European Journal of Pharmacology (v.604, #1-3)

Identification of a novel 5-HT4 receptor splice variant (r5-HT4c1) and preliminary characterisation of specific 5-HT4a and 5-HT4b receptor antibodies by Alison M. Ray; Rosemary E. Kelsell; Jennifer A. Houp; Fiona M. Kelly; Andrew D. Medhurst; Helen M. Cox; Andrew R. Calver (1-11).
The human 5-hydroxytryptamine (5-HT4) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT4a (r5-HT4a), 5-HT4b (r5-HT4b) and 5-HT4e (r5-HT4e). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT4c1) and present the first comprehensive analysis of 5-HT4 splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT4 splice variant specific antibodies. In transfected cells, r5-HT4c1 receptor exhibited similar binding properties to r5-HT4a and r5-HT4b. Functional studies showed that 5-HT4 agonists prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (±)-endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT4c1, but full agonists at r5-HT4a and r5-HT4b. Moreover, in contrast to r5-HT4a and r5-HT4b, r5-HT4c1 was not constitutively active. TaqMan mRNA analysis showed that r5-HT4a expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT4b and r5-HT4c1. mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT4a and r5-HT4b specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT4b immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT4 splice variant function.
Keywords: 5-HT4 receptor; Novel splice variant; Selective antibody;

We investigated the effects of tumor necrosis factor (TNF)-α on DNA synthesis and proliferation, and its signal transduction pathways in primary cultures of adult rat hepatocytes. TNF-α induced time- and dose-dependent increases in hepatocyte DNA synthesis and proliferation. The hepatocyte proliferation stimulated by 30 ng/ml TNF-α was significantly inhibited by anti-TNF receptor 2 antibody, but not by anti-TNF receptor 1 antibody. TNF-α-induced hepatocyte DNA synthesis and proliferation were blocked by AG1478 (10− 7 M), PD98059 (10− 6 M), LY 294002 (10− 7 M), and rapamycin (100 ng/ml). TNF-α at 30 ng/ml significantly increased phosphorylation of receptor tyrosine kinase (175 kDa) and p42 mitogen-activated protein (MAP) kinase. This data suggests that the proliferative signal for primary cultured hepatocytes induced by TNF-α is mediated by TNF receptor 2 and the receptor tyrosine kinase/MAP kinase pathway. In addition, TNF-α-induced hepatocyte mitogenesis was significantly blocked by somatostatin (10− 6 M), adenylate cyclase inhibitor dideoxyadenosine (10− 7 M), protein kinase A inhibitor H-89 (10− 7 M), and neutralizing antibody to transforming growth factor (TGF)-α in culture. Indeed, 30 ng/ml TNF-α was found to rapidly stimulate secretion of TGF-α, and this secretion was also blocked by anti-TNF receptor 2 antibody. Moreover, TGF-α secretion induced by TNF-α was suppressed by dideoxyadenosine, H-89, and somatostatin. Together, these results indicate that stimulation of TNF receptor 2 by 30 ng/ml TNF-α induces autocrine secretion of TGF-α via the adenylate cyclase/protein kinase A pathway, after which TGF-α induces hepatocyte DNA synthesis and proliferation through the TGF-α receptor-linked tyrosine kinase (175 kDa)/MAP kinase signaling system.
Keywords: Tumor necrosis factor-α; DNA synthesis; Proliferation; Signal transduction; Transforming growth factor-α; Hepatocyte; (Adult rat);

Effects of (−) epigallocatechin-3-gallate on Na+ currents in rat dorsal root ganglion neurons by Tae Hoon Kim; Jae-Min Lim; Sung Su Kim; Jungho Kim; Mijung Park; Jin-Ho Song (20-26).
The natural product (−) epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent found in green tea. Dorsal root ganglion neurons are primary sensory neurons, and express tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ currents, which are both actively involved in the generation and propagation of nociceptive signals. Effects of EGCG on tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ currents in rat dorsal root ganglion neurons were investigated using the whole-cell variation of the patch-clamp techniques. EGCG inhibited both types of Na+ currents potently and in a concentration-dependent manner. The apparent dissociation constant, K d, was estimated to be 0.74 and 0.80 µM for tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ currents, respectively. (−) Epigallocatechin (EGC) was far less potent to inhibit Na+ currents than EGCG, suggesting that gallate moiety of EGCG is an important functional group to modulate Na+ currents. EGCG had little or no effect on the activation or steady-state inactivation voltage of either type of Na+ current. EGCG simply reduced the availability of Na+ channels for activation. Thus, EGCG appears to bind to resting Na+ channels to inhibit them. EGCG slowed the recovery of tetrodotoxin-sensitive Na+ current from inactivation. The property of EGCG to inhibit sensory Na+ currents can be utilized to develop an analgesic agent.
Keywords: Catechins; Dorsal root ganglion; (−) Epigallocatechin-3-gallate; Green tea; Na+ current; Tetrodotoxin-resistant; Tetrodotoxin-sensitive;

