European Journal of Pharmacology (v.650, #2-3)

eNOS/Hsp70 interaction on rosuvastatin cytoprotective effect in neonatal obstructive nephropathy by Walter Manucha; Fernando Kurbán; Luciana Mazzei; María Eugenia Benardón; Victoria Bocanegra; Martín Rinaldi Tosi; Patricia Vallés (487-495).
There is growing evidence that statins may exert renoprotective effects beyond cholesterol reduction. The cholesterol-independent or “pleiotropic” effects of statins include the upregulation of endothelial nitric oxide synthase (eNOS). Here we determined whether eNOS associated with Hsp70 expression is involved in rosuvastatin resistance to obstruction-induced oxidative stress and cell death. Neonatal rats subjected to unilateral ureteral obstruction (UUO) within two days of birth and controls were treated daily with vehicle or rosuvastatin (10 mg/kg/day) for 14 days. Decreased endogenous nitric oxide (NO) and lower mRNA and protein eNOS expression associated with downregulation of heat shock factor 1 (Hsf1) mRNA and Hsp70 protein levels were observed in the obstructed kidney cortex. Increased nicotinamide adenine dinucleotide phosphate (NADHP) oxidase activity and apoptosis induction, regulated by mitochondrial signal pathway through an increased pro-apoptotic Bax/BcL2 ratio and caspase 3 activity, were demonstrated. Conversely, in cortex membrane fractions from rosuvastatin-treated UUO rats, marked upregulation of eNOS expression at transcriptional and posttranscriptional levels linked to increased Hsf1 mRNA expression and enhanced mRNA and protein Hsp70 expression, were observed. Consequently, there was an absence of apoptotic response and transiently decreased NADPH oxidase activity. In addition, interaction between eNOS and Hsp70 was determined by communoprecipitation in cortex membrane fractions, showing an increased ratio of both proteins, after rosuvastatin treatment in obstructed kidney. In summary, our data demonstrate that the effect of rosuvastatin on eNOS interacting with Hsp70, results in the capacity of both to prevent mitochondrial apoptotic pathway and oxidative stress in neonatal early kidney obstruction.
Keywords: Rosuvastatin; Endothelial nitric oxide synthase; Heat shock protein 70; Neonatal obstructive nephropathy; Apoptosis;

Hypocrellin B-encapsulated nanoparticle-mediated rev-caspase-3 gene transfection and photodynamic therapy on tumor cells by Dingqun Bai; Xinshu Xia; Christine M.N. Yow; Ellie S.M. Chu; Chuanshan Xu (496-500).
Gene therapy and photodynamic therapy are two kinds of important therapeutic strategies for treating malignant tumors. In order to explore the combined effects of gene therapy and PDT on tumor cells, rev-caspase-3 gene was transfected into the tumor model CNE2 cells using hypocrellin B-encapsulated nanoparticle (nano-HB) as a carrier. The transfected CNE2 cells were then irradiated by light from a LED source and the survival rate was investigated 18 h after PDT. Apoptosis was analyzed by a flow cytometer with propidium iodine (PI) staining and the active caspase-3 expression was measured using flow cytometry with phycoerythrin (PE)-conjugated anti-active caspase-3 antibody. The result from the flow cytometer showed that the level of the activated caspase-3 significantly increased up to 63.10% in the transfected CNE2 cells. The survival rate 18 h after gene transfection alone and nano-HB-mediated PDT was 96.6 ± 2.07%, 72.6 ± 4.15%, respectively. However, the survival rate of the transfected CNE2 cells 18 h after LED exposure significantly decreased to 50.6 ± 5.98% under the light energy of 4 J/cm2. Apoptotic rate 18 h after the combination of gene transfection and PDT increased up to 24.65%. Our findings demonstrated that nano-HB could significantly enhance the tranfection efficiency of rev-caspase-3 gene in the CNE2 cells. LED irradiation could effectively kill the treated CNE2 cells and induce apoptosis, suggesting hypocrellin B-encapsulated nanoparticle as an efficient gene carrier and a novel photosensitizer. The combination of gene therapy and PDT using nanoparticle as a mediator can be developed for treating nasopharyngeal carcinoma.
Keywords: Hypocrellin; Nanoparticle; Rev-caspase-3; Gene transfection; Photodynamic therapy; Tumor;

Phospholipase A2 releases the fatty acid arachidonic acid from membrane phospholipids. We used the purported phospholipase A2 stimulator, melittin, to examine the effects of endogenous arachidonic acid signaling on dopamine transporter function and trafficking. In HEK-293 cells stably transfected with the dopamine transporter, melittin reduced uptake of [ 3 H]dopamine. Additionally, measurements of fatty acid content demonstrated a melittin-induced release of membrane-incorporated arachidonic acid, but inhibitors of phospholipase C, phospholipase D, and phospholipase A2 did not prevent the release. Subsequent experiments measuring [125I]RTI-55 binding to the dopamine transporter demonstrated a direct interaction of melittin, or a melittin-activated endogenous compound, with the transporter to inhibit antagonist binding. This effect was not specific to the dopamine transporter, as [3H]spiperone binding to the recombinant dopamine D2 receptor was also inhibited by melittin treatment. Finally, melittin stimulated an increase in internalization of the dopamine transporter, and this effect was blocked by pretreatment with cocaine. Thus, melittin acts through multiple mechanisms to regulate cellular activity, including release of membrane-incorporated fatty acids and interaction with the dopamine transporter.
Keywords: Endocytosis; Arachidonic acid; Phospholipase; Dopamine transporter; Dopamine receptor;

