European Journal of Pharmacology (v.621, #1-3)

Curcumin is a potent inhibitor of the transcription factor activator protein-1 which plays an essential role in osteoclastogenesis. However, the effects of curcumin on bone metabolism have not been clarified in vivo. We reported herein the inhibitory effects of curcumin on the stimulated osteoclastic activity in insulin-dependent diabetes mellitus using rats with streptozotocin-induced diabetes. A dietary supplement of curcumin reversed the increase in levels of activity and mRNA of tartrate-resistant acid phosphatase (TRAP) and cathepsin K to control values. A histochemical analysis showed that the increase in TRAP-positive cells in the distal femur of the diabetic rats was reduced to the control level by the supplement. These results suggested that curcumin reduced diabetes-stimulated bone resorptive activity and the number of osteoclasts.When bone marrow cells were cultured with macrophage colony stimulating factor and receptor activator NF-κB ligand (RANKL), the increased activity to form TRAP-positive multinucleated cells and the increased levels of mRNA and protein of c-fos and c-jun in the cultured cells from diabetic rats decreased to control levels in the curcumin-supplemented rats. Similarly, the increased expression of c-fos and c-jun in the distal femur of the diabetic rats was significantly reduced by the supplement. These results suggested that curcumin suppressed the increased bone resorptive activity through the prevention of osteoclastogenesis associated with inhibition of the expression of c-fos and c-jun in the diabetic rats.
Keywords: Curcumin; Diabetes mellitus; Osteoclastogenesis; AP-1 (activator protein-1); c-fos; c-jun;

The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are metabolised by cells by hydrolysis to arachidonic acid followed by esterification into phospholipids. Here, we report that nitric oxide (NO) donors significantly increase the amount of tritium accumulated in the cell membranes of RBL2H3 rat basophilic cells, 3T3-L1 mouse fibroblast cells and b.End5 mouse brain endothelioma cells following incubation of the intact cells with AEA labelled in the arachidonate part of the molecule. Similar results were seen with 2-AG and with arachidonic acid, whilst the NO donors reduced the accumulation of tritium after incubation of RBL2H3 cells with AEA labelled in the ethanolamine part of the molecule. Pretreatment of intact cells with NO donors did not increase the activity of the enzyme mainly responsible for metabolism of AEA, fatty acid amide hydrolase (FAAH). Furthermore, inhibition of FAAH completely blocked the effect produced by NO donors in cells with a large FAAH component, suggesting that for AEA, the effects were downstream of the enzyme. These data raise the possibility that the cellular processing of endocannabinoids following its uptake can be regulated by nitric oxide.
Keywords: Endocannabinoid; Anandamide; Arachidonic acid; Nitric oxide; Membrane accumulation; Fatty acid amide hydrolase;

Pharmacological effects of carvedilol on T-type calcium current in murine HL-1 cells by Chunyu Deng; Fang Rao; Shulin Wu; Sujuan Kuang; Xiaoying Liu; Zhiling Zhou; Zhixin Shan; Qiuxiong Lin; Weimin Qian; Min Yang; Qingshan Geng; Youyi Zhang; Xiyong Yu; Shuguang Lin (19-25).
Carvedilol is widely used in the treatment of cardiovascular diseases including atrial fibrillation. T-type Ca2+ channels have been recognized recently in the mechanisms underlying atrial arrhythmias. However, it is unclear whether carvedilol may affect the T-type Ca2+ channel. The present study evaluated the pharmacological effects of carvedilol on T-type calcium current (I Ca,T) in the murine HL-1 cell line. I Ca ,T was recorded by the whole-cell patch–clamp technique. Calcium transient was monitored by the fluorescent dye Fluo-4/AM and confocal laser scanning microscopy. Carvedilol reversibly inhibited I Ca ,T in a concentration-dependent manner, with an IC50 of 2.1 µM. 3 µM carvedilol was found to decrease the peak I Ca ,T amplitude at − 20 mV from 20.1 ± 1.8 pA/pF to 10.9 ± 2.1 pA/pF. Carvedilol significantly shifted the steady-state inactivation curve of I Ca ,T towards more negative potential by 12.8 mV, while the activation curve was not significantly altered. Carvedilol delayed recovery from inactivation of I Ca ,T, time constant (τ) was 112.4 ± 3.5 ms in control and 270.1 ± 4.7 ms in carvedilol. Carvedilol-induced inhibition rate in I Ca ,T was enhanced with the increase in stimuli frequency, the inhibitory rate was 23.2 ± 4.1% at 0.2 Hz and 47.2 ± 0.6% at 2 Hz. Carvedilol still produced the significant decrease in the amplitude of I Ca ,T in the presence of H-89, PKA inhibitor. Carvedilol significantly inhibited the amplitude of the calcium transient in a concentration-dependent manner. These findings indicate that carvedilol inhibits I Ca ,T in atrial cells by mechanisms involving preferential interaction with the inactivated state of T-type Ca2+ channel.
Keywords: Carvedilol; T-type Calcium Current (I Ca ,T); HL-1 cells; Whole-cell patch–clamp technique;