Mitogen-activated protein kinase 3/mitogen-activated protein kinase 1 activates apoptosis during testicular ischemia–reperfusion injury in a nuclear factor-κB-independent manner by Letteria Minutoli; Pietro Antonuccio; Francesca Polito; Alessandra Bitto; Francesco Squadrito; Vincenzo Di Stefano; Piero Antonio Nicotina; Carmine Fazzari; Daniele Maisano; Carmelo Romeo; Domenica Altavilla (27-35).
Nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase3/MAPK1 and MAPK8 are involved in testicular ischemia reperfusion injury (testicular-I/R). NF-κB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 activation and enhanced MAPK3/MAPK1 activity. To better understand the role of MAPK3/MAPK1 up-regulation during testicular-I/R, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO mice during testicular-I/R.KO and wild-type (WT) animals underwent 1 h testicular ischemia followed by 24 h reperfusion or a sham testicular-I/R. Animals received either PD98059 (5 mg/kg/ip) or its vehicle. MAPK3/MAPK1, BAX, caspase-3 and -9 and TNF-α expression were assessed along with histological examination and an immunostaining for protein of apoptosis.Testicular-I/R caused a greater increase in MAPK3/MAPK1 in KO than in WT animals in both testes. KO mice had a lower expression of the apoptotic proteins and TNF-α as well as reduced histological damage compared to WT. Immunostaining confirmed the lower expression of BAX in the Leydig cells of KO mice. Administration of PD98059, abrogated MAPK3/MAPK1 expression and slightly reduced TNF-α but did not improve or reverse the histological damage in KO. PD98059 significantly reduced the histological damage in WT mice and markedly reduced the apoptotic proteins in KO and WT mice.These results suggest that testicular-I/R triggers also a pathway of organ damage involving MAPK3/MAPK1, TNF-α, BAX, caspase-3 and -9 that activates an apoptotic machinery in an NF-κB independent manner. These findings should contribute to better understand testicular torsion-induced damage.
Keywords: MAPK3/MAPK1; BAX; Caspase 3 and 9; NF-κB; Testis torsion;

Treatment of depression may ameliorate the cognitive disability and motor slowness in Parkinson's disease. It has been shown that antidepressants, including fluoxetine, may attenuate or exacerbate neuronal cell death. The present study assessed the effect of antidepressants (amitriptyline, tranylcypromine and fluoxetine) against the toxicity of 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process in differentiated PC12 cells. Amitriptyline and tranylcypromine attenuated the MPP+-induced cell death that may be associated with mitochondrial membrane permeability change and oxidative stress. Both compounds prevented the loss of the mitochondrial transmembrane potential, over-expression of Bax, reduction in Bcl-2 level, cytochrome c release, caspase-3 activation, formation of reactive oxygen species and depletion of GSH. The inhibitory effect of tranylcypromine was greater than that of amitriptyline on the basis of concentration. In contrast, fluoxetine revealed a toxic effect and exhibited an additive effect against the toxicity of MPP+. Results show that amitriptyline and tranylcypromine may attenuate the MPP+ toxicity by suppressing the mitochondrial membrane permeability change that leads to cytochrome c release and subsequent caspase-3 activation. The effects seem to be associated with the inhibitory action on the formation of reactive oxygen species and the depletion of GSH. In contrast, fluoxetine seems to exert an additive toxic effect against neuronal cell damage by increasing mitochondrial damage and oxidative stress.
Keywords: Antidepressant; 1-Methyl-4-phenylpyridinium; Mitochondrial membrane permeability change; Cell death; Different effect;