Harmine, a β-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo by Takayuki Yonezawa; Shin-ichi Hasegawa; Midori Asai; Tadashi Ninomiya; Toshinori Sasaki; Byung-Yoon Cha; Toshiaki Teruya; Hidehiro Ozawa; Kazumi Yagasaki; Kazuo Nagai; Je-Tae Woo (511-518).
Bone homeostasis is controlled by the balance between osteoblastic bone formation and osteoclastic bone resorption. Excessive bone resorption is involved in the pathogenesis of bone-related disorders such as osteoporosis, arthritis and periodontitis. To obtain new antiresorptive agents, we searched for natural compounds that can inhibit osteoclast differentiation and function. We found that harmine, a β-carboline alkaloid, inhibited multinucleated osteoclast formation induced by receptor activator of nuclear factor-κB ligand (RANKL) in RAW264.7 cells. Similar results were obtained in cultures of bone marrow macrophages supplemented with macrophage colony-stimulating factor and RANKL, as well as in cocultures of bone marrow cells and osteoblastic UAMS-32 cells in the presence of vitamin D3 and prostaglandin E2. Furthermore, harmine prevented RANKL-induced bone resorption in both cell and bone tissue cultures. Treatment with harmine (10 mg/kg/day) also prevented bone loss in ovariectomized osteoporosis model mice. Structure–activity relationship studies showed that the C3–C4 double bond and 7-methoxy group of harmine are important for its inhibitory activity on osteoclast differentiation. In mechanistic studies, we found that harmine inhibited the RANKL-induced expression of c-Fos and subsequent expression of nuclear factor of activated T cells (NFAT) c1, which is a master regulator of osteoclastogenesis. However, harmine did not affect early signaling molecules such as ERK, p38 MAPK and IκBα. These results indicate that harmine inhibits osteoclast formation via downregulation of c-Fos and NFATc1 induced by RANKL and represses bone resorption. These novel findings may be useful for the treatment of bone-destructive diseases.
Keywords: Harmine; Osteoclast; Bone resorption; Osteoporosis;

Role of JNK and c-Jun signaling pathway in regulation of human serum paraoxonase 1 gene transcription by berberine in human HepG2 cells by Chi-Chih Cheng; Chi-Mei Hsueh; Kae-Woei Liang; Chih-Tai Ting; Chi-Luan Wen; Shih-Lan Hsu (519-525).
Human serum paraoxonase 1 (PON1), an arylesterase, is associated with high-density lipoprotein (HDL) and can inhibit the oxidative modification of low-density lipoprotein (LDL), implying that PON1 may prevent atherosclerosis. Berberine, a botanical alkaloid, lowers the cholesterol level in serum and is thought to display cardioprotective properties. However, the effect of berberine on PON1 gene expression remains unclear. Thus, we evaluated how berberine regulates PON1 gene expression. In human hepatoma HepG2 and Huh7 cells, the PON1 protein levels were increased by berberine in a dose- and time-dependent manner. Data from real time PCR analysis indicated that berberine could up-regulate PON1 expression at the transcriptional level. Additionally, treating HepG2 cells with berberine increased the levels of phosphorylated JNK and its downstream target c-Jun. The PON1 upstream region contained a consensus binding site for AP1, and the electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated that the AP1 factors, especially c-Jun, bind to the upstream sequence of the PON1 promoter upon berberine treatment. Moreover, pretreatment with SP600125 (JNK inhibitor) or curcumin (AP-1 inhibitor) markedly attenuated the berberine-induced PON1 promoter activity and protein expression. This is the first study to suggest that JNK/c-Jun signalling pathway plays a crucial role in berberine-regulated PON1 transcription in human hepatoma cells. The induction of PON1 by berberine elucidates a potential mechanism through which berberine may protect against atherosclerosis.
Keywords: Paraoxonase 1; Berberine; JNK; c-Jun; AP-1;

Inactivation of Nuclear Factor-κB by citrus flavanone hesperidin contributes to apoptosis and chemo-sensitizing effect in Ramos cells by Maryam Nazari; Asghar Ghorbani; Azita Hekmat-Doost; Mahmood Jeddi-Tehrani; Hamid Zand (526-533).
Hesperidin, a flavonoid present in fruits and vegetables, possesses anti-inflammatory and chemopreventive effects. Some evidence implies that hesperidin may recruit Peroxisome Proliferator-Activated Receptor-gamma (PPARγ) to exert biological action. It has been shown also that inactivation of Nuclear Factor-kappaB is another mechanism of flavonoids' action. Therefore, we wished to investigate the role of these transcription factors in apoptosis induced by hesperidin in Ramos Burkitt's lymphoma cells. We found that incubation of Ramos cells with hesperidin abrogates constitutive and doxorubicin-induced NF-κB activation through inhibition of phosphorylation of its inhibitory subunits (IκB). Moreover, our results showed that incubation of Ramos cells with hesperidin resulted in inhibition of proliferation and apoptosis in a PPARγ-independent way. Our results demonstrated that hesperidin inhibits proliferation of Ramos cells and sensitizes them to doxorubicin-induced apoptosis through inhibition of both constitutive and doxorubicin-mediated NF-κB activation in a PPARγ-independent manner.
Keywords: Hesperidin; Flavonoid; IkappaB; NF-kappaB; PPARγ;

