhibitory action upon the synthesis of NO and we have shown that this effect was due to suppression of both iNOS gene and enzyme expression without any effects upon scavenging of nitrite. We also demonstrated that BHMC has a very minimal effect upon iNOS activity with no effect at all upon the secretion of PGE2 but has a strong inhibitory effect upon MCP-1 and IL-10 secretion and gene expression. Secretion and gene expression of TNF-α and IL-6 were moderately inhibited whereas IL-8 and IL-1β were not altered. We conclude that BHMC selectively inhibits the synthesis of several inflammatory mediators. BHMC should be considered a promising drug lead for preclinical and further pharmacological studies.
Keywords: Cytokine; Chemokine; iNOS; Curcuminoid; Macrophage;

Anti-glycative effects of oleanolic acid and ursolic acid in kidney of diabetic mice by Zhi-hong Wang; Cheng-chin Hsu; Chien-ning Huang; Mei-chin Yin (255-260).
Inhibitory effects of oleanolic acid (OA) and ursolic acid (UA) on aldose reductase (AR) and glycative products in kidney of diabetic mice were examined. OA or UA at 0.05, 0.1 or 0.2% was supplied for 10 weeks. Diabetic mice with 0.1 or 0.2% OA or UA treatments had significantly higher body weight and lower kidney weight at weeks 5 and 10 (P  < 0.05). OA or UA intake at 0.1 or 0.2% increased their content in the kidney, dose-dependently decreased plasma glucose, HbA1c, renal N ε-(carboxymethyl)lysine, urinary glycated albumin and urinary albumin levels; elevated plasma insulin and renal creatinine clearance levels; as well as decreased renal sorbitol and fructose concentrations (P  < 0.05). OA or UA treatments at 0.1 and 0.2% also significantly diminished renal AR activity and dose-dependently down-regulated renal AR mRNA expression (P <  0.05). These two compounds at 0.2% significantly reduced renal sorbitol dehydrogenase activity (P  < 0.05). OA, not UA, treatments at 0.1 or 0.2% dose-dependently enhanced renal glyoxalase I (GLI) activity, up-regulated renal GLI mRNA expression and lowered renal methylglyoxal level (P <  0.05). Based on these marked anti-glycative effects, the supplement of OA or UA might be helpful for the prevention or alleviation of glycation associated renal diseases.
Keywords: Oleanolic acid; Ursolic acid; Glycation; Aldose reductase; Renal CML;

Biological activity of EXf, a peptide analogue of exendin-4 by Guang-ming Song; Yi Huan; Su-juan Sun; Yue-teng Chen; Quan Liu; Zhu-fang Shen (261-267).
Exendin-4 is an incretin mimetic that has been developed for the treatment of patients with type 2 diabetes. EXf is an available carboxy-terminal truncated fragment of exendin-4 with two amino acid substitutions. The purpose of these studies was to evaluate the biological activity of EXf. After a single subcutaneous injection, EXf significantly decreased plasma glucose concentration and glucose excursion following the administration of an oral glucose challenge both in non-diabetic (ICR), monosodium l-glutamate induced insulin resistance (MSG-IR) and diabetic KK-ay mice. Meanwhile, EXf resulted in an increase of first-phase insulin secretion in normal mice and KK-ay mice following the glucose challenge. EXf was also shown to inhibit small intestinal transit in rodent models. EXf activated the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1) GFP-construct in a dose-dependent manner in the cultured mouse insulinoma cell line, termed NIT-1, and this agonist activity was blocked by the glucagon-like peptide 1 (GLP-1) receptor antagonist exendin(9–39). In summary, EXf, an analogue of exendin-4, has agonist activity to GLP-1 receptor in vitro and glucoregulatory activities in vivo, thus it can be considered as a new candidate for the treatment of type 2 diabetes.
Keywords: Glucose tolerance; Insulin; NIT-1 cells; Incretin mimetic; (Mice);