We have recently observed that the corticotrophin releasing factor (CRF) related peptide urocortin reverses key features of nigrostriatal damage in two paradigms of Parkinson's disease. Here we have studied whether these effects are supported by a retention of striatal basal and evoked extracellular dopamine and the receptor(s) that may mediate this effect. Fourteen days following stereotaxic injections of 6-hydroxydopamine (6-OHDA) or lipopolysaccharide (LPS) and urocortin, extracellular dopamine levels in striata ipsilateral to injection sites of 6-OHDA/LPS and urocortin treated rats were comparable with sham injected rats, whilst rats given 6-OHDA/LPS and vehicle had considerably lower dopamine levels. Striatal dopamine levels in animals where urocortin injection was delayed by seven days were only modestly decreased compared to animals receiving 6-OHDA/LPS and urocortin concomitantly. Additionally, the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were also preserved in dialysates from urocortin treated rats. The effects of urocortin were entirely blocked by the non-selective CRF receptor antagonist α-helical CRF as well as the selective CRF1 antagonist NBI 27914 and were not replicated by the selective CRF2 ligand urocortin III. In the substantia nigra tissue dopamine changes mirrored those seen in striatal extracellular dopamine. Our data strongly suggest that urocortin is capable of maintaining adequate nigrostriatal function in vivo via CRF1 receptors following. neurotoxic challenge.
Keywords: Urocortin; 6-hydroxydopamine; Lipopolysaccaride; Dopamine; In vivo microdialysis;

Oxaloacetate restores the long-term potentiation impaired in rat hippocampus CA1 region by 2-vessel occlusion by Máté Marosi; János Fuzik; Dávid Nagy; Gabriella Rákos; Zsolt Kis; László Vécsei; József Toldi; Angela Ruban-Matuzani; Vivian I. Teichberg; Tamás Farkas (51-57).
Various acute brain pathological conditions are characterized by the presence of elevated glutamate concentrations in the brain interstitial fluids. It has been established that a decrease in the blood glutamate level enhances the brain-to-blood efflux of glutamate, removal of which from the brain may prevent glutamate excitotoxicity and its contribution to the long-lasting neurological deficits seen in stroke. A decrease in blood glutamate level can be achieved by exploiting the glutamate-scavenging properties of the blood-resident enzyme glutamate-oxaloacetate transaminase, which transforms glutamate into 2-ketoglutarate in the presence of the glutamate co-substrate oxaloacetate. The present study had the aim of an evaluation of the effects of the blood glutamate scavenger oxaloacetate on the impaired long-term potentiation (LTP) induced in the 2-vessel occlusion ischaemic model in rat. Transient (30-min) incomplete forebrain ischaemia was produced 72 h before LTP induction. Although the short transient brain hypoperfusion did not induce histologically identifiable injuries in the CA1 region (Fluoro-Jade B, S-100 and cresyl violet), it resulted in an impaired LTP function in the hippocampal CA1 region without damaging the basal synaptic transmission between the Schaffer collaterals and the pyramidal neurons. This impairment could be fended off in a dose-dependent manner by the intravenous administration of oxaloacetate in saline (at doses between 1.5 mmol and 0.1 μmol) immediately after the transient hypoperfusion. Our results suggest that oxaloacetate-mediated blood and brain glutamate scavenging contributes to the restoration of the LTP after its impairment by brain ischaemia.
Keywords: Glutamate neurotoxicity; Hypoperfusion; 2-vessel occlusion; Oxaloacetate; Glutamate-scavenging; LTP;

The selective non-peptidic delta opioid agonist SNC80 does not facilitate intracranial self-stimulation in rats by Gail Pereira Do Carmo; John E. Folk; Kenner C. Rice; Elena Chartoff; William A. Carlezon; S. Stevens Negus (58-65).
Delta opioid receptor agonists are under development for a variety of clinical applications, and some findings in rats raise the possibility that agents with this mechanism have abuse liability. The present study assessed the effects of the non-peptidic delta opioid agonist SNC80 in an assay of intracranial self-stimulation (ICSS) in rats. ICSS was examined at multiple stimulation frequencies to permit generation of frequency-response rate curves and evaluation of curve shifts produced by experimental manipulations. Drug-induced leftward shifts in ICSS frequency-rate curves are often interpreted as evidence of abuse liability. However, SNC80 (1.0–10 mg/kg s.c.; 10–56 mg/kg i.p.) failed to alter ICSS frequency-rate curves at doses up to those that produced convulsions in the present study or other effects (e.g. antidepressant effects) in previous studies. For comparison, the monoamine releaser d-amphetamine (0.1–1.0 mg/kg, i.p.) and the kappa agonist U69,593 (0.1–0.56 mg/kg, i.p.) produced dose-dependent leftward and rightward shifts, respectively, in ICSS frequency-rate curves, confirming the sensitivity of the procedure to drug effects. ICSS frequency-rate curves were also shifted by two non-pharmacological manipulations (reductions in stimulus intensity and increases in response requirement). Thus, SNC80 failed to facilitate or attenuate ICSS-maintained responding under conditions in which other pharmacological and non-pharmacological manipulations were effective. These results suggest that non-peptidic delta opioid receptor agonists have negligible abuse-related effects in rats.
Keywords: Delta opioid receptor agonists; SNC80; Amphetamine; U69,593; Intracranial self-stimulation;