We characterized agonist-induced internalization, recycling and downregulation of each muscarinic receptor subtype (M1–M5) stably expressed in Chinese hamster ovary (CHO) cells. The radioligands [3H]QNB and [3H]NMS were used to measure the total and plasma membrane populations of muscarinic receptors, respectively. Following carbachol treatment (1 mM), the rank orders for the rate of carbachol-induced internalization of the muscarinic subtypes were M2  > M4  = M5  > M3  = M1, respectively. Unlike the M2 receptor, M1, M3, M4 and M5 receptors recycled back to the plasma membrane after 1 h carbachol treatment. The receptor downregulation elicited to 24 h carbachol treatment was similar for M2, M3, M4 and M5 receptors, whereas that for the M1 receptor was greater. Our results indicate that there are subtype-specific differences in the rate and extent of agonist-induced muscarinic receptor internalization, recycling and downregulation in CHO cells.
Keywords: Muscarinic; Internalization; Recycling; Downregulation; Carbachol;

TEMPOL protects human neuroblastoma SH-SY5Y cells against ß-amyloid-induced cell toxicity by Pennapa Chonpathompikunlert; Junkyu Han; Kazuko Toh; Hiroko Isoda; Yukio Nagasaki (544-549).
Amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). It can cause cell death in Alzheimer's disease by evoking a cascade of oxidative damage to neurons. Antioxidant compounds may help to elucidate and develop a treatment for Alzheimer's disease. In the present study, we investigated the protective effect of TEMPOL (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy), a cyclic nitroxide which is particularly effective at reducing oxidative injury, on Aβ1–42-induced SH-SY5Y cell toxicity. Exposure of cells to 20 μM Aβ1–42 for 48 h caused viability loss and apoptotic increase, and pre-treatment with TEMPOL for 24 h significantly reduced the viability loss and apoptotic rate. In addition, TEMPOL inhibited Aβ1–42-induced superoxide anion generation and hydroxyl radical generation to a striking degree. Based on these results, it is concluded that TEMPOL effectively protects SH-SY5Y cells against β-amyloid-induced damage by suppressing the generation of reactive oxygen species especially, superoxide anion.
Keywords: Amyloid-β peptide; Neuroblastoma SH-SY5Y cell; Superoxide anion generation; Hydroxyl radical generation; Apoptosis; Alzheimer's disease;

Low dose of bupropion significantly enhances the anticonvulsant activity of felbamate, lamotrigine and topiramate in mice by Bartłomiej Barczyński; Grzegorz Buszewicz; Jarogniew J. Łuszczki; Krzysztof Bańka; Krzysztof Tutaj; Tomasz Mróz; Marian Wielosz; Roman Mądro; Piotr Tutka (550-555).
Experimental evidence indicates that bupropion hydrochloride, an antidepressant and a first-line smoking cessation aid, exerts dose-dependently anticonvulsant and convulsant effects. In this study, chronic bupropion pretreatment intraperitoneally (i.p.) for 14 days in a dose of 5 mg/kg reduced the ED50 (i.e. the dose protecting 50% of mice against electroconvulsions) of lamotrigine, topiramate, and felbamate from 4.58, 60.95, and 48.79 (antiepileptic + vehicle) to 3.01, 41.68, and 37.28 mg/kg (antiepileptic + bupropion), respectively, against maximal electroshock-induced seizures in mice. Bupropion significantly increased the plasma and brain concentrations of lamotrigine. Plasma concentration of topiramate was elevated, however, the brain concentration of the drug was not affected. Neither plasma nor brain concentrations of felbamate were elevated by bupropion administration. Bupropion did not exacerbate motor coordination impairment caused by the antiepileptic drugs in the rotarod test. Chronic administration of bupropion significantly potentiates the protective activity of lamotrigine, topiramate, and felbamate against maximal electroshock-induced seizures. A pharmacokinetic interaction is responsible for the effect of bupropion co-administered with lamotrigine.
Keywords: Antidepressant drug; Antiepileptic drug; Bupropion; Epilepsy; Seizure;

Role of the effect of inhibition of neutral endopeptidase on vascular and neural complications in streptozotocin-induced diabetic rats by Christine L. Oltman; Eric P. Davidson; Lawrence J. Coppey; Travis L. Kleinschmidt; Brian Dake; Mark A. Yorek (556-562).
We have previously shown that treating streptozotocin-induced diabetic rats, an animal model of type 1 diabetes, with Ilepatril (an inhibitor of neutral endopeptidase and angiotensin converting enzyme (ACE)) improves vascular and neural function. In this study we sought to determine the individual effect of inhibition of neutral endopeptidase and ACE on diabetes-induced vascular and neural dysfunction. After 4 weeks of untreated diabetes, rats were treated for 12 weeks with Ilepatril, Enalapril (ACE inhibitor) or Candoxatril (neutral endopeptidase inhibitor) followed by analysis of neural and vascular function. Diabetes caused slowing of motor and sensory nerve conduction, thermal hypoalgesia, reduction in intraepidermal nerve fiber density in the hindpaw and impairment in vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineural arterioles of the sciatic nerve and to atrial natriuretic peptide and calcitonin gene-related peptide in renal arteries. Inhibition of neutral endopeptidase or ACE improved neural function; however, dual inhibition of neutral endopeptidase and ACE with Ilepatril tended to have the greatest efficacy. Ilepatril and Candoxatril treatment of diabetic rats was more efficacious in improving vascular responsiveness in epineurial arterioles than treatment with Enalapril. Ilepatril, Enalapril or Candoxatril treatment of diabetic rats were all efficacious in renal arteries. These studies suggest that combination therapy may be the most effective approach for treatment of diabetic neural and vascular complications.
Keywords: Diabetes; Diabetic neuropathy; Diabetic nephropathy; Vascular relaxation; Neutral endopeptidase; Vasopeptidase inhibitor; Angiotensin converting enzyme;