Protection by taurine of rat brain cortical slices against oxygen glucose deprivation- and reoxygenation-induced damage by Lorenzo Ricci; Massimo Valoti; Giampietro Sgaragli; Maria Frosini (26-32).
Taurine neuroinhibitory features have suggested its potential for neuroprotection. The aim of the present study was to assess whether it prevents or counteracts brain ischemia and reperfusion-induced cell injury. Rat brain cortical slices were subjected to oxygen/glucose deprivation and reperfusion. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, taken as an index of cell swelling. When added during the reperfusion period taurine did not significantly affect oxygen/glucose deprivation-induced LDH and glutamate release, while it antagonised tissue water gain in a concentration-dependent manner (IC50  = 46.5 µM). The latter effect was antagonised by 50% when a taurine transport inhibitor, 2-(guanidino)ethanesulphonic acid (GES), or a GABAA receptor antagonist, bicuculline, was added together with taurine, while it was completely abolished when both GES and bicuculline or the volume-sensitive outwardly rectifying (VSOR) Cl channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), was used. On the contrary, when present throughout the entire experiment, taurine significantly reduced oxygen/glucose deprivation-induced LDH and glutamate release with a maximal effect (45% reduction) between 5 and 20 mM. Taurine antagonised also tissue water gain according to a “U-shaped” concentration–response curve, which was significant within the range of 0.01–1.0 mM concentration. This effect was partially counteracted by GES as well as by bicuculline and fully reverted by NPPB. In conclusion, since brain edema is a major contributing factor to morbidity and mortality in stroke, the present findings give the rational basis for assessing taurine efficacy in reducing brain edema in vivo.
Keywords: Taurine; Brain ischemia; GABA-ergic system; Brain edema; Neuroprotection;

Modulatory effects of probenecid on the nitroglycerin-induced changes in the rat caudal trigeminal nucleus by Eniko Vamos; Arpad Pardutz; Annamaria Fejes; Janos Tajti; Jozsef Toldi; Laszlo Vecsei (33-37).
Four hours after systemic administration of the nitric oxide donor nitroglycerin (10 mg/kg bodyweight, s.c.), the neurons of the rat caudal trigeminal nucleus are activated, the area covered by calcitonin gene-related peptide (CGRP)-immunoreactive fibres is decreased and the neuronal nitric oxide synthase (nNOS)- and the calmodulin-dependent protein kinase II alpha (CamKIIα)-immunopositive neurons in the same area are increased. Probenecid is a non-selective inhibitor of multidrug-resistance associated proteins and organic anion transporters thus it can modulate the transport functions in the central nervous system influencing nociception. Accordingly, the aim of the present experiments was to examine the effects of probenecid administration on the nitroglycerin-induced expressions of nNOS, CamKIIα and CGRP in the rat caudal trigeminal nucleus. Probenecid (200 mg/kg bodyweight, i.p.) pretreatment proved to mitigate the nitroglycerin-induced changes in expression in the rat caudal trigeminal nucleus. The data suggest that the changes caused by nitroglycerin in the expressions of CGRP, nNOS and CamKIIα can be influenced by probenecid modulating the inflammatory functions in the nervous system. These data may be of relevance for the pathogenesis of migraine headache.
Keywords: Probenecid; Nitroglycerin; Neuronal nitric oxide synthase; Calmodulin-dependent protein kinase II alpha; Calcitonin gene-related peptide; Caudal spinal trigeminal nucleus;