Glucocorticoids, secreted by the adrenals in response to stress, have profound effects on behavioural responsiveness to psychostimulant drugs. We have studied the critical time-window for the influence of corticosterone on behavioural sensitisation to cocaine in relation to i) the stage of behavioural sensitisation, and ii) the time of drug exposure. Previously, we have identified a mouse strain (DBA/2) in which surgical removal of the adrenals (adrenalectomy) fully prevented locomotor sensitisation to cocaine. To investigate the role of corticosterone in expression of behavioural sensitisation, the glucocorticoid receptor antagonist mifepristone (RU38486) was administered to previously sensitised mice prior to a cocaine challenge. Furthermore, adrenalectomised mice were given corticosterone replacement at different intervals prior to each drug administration, to investigate the role of the glucocorticoid in initiation of behavioural sensitisation, and in relation to the time of drug exposure. Administration of mifepristone to previously sensitised animals failed to block expression of cocaine-induced behavioural sensitisation. In adrenalectomised mice, intermittent replacement of corticosterone (1 mg/kg i.p., either 2 h or 5 min prior to each cocaine administration), did not reverse the sensitisation deficit. By contrast, chronic corticosterone replacement (20% pellet) partially restored initiation of behavioural sensitisation. These data indicate that the presence of corticosterone facilitates the initiation rather than the expression of behavioural sensitisation to cocaine. However, because high corticosterone concentrations only partially reversed the sensitisation deficit of adrenalectomised mice, the adrenal glucocorticoid seems necessary, but not sufficient, for full behavioural sensitisation to cocaine in the DBA/2 strain.
Keywords: Corticosterone; HPA axis; Adrenalectomy; Stress; Psychostimulant; Locomotion;

Serotonin 1B (5-HT1B) receptors may play a role in regulating motivation and reward-related behaviours. To date, no studies have investigated the effects of the highly selective 5-HT1B receptor agonist CP 94253, on the reward model of ventral tegmental area intracranial self-stimulation. The current study investigated the hypothesis that 5-HT1B receptors play an inhibitory role in ventral tegmental area ICSS. Using Sprague–Dawley rats, the effects of the selective 5-HT1B receptor agonist CP 94253 (0–5.0 mg/kg) and the 5-HT1B/1D receptor antagonist GR 127935 (10.0 mg/kg) were investigated in rats trained to respond for ventral tegmental area ICSS; results were compared using rate-frequency threshold analysis. The highest dose of CP 94253 (5.0 mg/kg) tested in ventral tegmental area ICSS produced an increase in rate-frequency thresholds without affecting maximal response rates. This effect was attenuated by GR 127935 which did not show any effects when administered alone. These results suggest that 5-HT1B receptors play an inhibitory role in regulating ventral tegmental area ICSS.
Keywords: Reward; Mesolimbic; Serotonin; Intracranial Self-stimulation; 5-HT1B receptor; (Rat);