Stress-induced analgesia and endogenous opioid peptides: The importance of stress duration by Drupad Parikh; Abdul Hamid; Theodore C. Friedman; Khanh Nguyen; Andy Tseng; Paul Marquez; Kabirullah Lutfy (563-567).
Stress is known to elicit pain relief, a phenomenon referred to as stress-induced analgesia. Based on stress parameters, opioid and non-opioid intrinsic pain inhibitory systems can be activated. In the present study, we assessed whether changing the duration of stress would affect the involvement of endogenous opioids in antinociception elicited by swim in warm water (32 °C), known to be opioid-mediated. Using mice lacking beta-endorphin, enkephalins or dynorphins and their respective wild-type littermates, we assessed the role of each opioid peptide in antinociception induced by a short (3 min) vs. long (15 min) swim. Mice were tested for baseline hot plate latency, exposed to swim (3 or 15 min) in warm water (32 °C) and then tested for antinociception at 5, 15 and 30 min. Our results revealed that both swim paradigms induced significant antinociception in wild-type mice. However, the short swim failed to induce antinociception in beta-endorphin-deficient mice, illustrating that beta-endorphin is important in this form of stress-induced antinociception. On the other hand, antinociception elicited by the long swim was only slightly reduced in beta-endorphin-deficient mice despite pretreatment with naloxone, a non-selective opioid receptor antagonist, significantly attenuated the antinociception elicited by the long swim. Nevertheless, a delayed hyperalgesic response developed in mice lacking beta-endorphin following exposure to either swim paradigm. On the other hand, mice lacking enkephalins or dynorphins and their respective wild-type littermates expressed a comparable antinociceptive response and did not exhibit the delayed hyperalgesic response. Together, our results suggest that the endogenous opioid peptide beta-endorphin not only mediates antinociception induced by the short swim but also prevents the delayed hyperalgesic response elicited by either swim paradigm.
Keywords: Stress-induced analgesia; Endogenous opioid peptides; Knockout mouse; Swim duration; Hot plate test;

The simple diamine diaminoethane (ethylenediamine, EDA) has been shown to activate GABA receptors in the central and peripheral nervous systems, partly by a direct action and partly by releasing endogenous GABA. These effects have been shown to be produced by the complexation of EDA with bicarbonate to form a carbamate. The present work has compared EDA, GABA and β-alanine responses in rat CA1 neurons using extracellular and intracellular recordings, as well as neocortical evoked potentials in vivo. Superfusion of GABA onto hippocampal slices produced depolarisation and a decrease of field epsps, both effects fading rapidly, but showing sensitivity to blockade by bicuculline. EDA produced an initial hyperpolarisation and increase of extracellular field epsp size with no fade and only partial sensitivity to bicuculline, with subsequent depolarisation, while β-alanine produces a much larger underlying hyperpolarisation and increase in fepsps, followed by depolarisation and inhibition of fepsps. The responses to β-alanine, but not GABA or EDA, were blocked by strychnine. In vivo experiments, recording somatosensory evoked potentials, confirmed that EDA produced an initial increase followed by depression, and that this effect was not fully blocked by bicuculline. Overall the results indicate that EDA has actions in addition to the activation of GABA receptors. These actions are not attributable to activation of β-alanine-sensitive glycine receptors, but may involve the activation of sites sensitive to adipic acid, which is structurally equivalent to the dicarbamate of EDA. The results emphasise the complex pharmacology of simple amines in bicarbonate-containing solutions.
Keywords: Ethylenediamine; 1,2-diaminoethane; GABA; Bicuculline; Kynurenic acid; Hippocampus; Cortical evoked potentials;

The impairment of N-Methyl-d-Aspartate receptors is thought to contribute to negative symptoms and cognitive deficits. In vitro studies suggest that atypical antipsychotic drugs like clozapine may help to alleviate these deficits by enhancing glutamatergic function. The present study examined the in vivo interaction of clozapine with N-Methyl d-aspartate by training one group of C57BL/6 mice to discrimination 2.5 mg/kg clozapine from vehicle and another group to discriminate 30 mg/kg N-Methyl d-aspartate from vehicle in a two-lever drug discrimination task. Cross-generalization testing revealed that N-Methyl d-aspartate (3–56 mg/kg) failed to substitute for clozapine in the clozapine-trained mice, while clozapine (0.625 mg/kg) produced partial substitution in the N-Methyl d-aspartate-trained mice. Interestingly, administration of a low, non-generalizing dose of each training drug in combination with the full range of doses of the alternate training drug produced full and dose–dependent substitution in both clozapine- and N-Methyl d-aspartate-trained mice. The α1 antagonist prazosin fully and dose-dependently substituted for both clozapine and N-Methyl d-aspartate. These results suggest that the shared discriminative stimulus properties between clozapine and N-Methyl d-aspartate may be mediated through indirect mechanisms, possibly in part through α1 adrenergic antagonism.
Keywords: Clozapine; N-Methyl-d-Aspartate (N-Methyl d-aspartate); Prazosin; Glutamate; Drug discrimination; C57BL/6 mouse;