l-3-n-butylphthalide improves cognitive impairment induced by intracerebroventricular infusion of amyloid-β peptide in rats by Ying Peng; Changhong Xing; Shaofeng Xu; Cynthia A. Lemere; Guiquan Chen; Bin Liu; Ling Wang; Yipu Feng; Xiaoliang Wang (38-45).
Alzheimer's disease is the most common form of dementia. Amyloid-β protein is considered as a key factor of pathogenesis of Alzheimer's disease. l-3-n-butylphthalide (L-NBP), an anti-cerebral ischemia drug, has been shown to have therapeutic effects in vascular dementia animal models. In the present study, we investigated the potential of L-NBP to protect against cognitive impairment, oxidative damage and neuropathological changes induced by intracerebroventricular infusion of amyloid-β peptide in rats. Daily treatments of 10 and 30 mg/kg L-NBP significantly improved spatial learning deficits and attenuated working memory deficits in Morris water maze task. L-NBP partially reversed the reduction of glutathione peroxidase activities and decreased malondialdehyde levels in the cortex and hippocampus. Furthermore, L-NBP markedly inhibited amyloid-β-induced neuronal apoptosis, possibly by blocking caspase-3 activation. In addition, L-NBP reduced activation of glycogen synthase kinase-3β and tau protein phosphorylation. Our results demonstrate that L-NBP protects against amyloid-β-induced neurodegeneration and cognitive decline in a rat model, suggesting that it may have potential as a therapy for Alzheimer's disease.
Keywords: l-3-n-butylphthalide (L-NBP); Amyloid-β; Learning and memory deficits; Alzheimer's disease; Tau protein phosphorylation;

Flavonoid quercetin protects against swimming stress-induced changes in oxidative biomarkers in the hypothalamus of rats by Nagaraja Haleagrahara; Ammu Radhakrishnan; Nagarajah Lee; Ponnusamy Kumar (46-52).
Quercetin is a bioflavonoid abundant in onions, apples, tea and red wine and one of the most studied flavonoids. Dietary quercetin intake is suggested to be health promoting, but this assumption is mainly based on mechanistic studies performed in vitro. The objective of this study was to investigate the effect of quercetin on stress-induced changes in oxidative biomarkers in the hypothalamus of rats. Adult male Sprague Dawley rats were subjected to forced swimming stress for 45 min daily for 14 days. Effect of quercetin at three different doses (10, 20 and 30 mg/kg body weight) on serum corticosterone and oxidative biomarkers (lipid hydroperoxides, antioxidant enzymes and total antioxidants) was estimated. Swimming stress significantly increased the serum corticosterone and lipid hydroperoxide levels. A significant decrease in total antioxidant levels and super oxide dismutase, glutathione peroxidase and catalase levels was seen in the hypothalamus after stress and treatment with quercetin significantly increased these oxidative parameters and there was a significant decrease in lipid hydroperoxide levels. These data demonstrate that forced swimming stress produced a severe oxidative damage in the hypothalamus and treatment with quercetin markedly attenuated these stress-induced changes. Antioxidant action of quercetin may be beneficial for the prevention and treatment of stress-induced oxidative damage in the brain.
Keywords: Stress; Quercetin; Oxidative stress; Antioxidant; Hypothalamus;

Effects of ST2742, a novel antipsychotic, on prepulse inhibition by Katia Lombardo; Maria Antonietta Stasi; Franco Borsini (53-60).
ST2472, namely (9-piperazin-1-ylpyrrolo [2,1]-b[1,3]benzothiazepine), was previously shown to have antipsychotic activity in the conditioned avoidance response (CAR) test. In the present work we aimed at evaluating the antipsychotic potential of ST2472, administered orally at doses ranging from 0.75 to 6 mg/kg, in the prepulse inhibition (PPI) test. Apomorphine and MK801, namely (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, were used as reference PPI disruption compounds, and were both administered subcutaneously. The typical antipsychotic haloperidol as well as the atypical antipsychotics clozapine and olanzapine was used as reference antipsychotics and administered orally. In the apomorphine-induced disruption of PPI, we found that ST2472, haloperidol, clozapine, and olanzapine were able to antagonise the effect of apomorphine. In the MK801-induced disruption of PPI, conversely, ST2472, haloperidol and clozapine failed to antagonise the effect of MK801, while the antagonistic effects of olanzapine were variable. These results confirm and further extend the antipsychotic potential of ST2472 and warrant future translational studies in humans.
Keywords: Prepulse inhibition; Antipsychotic; ST2472; Apomorphine; MK801; (Rat);