The mechanisms that subserve ghrelin-evoked vasodilatation have not been elucidated in previous studies. Changes in perfusion pressure evoked by ghrelin and its N-terminal fragments were examined ex vivo in phenylephrine-constricted perfused mesenteric vascular beds of male Sprague Dawley rats maintained at a constant flow rate. Both ghrelin (maximum effect [E max] 45%) and des-acyl ghrelin (E max 43%) evoked vasodilatation at concentrations between 10 pM and 1 nM, compared to acetylcholine (median effective concentration [EC50] 3 nM; E max 93%). Those responses were abolished in endothelium-denuded preparations, and in endothelium-intact preparations exposed to either calcium-activated potassium channel, or a depolarizing stimulus, or in the presence of a combination of either apamin and 1,2-chlorophenyl diphenylmethyl-1 H-pyrazole (triarylmethane-34 [TRAM-34]), or ouabain and barium. ATP-activated potassium channel blockade, or a combination of nitric oxide synthase and cyclooxygenase inhibition had no effect. The classical growth hormone secretagogue antagonist, [d-Lys3]-growth hormone-releasing peptide (10 nM), or several N-terminal fragments of des-acyl ghrelin, including the tripeptide glycine–serine–serine (G–S–S [1 nM]), showed endothelium-dependent vasodilatation like des-acyl ghrelin, while responses to glycine–serine or serine–serine were relatively lower. A higher concentration (100 μM) of l-serine, but not glycine, evoked vasodilatation of similar magnitude. The serine dense N-terminal sequence of des-acyl ghrelin mediates endothelium-dependent vasodilatation via activation of apamin + TRAM-34 sensitive small- and intermediate-conductance calcium-activated potassium channels present on the mesenteric endothelium. Thus, the vasodilator response to ghrelins in the perfused rat mesenteric vascular bed is not mediated by the classical growth hormone secretagogue receptor type 1a.
Keywords: Acetylcholine; Calcium-activated potassium channel; Des-acyl ghrelin; Endothelium; Ghrelin; l-serine; Mesenteric vascular bed; Phenylephrine; Vasodilatation;

Administration of angiotensin II, but not catecholamines, induces accumulation of lipids in the rat heart by Makiko Hongo; Nobukazu Ishizaka; Kyoko Furuta; Naoya Yahagi; Kan Saito; Ryota Sakurai; Gen Matsuzaki; Kazuhiko Koike; Ryozo Nagai (87-92).
Accumulation of lipids in the heart may cause cardiac dysfunction in various disorders, such as obesity and diabetes. In the current study, we have investigated whether administration of angiotensin II or norepinephrine induces accumulation of lipids and/or changes in the expression of genes related to lipid metabolism in the rat heart. Lipid deposition was found in myocardial, vascular wall, and perivascular cells of the angiotensin II-infused rat heart, and superoxide generation was increased in these lipid-positive cells. By contrast, intracardiac lipid deposition was not found in the heart of norepinephrine-induced hypertensive rats. Triglyceride content in the heart tissue of angiotensin II-infused rats increased more than 3-fold as compared with untreated controls. Losartan completely, but hydralazine only partially, suppressed the angiotensin II-induced intracardiac lipid deposition and increase in tissue triglyceride content. Administration of angiotensin II upregulated the mRNA expression of sterol regulatory element-binding protein-1c and fatty acid synthase, but downregulated that of uncoupling protein 2 and 3, in a manner dependent on the angiotensin AT1 receptor. Collectively, these results suggest that angiotensin II may be involved in modulating both intracardiac lipid content and lipid metabolism-related gene expression, in part via an angiotensin AT1 receptor-dependent and pressor-independent mechanism.
Keywords: Angiotensin II; Lipid accumulation; Lipotoxicity; Gene expression;

Stabilizing effects of eicosapentaenoic acid on Kv1.5 channel protein expressed in mammalian cells by Shunya Koshida; Yasutaka Kurata; Tomomi Notsu; Yutaka Hirota; Ting Y. Kuang; Peili Li; Udin Bahrudin; Shingo Harada; Junichiro Miake; Yasutaka Yamamoto; Yoshiko Hoshikawa; Osamu Igawa; Katsumi Higaki; Masaaki Soma; Akio Yoshida; Haruaki Ninomiya; Goshi Shiota; Yasuaki Shirayoshi; Ichiro Hisatome (93-102).
We investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the stability of Kv1.5 channel protein. The expression and function of Kv1.5 (Kv1.5-FLAG) in transfected African green monkey kidney fibroblast cells as well as rat atrium were estimated by immunoblotting, immunoprecipitation, immunofluorescence and patch-clamp techniques. Both EPA and DHA immediately blocked Kv1.5 channel current in a dose-dependent manner, accompanied by reduction of their phosphorylation. Chronic treatment (for 12 h) with EPA at lower concentrations (0.3–10 μM) increased the level of Kv1.5-FLAG protein as well as Kv1.5 channel current without changes in its gating kinetics, prolonging its half-life; in contrast, both EPA and DHA at higher concentrations (30–100 μM) decreased the expression of Kv1.5-FLAG. EPA at the higher concentrations also decreased mRNA of Kv1.5 and synapse-associated protein 97 expression. EPA at the lower concentrations increased Kv1.5 expression in the endoplasmic reticulum, Golgi apparatus and cell membrane. EPA-induced increase of Kv1.5 channel expression and current was abolished by pretreatment with the protein transport inhibitor brefeldin A or colchicines, and by the Kv1.5 channel blocker 4-aminopyridine. Oral administration of EPA (30 mg/kg) increased the level of endogenous Kv1.5 in rat atria. These results indicate that chronic treatment with EPA at lower concentrations stabilizes Kv1.5 channel protein in the endoplasmic reticulum and Golgi apparatus thereby enhancing the Kv1.5 channel current on the cell membrane.
Keywords: EPA; DHA; Kv1.5; Ultra-rapid delayed-rectifier K+ channel current;