Hyperactivity of the hypothalamus–pituitary–adrenal axis in lipopolysaccharide-induced neurodevelopmental model of schizophrenia in rats: Effects of antipsychotic drugs by Agnieszka Basta-Kaim; Bogusława Budziszewska; Monika Leśkiewicz; Katarzyna Fijał; Magdalena Regulska; Marta Kubera; Krzysztof Wędzony; Władysław Lasoń (586-595).
Recent data indicate that a significant number of schizophrenic patients are hypercortisolemic and that glucocorticoids are involved in the pathogenesis of schizophrenia. The aim of the present study was to evaluate whether behavioural schizophrenia-like changes in the lipopolysaccharide (LPS)-induced neurodevelopmental model of this brain disorder are associated with alterations in the level of plasma corticosterone, the concentration of glucocorticoid receptors and the amount of the immunophilin FKBP51, the glucocorticoid receptor co-chaperone, in the hippocampus and frontal cortex. We found that the adult offspring of prenatally LPS-treated rats showed a deficit in prepulse inhibition (PPI), an enhancement of amphetamine-induced locomotor activity, an elevated plasma level of corticosterone and a decrease in both the glucocorticoid receptor level in the hippocampus and the FKBP51 concentration in the frontal cortex. Most of these changes were reversed by the atypical antipsychotic drug clozapine, whereas chlorpromazine had no effect on PPI but attenuated the amphetamine-induced hyperactivity and normalised the hippocampal level of glucocorticoid receptors. The changes in the level of plasma corticosterone and cortical FKBP51 were attenuated by chlorpromazine in female offspring only. This study supports the hypothesis of hypothalamic–pituitary–adrenal (HPA) axis hyperactivity in schizophrenia and suggests that this hyperactivity results from a decrease in the hippocampal glucocorticoid receptor level and a decrease in FKBP51 in the frontal cortex.
Keywords: Neurodevelopmental model of schizophrenia; HPA axis activity; Glucocorticoid receptor; FKBP51; Sensorimotor gating; Locomotor activity;

Muscarinic acetylcholine receptors in the nucleus accumbens core and shell contribute to cocaine priming-induced reinstatement of drug seeking by Judy Yee; Katie R. Famous; Thomas J. Hopkins; Michael C. McMullen; R. Christopher Pierce; Heath D. Schmidt (596-604).
Muscarinic acetylcholine receptors in the nucleus accumbens play an important role in mediating the reinforcing effects of cocaine. However, there is a paucity of data regarding the role of accumbal muscarinic acetylcholine receptors in the reinstatement of cocaine-seeking behavior. The goal of these experiments was to assess the role of muscarinic acetylcholine receptors in the nucleus accumbens core and shell in cocaine and sucrose priming-induced reinstatement. Rats were initially trained to self-administer cocaine or sucrose on a fixed-ratio schedule of reinforcement. Lever-pressing behavior was then extinguished and followed by a subsequent reinstatement phase during which operant responding was induced by either a systemic injection of cocaine in cocaine-experienced rats or non-contingent delivery of sucrose pellets in subjects with a history of sucrose self-administration. Results indicated that systemic administration of the muscarinic acetylcholine receptor antagonist scopolamine (5.0 mg/kg, i.p.) dose-dependently attenuated cocaine, but not sucrose, reinstatement. Furthermore, administration of scopolamine (36.0 μg) directly into the nucleus accumbens shell or core attenuated cocaine priming-induced reinstatement. In contrast, infusion of scopolamine (36.0 μg) directly into the accumbens core, but not shell, attenuated sucrose reinstatement, which suggests that muscarinic acetylcholine receptors in these two subregions of the nucleus accumbens have differential roles in sucrose seeking. Taken together, these results indicate that cocaine priming-induced reinstatement is mediated, in part, by increased signaling through muscarinic acetylcholine receptors in the shell subregion of the nucleus accumbens. Muscarinic acetylcholine receptors in the core of the accumbens, in contrast, appear to play a more general (i.e. not cocaine specific) role in motivated behaviors.
Keywords: Relapse; Addiction; Psychostimulant; Sucrose seeking; Acetylcholine; Scopolamine;

Ibudilast, a mixed phosphodiesterase (PDE) 3/4 inhibitor, is a cerebral vasodilator widely used in Japan for treating post-stroke dizziness. However, little studies have been conducted on the vasorelaxant effects of PDE inhibitors in the vertebrobasilar artery associated with dizziness onset. The in vitro vasorelaxant properties of ibudilast were, therefore, investigated by comparing with known selective PDE inhibitors, using vertebrobasilar arteries. Vasorelaxant activities of PDE3, PDE4, PDE5 inhibitors, and ibudilast were assessed in 5-hydroxytryptamine precontracted ring preparations from rabbit intracranial and extracranial vertebrobasilar arteries. Ibudilast more selectively relaxed the intracranial than extracranial artery. Similarly, selective PDE3 and PDE4 inhibitors showed higher selectivity for intracranial arteries. Furthermore, like selective PDE4 inhibitor, the vasorelaxation by ibudilast accompanied by increase in cAMP levels was inhibited by the adenylyl cyclase inhibitor SQ22536 in intracranial arteries. Next, it was examined whether nitric oxide (NO)/cGMP signaling is involved in this vasorelaxation in intracranial arteries. The suppression of NO/cGMP signaling by an NO synthase inhibitor or a guanylyl cyclase inhibitor potentiated the vasorelaxion by a PDE3 inhibitor and reduced that by a PDE4 inhibitor, while either suppression of the signaling had little influence on that by ibudilast. These results suggest that ibudilast has the high vasoselectivity for intracranial artery based on a mixed PDE3 and PDE4-inhibition, and effectively relaxes intracranial arteries independently of NO/cGMP signaling because of its vasorelaxation compensated by either PDE3- or PDE4-inhibition depending on the state of NO/cGMP signaling change.
Keywords: Ibudilast; Mixed phosphodiesterase (PDE) 3/4 inhibitor; Vertebrobasilar artery; NO/cGMP signaling; cAMP; Cerebral vasodilator;