Cardiovascular effects of intravenous administered 26RFa, a novel RFamide peptide ligand for GPR103, in anaesthetised rats by Quan Fang; Qian Liu; Ning Li; Tian-nan Jiang; Yu-lin Li; Xiang Yan; Rui Wang (61-66).
26RFa, a novel RFamide-related peptide, has been identified as the endogenous ligand for GPR103, and the biological functions of this neuropeptide have not been well investigated. In the present study, the cardiovascular effects of intravenous administration of rat/mouse 26RFa were tested in rats. Intravenous administered 26RFa (100–800 nmol/kg, i.v.) caused a biphasic change in blood pressure, and an increase in heart rate in urethane-anaesthetised rats. The pressor effects induced by 26RFa were significantly inhibited by pretreatment with the α- and β-adrenoreceptor antagonists. In contrast, the tachycardiac responses to 26RFa were significantly attenuated by bilateral cervical vagotomy and β-adrenoreceptor antagonist propranolol. These data imply that the peripheral cardiovascular regulation of 26RFa might be involved in vagal components and catecholaminergic pathway. Furthermore, in order to evaluate the importance of the regions of 26RFa molecule in its cardiovascular regulation, the two C-terminal fragments of rat/mouse 26RFa, 26RFa(8–26) and 26RFa(19–26), were synthesized and investigated to address their peripheral cardiovascular responses in rats. Surprisingly, intravenous injections of 26RFa(8–26) and 26RFa(19–26) (50–300 nmol/kg, i.v.) produced dose-dependent increases in blood pressure and heart rate, which exerted different sensitivities to bilateral vagotomy and β-adrenergic receptor antagonist. The results indicate that intravenous administrations of 26RFa and its fragments induced their cardiovascular effects via different pathways, which further suggest that the N-terminal residues of 26RFa are required for its cardiovascular activities.
Keywords: 26RFa; MAP (mean arterial pressure); HR (heart rate); Fragment; (Rat);

Lipopolysaccharide alters vasodilation to atrial natriuretic peptide via nitric oxide and endothelin-1: Time-dependent effects by Johann K. Scicluna; Arnaud Mansart; Jonathan J. Ross; Charles S. Reilly; Nicola J. Brown; Zoë L.S. Brookes (67-70).
Nitric oxide (NO) induces vascular relaxation via cGMP in vascular smooth muscle (VSM) and is an important mediator of vascular tone during sepsis, as endothelial NO synthase (eNOS) may be upregulated during the early stages. Atrial natriuretic peptide (ANP) also stimulates cGMP via eNOS hence, this study aimed to investigate the role of NO in time-dependent altered vascular responses to ANP during the first 4 h of exposure to bacterial lipopolysaccharide (LPS). We used male rat saphenous arteries [internal relaxed diameter 63–152 µm, n  = 48], mounted on a wire myograph and pre-constricted with phenylephrine. At 2 h in the presence of LPS, there was increased relaxation to ANP in arteries exposed to LPS [16.3 ± 2.4%, P  < 0.05]. However the response to ANP was not altered by the NOS inhibitor Nω-nitro-l-arginine methyl ester (L-NAME, 10− 4  M) and following denudation (vessels without endothelium). At 4 h there was no longer increased relaxation to ANP in the presence of LPS. Moreover the vasodilator response to ANP was significantly reduced following L-NAME or denudation [4.4 ± 1.0% and 4.3 ± 1.1% respectively, P  < 0.05]. However, the non-specific endothelin-1 (ET-1) receptor antagonist Bosentan [10− 5  M] increased dilatation in LPS exposed arteries at 1 and 2 h, reaching significance at 4 h [14.0 ± 3.4%, P  < 0.05]. In summary, an endothelial and NO dependent mechanism is responsible for increased relaxation to ANP following 2 h exposure to LPS. However after 4 h an endothelial and NO independent process involving ET-1 is responsible for decreased relaxation to ANP. The enhanced response to ANP may exacerbate early systemic vasodilatation during early sepsis.
Keywords: Atrial natriuretic peptide; Nitric oxide; Endothelin-1; Lipopolysaccharide;