Androgens elicit an acute cardiotonic effect in cardiac preparations of rats. This effect is produced via an extracellular interaction that may be coupled to pertussis-sensitive G-proteins and is associated with an increase in cAMP, polyamine synthesis and intracellular calcium. The nature of the targets and the existence of a dimorphic effect in this nongenomic effect of androgens are unknown. The purpose of this study was to characterize a possible gender and sex hormone influence on the 5α-dihydrotestosterone-elicited cardiotonic effect, taking into account the possible role of the β-adrenoceptors and ornithine decarboxylase activity on this response. [Float1]Regarding this, the effect of 5α-dihydrotestosterone on isolated left atria from male, estrogenized female and gonadectomized male and female rats was studied. The results showed that 5α-dihydrotestosterone-elicited cardiotonic effect was preserved independent of gender and sex hormones, being higher in control males than in the rest of the groups. This correlated with the testosterone plasma levels, except in estrogenized females, suggesting that the androgens positively and the estrogens negatively regulated the response. In all groups, 5α-dihydrotestosterone produced an increase in cAMP levels, but only in control males did it produce an increase in ornithine decarboxylase activity. In the other groups, the absence of an effect on ornithine decarboxylase might limit the capability of the response to the androgen. Altogether, androgens may help to control cardiac performance by a direct interaction on the heart in both sexes. Gender and sex differences in the magnitude of inotropism being due mainly to changes in β-adrenoceptors and cAMP production and in intracellular polyamine synthesis.
Keywords: Androgens; 5α-dihydrotestosterone; Atria; Heart; β-adrenoceptors; cAMP; Ornithine decarboxylase;

Resveratrol pretreatment can protect the heart by inducing pharmacological preconditioning. Whether resveratrol protects the heart when applied at reperfusion remains unknown. We examined the effect of resveratrol on myocardial infarct size when given at reperfusion and investigated the mechanism underlying the effect. Isolated rat hearts were subjected to 30 min ischemia followed by 2 h of reperfusion, and myocardial samples were collected from the risk zone for Western blot analysis. Mitochondrial swelling was spectrophotometrically measured as a decrease in absorbance at 520 nm (A 520). Resveratrol reduced infarct size and prevented cardiac mitochondrial swelling. Resveratrol enhanced GSK-3β phosphorylation upon reperfusion, an effect that was mediated by the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway. Resveratrol translocated GSK-3β from cytosol to mitochondria via the cGMP/PKG pathway. Further studies showed that mitochondrial GSK-3β was co-immunoprecipitated with cyclophilin D but not with VDAC (voltage dependent anion channel) or ANT (adenine nucleotide translocator). These data suggest that resveratrol prevents myocardial reperfusion injury presumably by targeting the mPTP through translocation of GSK-3β from cytosol to mitochondria. Translocated GSK-3β may ultimately interact with cyclophilin D to modulate the mPTP opening.
Keywords: Resveratrol; Reperfusion injury; Glycogen synthase kinase 3β; Mitochondrial permeability transition pore;