Olprinone, a PDE3 inhibitor, modulates the inflammation associated with myocardial ischemia–reperfusion injury in rats by Rosanna Di Paola; Emanuela Mazzon; Irene Paterniti; Daniela Impellizzeri; Placido Bramanti; Salvatore Cuzzocrea (612-620).
Coronary ischemia and subsequent reperfusion result in deleterious effects, one of the principal ones being vascular and myocardial inflammation. Olprinone hydrochloride, a specific phosphodiesterase III inhibitor, has anti-inflammatory effects in addition to its inotropic and vasodilator effects. The purpose of this study was to examine the beneficial effects of olprinone on myocardial ischemia–reperfusion injury. Myocardial ischemia–reperfusion injury was caused by clamping the LAD (left anterior descending) coronary artery for 25 min followed by a release of the clamp allowing reperfusion for 1 h. Olprinone i.p. (0.2 mg/kg, i.p.) was administrated 15 min after ischemia. The olprinone administration significantly reduced the: (1) histological evidence of myocardial injury, (2) pro-inflammatory cytokines: tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β), (3) adhesion molecules: Inter-Cellular Adhesion Molecule 1 (ICAM-1) and P-Selectin, (4) nitrotyrosine formation, (5) nuclear factor kappa-B (NF-κB) expression, (6) Poly (ADP-ribose) (PAR) formation, and (7) apoptosis (Bax, Bcl-2, Fas-L and terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL). Based on these findings this study provides the evidence that treatment with olprinone ameliorated the inflammatory process associated with myocardial ischemia–reperfusion in rats and suggests that this drug may have potential in the treatment of various ischemia and reperfusion diseases.
Keywords: Inflammation; Olprinone; Phosphodiesterase III inhibitor; Myocardial ischemia–reperfusion; Pro-inflammatory cytokine; Apoptosis;

In the present study, we investigated the vasculoprotective effect of sigma-1 receptor stimulation with fluvoxamine on pressure overload hypertrophy-induced vascular injury in the thoracic aorta and defined mechanisms underlying that activity. Wistar rats underwent bilateral ovariectomy, and two weeks later were further treated with abdominal aortic stenosis. To confirm the vasculoprotective role of sigma-1 receptor signaling, we treated rats with the agonist fluvoxamine (at 0.5 and 1.0 mg/kg) and with the antagonist NE-100 (at1.0 mg/kg) for 4 weeks orally once a day after the onset of aortic banding. Interestingly, sigma-1 receptor expression in the thoracic aorta decreased significantly 4 weeks after pressure overload-induced hypertrophy in vehicle treated ovariectomized rats. Fluvoxamine administration significantly attenuated pressure overload-induced vascular injury with concomitant increase in receptor expression and subsequent decrease in IP3 receptor expression. Fluvoxamine treatment also significantly restored pressure overload-induced impaired Akt phosphorylation and stimulated eNOS protein expression as well as Akt-mediated eNOS phosphorylation (Ser1177). Fluvoxamine's vasculoprotective effect was nullified by co-administration of a sigma-1 receptor antagonist. No changes in phosphorylation of ERK1/2 or PKCα in the aorta were observed following pressure overload and after fluvoxamine treatment. Our findings confirm, for the first time, a potential role for sigma-1 receptor expression and signaling in the thoracic aorta in attenuating hypertrophy-induced vascular injury in ovariectomized rats. Thus, we demonstrate, for the first time, a potential role in the thoracic aorta for sigma-1 receptor expression and signaling via Akt–eNOS in attenuating hypertrophy-induced vascular injury in ovariectomized rats.
Keywords: Sigma-1 receptor; Thoracic aorta; Myocardial hypertrophy; Protein kinase B (Akt); Endothelial nitric oxide synthase (eNOS);

Thromboxane is a key inflammatory mediator and potent airway constrictor. It acts on thromboxane A2 (TP) receptors and contributes to airway inflammation and airway hyperresponsiveness that is the characteristic feature of asthma. The present study was designed to study TP receptor signaling in airway smooth muscle cells by using an organ culture model and a set of selective pharmacological inhibitors for mitogen-activated protein kinase (MAPK) and calcium signal pathways. Western-blot, immunohistochemistry, myograph and a selective TP receptor agonist U46619 were used for examining TP receptor signal proteins and function. Organ culture of rat bronchial segments for up to 48 h induces a time-dependently increased airway contractile response to U46619. This indicates that organ culture increases TP receptor signaling in the airway smooth muscle cells. The enhanced bronchial contraction was attenuated by the inhibition of c-Jun N-terminal kinase (JNK) MAPK activity, chelation of extracellular calcium and calcium channel blocker nifedipine, suggesting that JNK MAPK activity and elevated intracellular calcium level are required for the TP receptor signaling. In conclusion, airway smooth muscle cell TP receptor signaling occurs via JNK MAPK activity and the elevation of extracellular calcium influx, which may provide knowledge for understanding the signaling pathway responsible for the modulation of TP receptor mediated airway hyperresponsiveness to thromboxane.
Keywords: TP receptor; Signaling; JNK MAPK; Calcium influx; Airway hyperresponsiveness;