Preservation of cardiac contractility after long-term therapy with oxypurinol in post-ischemic heart failure in mice by Zhen Tan; Tieying Dai; Xin Zhong; Ye Tian; Michelle K. Leppo; Wei Dong Gao (71-77).
Previously, we showed that oral allopurinol increased survival in mice with post-ischemic cardiomyopathy and attributed this outcome to an improvement of excitation–contraction coupling that boosted contractility. In this study, we tested the sustainability of this enhanced contraction associated with decreased oxidative damage over an extended time. Mice were divided into three groups: sham-operated control, myocardial infarction-heart failure (MI-HF), and oxypurinol-treated heart failure (Oxy-HF). After 9–11 months, echocardiography showed that mice treated with oxypurinol (1 mM in drinking water) had significantly higher left ventricle fractional contraction and fractional wall thickening during systole than did mice in the MI-HF group (left ventricle fractional contraction: 28.4 ± 2.2% vs. 19.9 ± 2.3%, P  < 0.05; left ventricle fractional wall thickening: 45.0 ± 4.0% vs. 23.5 ± 2.0%, P  < 0.05). Left ventricular diastolic dimension, however, remained enlarged (0.50 ± 0.04 vs. 0.54 ± 0.05 cm, not significant). Twitch force was significantly higher at any given external Ca2+ concentration in the Oxy-HF group than in the MI-HF group (P  < 0.01); amplitudes of intracellular Ca2+ transients were also higher in the Oxy-HF group but were not statistically different from those of the MI-HF group. Force–frequency relation was improved in the Oxy-HF group. Muscle in the Oxy-HF group exhibited increases in myofilament Ca2+ responsiveness, as evidenced by significantly higher maximal Ca2+-activated force (77.8 ± 12.7 vs. 36.4 ± 4.4 mN/mm2, P  < 0.01). Finally, lipid peroxidation and myofilament oxidation were suppressed in the Oxy-HF group. These results indicate that the beneficial effects of antioxidation can be sustained by long-term treatment with oxypurinol after ischemic heart failure, with significantly improved cardiac contractility.
Keywords: Heart failure; Oxypurinol; Oxidative stress; Cardiac contraction; Calcium; (Mouse);

The effects of NMDA receptor antagonists over intestinal ischemia/reperfusion injury in rats by Carlos Rodrigo Cámara-Lemarroy; Francisco Javier Guzmán-de la Garza; Gabriela Alarcón-Galván; Paula Cordero-Pérez; Nancy Esthela Fernández-Garza (78-85).
Intestinal ischemia/reperfusion causes severe injury and alters motility. N-methyl-d-aspartate (NMDA) receptor antagonists have been shown to reduce ischemia/reperfusion injury in the nervous system, and in other organs. In this study, we set out to investigate the effects of NMDA receptor antagonists over intestinal ischemia/reperfusion injury. Male Wistar rats were randomly divided into four groups: (1) a control, sham-operated group; (2) an intestinal ischemia/reperfusion group subjected to 45 min ischemia and 1 h reperfusion; (3) a group treated with 10 mg/kg ketamine before ischemia/reperfusion; and (4) a group treated with 10 mg/kg memantine before ischemia/reperfusion. Intestinal samples were taken for histological evaluation. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), malondialdehyde (MDA), total antioxidant capacity, tumor necrosis factor alpha (TNF-alpha), P-selectin and antithrombin III (ATIII) were measured. Intestinal transit time was determined to evaluate intestinal motility. Fecal pellet output and animal weight were also registered daily for 7 days post-ischemia. After reperfusion, AST, LDH, TNF-alpha and P-selectin levels were elevated, ATIII levels were depleted, and ALT levels were unchanged in serum. Additionally, levels of MDA were increased and total antioxidant capacity was reduced in serum, indicating oxidative stress. Intestinal mucosa showed severe injury. Ketamine, but not memantine, diminished these alterations. Intestinal motility and fecal pellet output were also altered after ischemia/reperfusion. Both drugs abolished the alterations in motility. In conclusion, ketamine's protective effects over ischemia/reperfusion do not appear to be NMDA mediated, but they could be playing a role in protecting the intestine against ischemia-induced functional changes.
Keywords: Ischemia/reperfusion; Intestine; NMDA (N-methyl-d-aspartate); Ketamine; Memantine;