S 35171 exerts protective effects in spontaneously hypertensive stroke-prone rats by preserving mitochondrial function by Paolo Gelosa; Cristina Banfi; Maura Brioschi; Elena Nobili; Anita Gianella; Uliano Guerrini; Alice Pignieri; Elena Tremoli; Luigi Sironi (117-124).
S 35171 is one of a family of compounds that have been designed to protect mitochondrial function. We tested the hypothesis that S 35171 exerts protective effects in spontaneously hypertensive stroke-prone rats (SHRSPs), an animal model developing spontaneous brain damage preceded by proteinuria and systemic inflammation revealed by the urinary accumulation of acute-phase proteins (APPs) originating in the liver. Male SHRSPs fed a permissive diet received vehicle or S 35171 (10 mg/kg/day) started simultaneously with a high-sodium diet (group A) or after the establishment of proteinuria (group B). The drug delayed urinary APPs accumulation and the appearance of magnetic resonance imaging (MRI)-monitored brain lesions (after 62 ± 3 days in group A, and 51 ± 2 days in controls, P  < 0.01). The delay was more pronounced in group B as 30% of the animals survived the entire 90-day experimental period without brain abnormality. Proteomic analysis showed no significant alteration in the expression pattern of brain mitochondrial proteins, but the liver mitochondrial levels of carbamoylphosphate synthase I (CPS-I), an enzyme involved in urea metabolism) and the antioxidant peroxiredoxin-3 spot were affected by hypertension and S 35171. Stress reduces CPS-I and induces the peroxiredoxin-3 spot, whereas S 35171 brought about normal CPS-I expression and a 12-fold higher level of the peroxiredoxin-3 spot. As both enzymes are involved in maintaining mitochondrial functions, their increased expression after S 35171 treatment may be responsible for delaying the pathological condition that leads to the development of brain damage in SHRSPs.
Keywords: Cerebral ischemia; Stroke-prone rats; Mitochondria; Proteome;

Comparative therapeutic effects of metformin and vitamin E in a model of non-alcoholic steatohepatitis in the young rat by Giuseppina Mattace Raso; Emanuela Esposito; Anna Iacono; Maria Pacilio; Salvatore Cuzzocrea; Roberto Berni Canani; Antonio Calignano; Rosaria Meli (125-131).
Only in the last few years has non-alcoholic steatohepatitis been recognized as an important and relatively common liver disease. To date, the therapeutic options are limited, vitamin E and metformin have been proposed for the treatment of this condition, although their mechanisms are not completely clarified as yet. The aim of this study was to investigate the anti-inflammatory and anti-oxidative mechanisms of these drugs in an experimental model of non-alcoholic steatohepatitis in the young rat. Male rats, just after weaning, were divided into four groups: a control group that received a standard diet; a high fat diet group; two high fat diet fed groups treated with vitamin E or metformin, respectively. After 4 weeks, we evaluated in the liver the modification of lipid peroxidation, assessed by malondialdehyde, TNF-α levels, S-nitrosylated protein, inducible nitric oxide sinthase (iNOS), and peroxisome proliferators-activated receptors (PPAR) expression and metalloproteinase activity. High fat diet increased malondialdehyde, nitrotyrosilated proteins, and TNF-α tissue content. Moreover, a decrease of PPAR-α and an increase of PPAR-γ expression were observed. An increase of metalloproteinase activity was also shown. Among drug treatments, metformin reduced body weight gain and fat mass, metalloproteinase activity, and TNF-α tissue content, while it restored PPAR-α expression and downregulated PPAR-γ expression. Vitamin E reduced the oxidative damage, protein nitrotyrosilation, and tissue TNF-α levels. Moreover a decrease of PPAR-γ expression was also shown. These findings confirm the efficacy of both drugs as therapeutic tools in preventing the early onset of liver damage and non-alcoholic fatty liver disease progression.
Keywords: Steatohepatitis; High fat diet; Metformin; Vitamin E; Pro-inflammatory mediator; Peroxisome proliferator-activated receptor;

Effect of the ghrelin receptor agonist TZP-101 on colonic transit in a rat model of postoperative ileus by Graeme L. Fraser; Kalina Venkova; Hamid R. Hoveyda; Helmut Thomas; Beverley Greenwood-Van Meerveld (132-137).
Ghrelin, the natural ligand of the growth hormone secretagogue receptor (ghrelin receptor), is an orexigenic gut hormone with prokinetic action in the upper gastrointestinal tract. Previously we have shown in a rodent model of postoperative ileus that the synthetic ghrelin receptor agonist TZP-101 prevents the delay in gastric emptying and improves small intestinal transit. The goal of the present study was to investigate whether TZP-101 affects colonic transit and food intake in rats with postoperative ileus. Fasted rats were treated with morphine and subjected to laparotomy under isoflurane anesthesia. Following surgery the animals were placed in clean home cages and fecal pellet output and food intake were monitored for 48 h. TZP-101 or vehicle were administered as 3 i.v. bolus infusions at 0 h, 2 h and 4 h post-surgery. TZP-101 (0.03–1 mg/kg) dose-dependently decreased the time to first bowel movement and increased fecal pellet output measured at 12 h and 24 h post-surgery compared to the vehicle. The administration of TZP-101 was not associated with a significant alteration in food intake. In conclusion, this study provides the first experimental evidence that a novel ghrelin receptor agonist improves large bowel function in rats with postoperative ileus, suggesting that TZP-101 may be useful in the clinic to accelerate upper gastrointestinal transit and to shorten the time to the first bowel movement following surgery.
Keywords: Ghrelin receptor; TZP-101; Postoperative ileus; Colonic transit; Fecal pellet output; (Rat);