Differential recruitment of high affinity A1 and A2A adenosine receptors in the control of colonic neuromuscular function in experimental colitis by Luca Antonioli; Matteo Fornai; Rocchina Colucci; Oriana Awwad; Narcisa Ghisu; Marco Tuccori; Mario Del Tacca; Corrado Blandizzi (639-649).
This study investigated the expression of A1 and A2A receptors in the rat colonic neuromuscular compartment, and characterized their roles in the control of motility during inflammation. Colitis was induced by 2,4-dinitrobenzenesulfonic acid. A1, A2A receptors, and ecto-5′-nucleotidase (CD73, adenosine producing enzyme) mRNA expression was examined by RT-PCR. The effects of DPCPX (A1 receptor antagonist), CCPA (A1 receptor agonist), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (A2A receptor antagonist), 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (A2A receptor agonist), AOPCP (CD73 inhibitor) were tested on electrically or carbachol-evoked contractions in colonic longitudinal muscle preparations. In normal colon, RT-PCR revealed the presence of A1 receptors, A2A receptors and CD73, and an increased expression of A2A receptors and CD73 was detected in inflamed tissues. In normal colon, DPCPX or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol enhanced electrically-induced contractions, while in inflamed preparations the effect of DPCPX no longer occurred. In normal colon, CCPA or 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride decreased electrically-induced contractions. Under inflammation, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride reduced electrically evoked contractions with higher efficacy, while the inhibition by CCPA remained unchanged. A1 and A2A receptor ligands did not affect carbachol-induced contractions. AOPCP enhanced electrically-induced contractions and prevented the contractile effects of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, without interfering with DPCPX, both in normal and inflamed colons. These results indicate that, in normal colon, both A1 and A2A receptors contribute to the inhibitory control of motor functions at neuronal level. Under bowel inflammation, A1 receptor loses its modulating actions, while the recruitment of A2A receptor by CD73-dependent endogenous adenosine drives an enhanced inhibitory control of colonic neuromotility.
Keywords: Adenosine; A1 receptor; A2A receptor; Colonic neuromuscular function; Colitis; Rat;

Thromboxane A2 induces contraction of human prostate smooth muscle by Rho kinase- and calmodulin-dependent mechanisms by Frank Strittmatter; Christian Gratzke; Philipp Weinhold; Christian J. Steib; Anna C. Hartmann; Boris Schlenker; Karl-Erik Andersson; Petter Hedlund; Christian G. Stief; Martin Hennenberg (650-655).
Thromboxane A2 (TXA2) induces contraction in different smooth muscle types via its receptor (TXA2 receptor). However, any motoric role of TXA2 in prostate smooth muscle tone has not been studied to date. Here, we investigated whether TXA2 induces contraction of human prostate tissue. After ethical approval, prostate tissue was obtained from 47 patients undergoing radical prostatectomy. Effects of the TXA2 analogue U46619 ((5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptonic acid) in isolated human prostate strips were studied in organ bath experiments with or without the Rho kinase inhibitor, Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), or the calmodulin antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide hydrochloride). Expression of TXA2 synthase and TXA2 receptors were examined by Western blot analysis and immunohistochemistry. Endogenous TXA2 was quantified by enzyme immunoassay. U46619 induced concentration-dependent contractions of human prostate strips, with a maximum contraction at 3 μM. U46619-induced prostate contraction was significantly inhibited by Y27632 (30 μM) and by W7 (100 μM). TXA2 synthase and TXA2 receptors were detected by Western blot analysis. Immunohistochemical stainings showed that expression of TXA2 synthase in prostate tissue was located to glandular cells, while prostate TXA2 receptors were located to smooth muscle and glandular cells. The stable TXA2 metabolite TXB2 was detected by enzyme immunoassay in the prostate. TXA2 induces contraction of isolated human prostate tissue by TXA2 receptor activation. Prostate smooth muscle TXA2 receptors are coupled to Rho kinase and Ca2+-dependent mechanisms. The distribution of TXA2 synthase and TXA2 receptors in the human prostate suggests TXA2-mediated paracrine epithelial–stromal interactions.
Keywords: Benign prostate hyperplasia; Lower urinary tract; Lower urinary tract symptoms; Smooth muscle; Thromboxane A2; U46619;

During the course of liver fibrogenesis, hepatic stellate cell (HSC) proliferation as well as subsequent synthesis of excessive extracellular matrix components is known to be the central events. Thus, factors that could limit HSC proliferation are potential anti-fibrotic agents. The aim of this study was to investigate the effects of indole-3-carbinol (I3C), a nutritional component derived from Brassica family vegetables, on the proliferation of cultured HSC and to clarify the underline molecular mechanism. HSC-T6, an activated rat HSC line, was treated with I3C (50, 100 and 200 μM) for 24 h. The results indicated that I3C can significantly inhibit HSC proliferation in a concentration-dependent manner with or without platelet-derived growth factor-BB (PDGF-BB) stimulation (P  < 0.01). I3C could also block HSC in the G0/G1 phase from entering the S phase. The expressions of α-smooth muscle actin in HSC treated with I3C, were significantly decreased at levels of protein and mRNA (P  < 0.01). In addition, the type I collagen level, cyclin D1 and cyclin-dependent kinase 4 mRNA expressions, intracellular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and reactive oxygen species generation were significantly decreased by I3C (P  < 0.05). We also observed that the phosphorylated p38 MAPK in HSC-T6 was inhibited by I3C in a concentration-dependent manner; however, the phosphorylated ERK1/2 was unaltered. In conclusion, I3C could inhibit the proliferation of HSC by blocking the NADPH oxidase/reactive oxygen species/p38 MAPK signal pathway. These findings suggest that dietary I3C might play a novel role in prevention and treatment of chronic liver diseases.
Keywords: Indole-3-carbinol; Hepatic stellate cell; Activation; Proliferation; Platelet-derived growth factor;