Much recent attention has focused on the GLP-1 receptor as a potential target for antidiabetic drugs. Enzyme resistant GLP-1 mimetics such as exenatide are now employed for the treatment of type 2 diabetes, but must be administered by injection. The present study has examined and compared the in vitro and in vivo metabolic actions of a small molecule GLP-1 receptor agonist 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB), with native GLP-1, exenatide and liraglutide. DMB significantly stimulated in vitro insulin secretion from BRIN-BD11 cells but with decreased molar potency compared to native GLP-1 or related mimetics. Administration of DMB in combination with glucose to mice significantly (P  < 0.05) decreased the overall glucose excursion compared to controls. Exenatide and liraglutide evoked similar (P  < 0.001) reductions of the overall glycaemic excursion, but were significantly (P  < 0.001 and P  < 0.05; respectively) more effective than DMB. These observations were associated with prominently (P  < 0.05) enhanced glucose-mediated insulin release by exenatide and liraglutide, but not by DMB. Combined injection of DMB with either liraglutide or exenatide did not substantially improve glucose-lowering or insulin-releasing responses. However, administration of DMB in combination with exendin(9–39) did not impair its glucoregulatory actions. These results provide evidence to support the development and potential use of low molecular weight GLP-1 receptor agonists for the treatment of type 2 diabetes.
Keywords: Glucagon-like peptide-1 (GLP-1); Glucose homeostasis; Small molecule; Insulin secretion; Diabetes;

Curcumin protects rats against acetaminophen-induced hepatorenal damages and shows synergistic activity with N-acetyl cysteine by Ehsan Kheradpezhouh; Mohammad-Reza Panjehshahin; Ramin Miri; Katayoun Javidnia; Ali Noorafshan; Ahmad Monabati; Ahmad-Reza Dehpour (274-281).
Acetaminophen is one of the most popular analgesic and antipyretic drugs and its overdose, which can cause severe damage to liver and kidneys, is one of the most common reasons of emergency admissions. In this study we investigated the effects of curcumin, derived from plant Curcuma longa, on acetaminophen toxicity, and the possibility of combining therapy of curcumin and N-acetyl cysteine (NAC) to treat this toxicity. The experiments were conducted on 72 male Sprague–Dawley rats randomly divided into 12 groups. Control group was left without treatment, and the other groups were treated with different combinations of acetaminophen, curcumin and NAC. 15 min after intraperitoneal injection, the blood level of curcumin was measured using HPLC. Blood levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase), blood urea nitrogen and creatinine were determined 18 and 42 h after acetaminophen injection. One week later, the left kidney and the caudate lobe of the liver were harvested to assay glutathione peroxidase, catalase and malondialdehyde. The right kidney and the remaining lobes of the liver were used for histopathology. Analysis of organ function and oxidation parameters showed that curcumin significantly reduced toxic effects of acetaminophen on the liver and kidneys in a dose-dependent manner and significantly potentiated the protective effects of NAC. These findings were confirmed by histopathology. It is concluded that curcumin can protect the liver and kidney from the damage caused by acetaminophen overdose. Moreover, curcumin has the potential to be used in a combination therapy with NAC, significantly decreasing the therapeutic dose of NAC and therefore its side-effects.
Keywords: Acetaminophen; N-acetyl cysteine; Curcumin; Hepatorenal toxicity; (Rat);

Melatonin treatment is beneficial in pancreatic repair process after experimental acute pancreatitis by Shabir Sidhu; Promila Pandhi; Samir Malhotra; Kim Vaiphei; K.L. Khanduja (282-289).
Current treatment options for acute pancreatitis are supportive and symptomatic. Due to lack of agents targeting the underlying pathophysiology a large amount of experimental work is going on to identify novel therapeutic agents. The present study was carried out to explore if melatonin can modulate the spontaneous regeneration process of the pancreas after experimentally induced acute pancreatitis. Rats were given two i.p. injections of l-arginine in a dose of 200 mg/100 g at an interval of 1 h for induction of pancreatitis. After this rats were randomly divided into three groups i.e. saline, CCK-8 and melatonin. Drug treatment was started 2 h after the last l-arginine injection and continued till the day of sacrifice. An additional only saline treated control group was included for comparison. Animals in each group were sacrificed at 24 h, days 3, 14 and 28 after pancreatitis induction for determination of biochemical parameters (serum amylase, lipase and IL-10 and pancreatic amylase, total proteins and nucleic acid content) and histological examination. For rate of DNA synthesis and immunohistochemical studies animals were sacrificed at day 3 and day 7. Melatonin treatment was found to be beneficial in acute pancreatitis. Severity of acute pancreatitis was significantly reduced in melatonin group. Nucleic acid content, rate of DNA synthesis, pancreatic proteins and pancreatic amylase content were significantly improved. Histopathological examination showed significantly lower total scores in melatonin group. Results of melatonin group were comparable to that of positive control, CCK-8 group. Thus melatonin treatment was found to promote the spontaneous regeneration process of pancreatic tissue.
Keywords: Acute pancreatitis; Melatonin; Regeneration; Pancreatic stellate cell; Transforming growth factor-β; IL-10;