Erythropoietin suppresses peritoneal fibrosis in rat experimental model by Stefania Mondello; Emanuela Mazzon; Rosanna Di Paola; Concetta Crisafulli; Domenico Italiano; Michele Buemi; Calmela Aloisi; Salvatore Cuzzocrea (138-149).
Peritoneal dialysis (PD) is an alternative treatment of patients with end-stage renal disease. Unfortunately, long term peritoneal dialysis causes injury of the peritoneum associated with ultra filtration failure. Erythropoietin (EPO) is a potent stimulator of elytroid progenitor cells and its expression is enhanced by hypoxia. The aim of the present study was to evaluate the effect of EPO on the development of experimental peritoneal fibrosis induced by chlorhexidine gluconate (CG). A peritoneal fibrosis model was established using rats treated intraperitoneally with injection of CG. EPO was administered at the dose of 5000 U/kg i.p. three time per week for three weeks. When compared to CG-treated rats, EPO (5000 U/kg i.p. three time per week for three weeks) treated rats subjected to GC-induced peritoneal fibrosis experienced a significantly lower rate in the extent and severity of the histological signs of peritoneal injury. EPO also caused a substantial reduction of (i) the rise in myeloperoxidase activity (mucosa), (ii) the expression in the tissue of TNF-α, TGFβ and VEGF (iii) the increase in staining (immunohistochemistry) for nitrotyrosine and for PAR, as well as (iv) the NF-κB activation caused by CG in the peritoneum. Thus, EPO treatment reduces the degree of peritoneal fibrosis caused by CG. We propose that this evidence may help to clarify the potential therapeutic actions of EPO in patients with peritoneal fibrosis.
Keywords: Peritoneal dialysis; Erythropoietin (EPO); Peritoneal fibrosis; Oxidative stress;

Mouse Beta-TC6 insulinoma cells possessing nicotinic receptor [Ohtani, M., Oka, T., Badyuk, M., Xiao, Y., Kellar, KJ., Daly, JW., 2006. Mouse β-TC6 insulinoma cells: high expression of functional α3β4 nicotinic receptors mediating membrane potential, intracellular calcium, and insulin release. Mol. Pharmacol. 69, 899–907.] also expressed M3 and M4 muscarinic receptors. Carbamylcholine, a mixed muscarinic/nicotinic receptor agonist, or oxotremorine M, a selective muscarinic agonist, elicited an elevation of cytoplasmic Ca2+ concentration ([Ca2+]i) and release of insulin. The maximal [Ca2+]i response induced by carbamylcholine was larger than that of oxotremorine M or that of nicotine, suggesting that carbamylcholine enhanced the [Ca2+]i response by stimulating two types of receptor. M3 and M4 muscarinic receptor antagonists inhibited the [Ca2+]i responses to carbamylcholine and oxotremorine M, suggesting the involvement of these muscarinic receptors in the regulation of [Ca2+]i. In addition, pretreatment with carbamylcholine inhibited the [Ca2+]i responses to oxotremorine M or nicotine, indicating that the effect of carbamylcholine on [Ca2+]i was mediated by both muscarinic and nicotinic receptors. A phospholipase C (PLC) inhibitor U73122, a protein kinase C (PKC) inhibitor chelerythrine and a phospholipase A2 (PLA2) inhibitor AACOCF3 inhibited the [Ca2+]i response to carbamylcholine or oxotremorine M, while these inhibitors did not block the effect of nicotine. Carbamylcholine induced a smaller extent of insulin secretion than oxotremorine M, suggesting that concomitant stimulation of muscarinic and nicotinic receptors by carbamylcholine resulted in the negative type of the receptor interaction.
Keywords: Calcium; Carbamylcholine; Muscarinic receptor; Insulin secretion; Oxotremorine M; Nicotine;