A novel coenzyme A:diacylglycerol acyltransferase 1 inhibitor stimulates lipid metabolism in muscle and lowers weight in animal models of obesity by Toshihiro Yamamoto; Hiroshi Yamaguchi; Hiroshi Miki; Shuji Kitamura; Yoshihisa Nakada; Thomas D. Aicher; Scott A. Pratt; Koki Kato (663-672).
Obesity is characterized by the accumulation of triacylglycerol in adipocytes. Coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final and only committed step in triacylglycerol synthesis. In this report, we describe the pharmacological effects of a novel selective DGAT1 inhibitor, Compound-A. This compound inhibited triacylglycerol synthesis in both adipocytes and skeletal myotubes, and increased fatty acid oxidation in skeletal myotubes at 1 μM. The repeated administration of Compound-A to diet-induced obese C57BL/6J and genetically obese KKAy mice (3–30 mg/kg for 3–4 weeks) significantly decreased the visceral fat pad weights and the hepatic lipid contents compared to controls without affecting food intake. In addition, fatty acid oxidation in skeletal muscle tissues was increased by the treatment of Compound-A in both mice strains. This is the first report demonstrating that a small synthetic DGAT1 inhibitor increases fatty acid oxidation in skeletal muscle in vitro and ex vivo. These results suggest that DGAT1 inhibition is a promising therapeutic approach for the treatment of obesity and lipid abnormalities such as hepatic steatosis.
Keywords: DGAT1; Obesity; Hepatic steatosis; Diabetes; Triacylglycerol; Fatty acid oxidation;

PAM-1616, a selective peroxisome proliferator-activated receptor γ modulator with preserved anti-diabetic efficacy and reduced adverse effects by Mi-Kyung Kim; Yu Na Chae; Song-hyen Choi; Ho Sang Moon; Moon-Ho Son; Myung-Ho Bae; Hyun-ho Choi; Youn Hur; Eunkyung Kim; Yoo Hoi Park; Chan Sun Park; Jae Gyu Kim; Joong In Lim; Chang Yell Shin (673-681).
Peroxisome proliferator-activated receptor (PPAR) γ is known to be a key regulator of insulin resistance. PAM-1616 is a novel, non-thiazolidinedione small molecule compound synthesized in Dong-A Research Center. In this study, we characterized the pharmacological and safety profiles of PAM-1616 as a selective PPARγ modulator. PAM-1616 selectively binds to human PPARγ (IC50, 24.1 ± 5.6 nM) and is a partial agonist for human PPARγ with an EC50 of 83.6 ± 43.7 nM and a maximal response of 24.9 ± 7.1% relative to the full agonist, rosiglitazone. PAM-1616 was selective for human PPARγ than for human PPARα (EC50, 2658 ± 828 nM) without activating human PPARδ, which makes it a selective modulator of PPARγ. Treatment of high fat diet-induced obese C57BL/6J mice with PAM-1616 for 21 days improved HOMA-IR. Furthermore, PAM-1616 significantly improved hyperglycemia in db/db mice with little side effect when orally administered at a dose of 1 mg/kg/day for 28 days. Intriguingly, PAM-1616 was seen to increase the gene expression of inducible glucose transporter (GLUT4), while it partially induced that of a fatty acid carrier, aP2 in 3T3-L1 adipocytes, and it also showed partial recruitment of an adipogenic cofactor, TRAP220 as compared to rosiglitazone. PAM-1616 did not cause a significant increase in plasma volume of ICR mice when orally administered at a dose of 10 mg/kg/day for 9 days. PAM-1616 increased the expression of fluid retention-inducing genes such as serum/glucocorticoid-regulated kinase (SGK)-1 to a lesser extent as compared to rosiglitazone in human renal epithelial cells. These results suggest that PAM-1616 acts as a selective modulator of PPARγ with excellent antihyperglycemic property. The differential modulation of target gene by PAM-1616 might contribute to the improved side effect profiles.
Keywords: Peroxisome proliferator-activated receptor gamma modulator; PAM-1616; Insulin resistance; Fluid retention; Serum/glucocorticoid-regulated kinase;

Alendronate and raloxifene affect the osteoprotegerin/RANKL system in human osteoblast primary cultures from patients with osteoporosis and osteoarthritis by Mercè Giner; Ma José Rios; Ma José Montoya; Ma Angeles Vázquez; Cristina Miranda; Ramón Pérez-Cano (682-687).
The osteoprotegerin/RANKL system modulates bone remodelling. Alendronate and raloxifene are anti-resorptive drugs effective in osteoporotic disease. They reduce fracture risk, the activity of bone remodelling and increase bone mineral density. It is not known if they can exert a direct effect in osteoblasts via the osteoprotegerin/RANKL system. Our objective was to assess the effects of alendronate and raloxifene among osteoprotegerin production (ELISA), as well as osteoprotegerin and RANKL expression (RT-PCR), in primary cultures of human osteoblasts (hOB). We compared 17 osteoporotic patients with 16 patients affected by osteoarthritis in basal conditions and after incubation with alendronate (10−6  M), raloxifene (10−7  M) or 17-β estradiol (10−7  M) for 24 h. The statistical analysis was determined by ANOVA. Osteoprotegerin protein secretion in hOB cultures was higher in patients with osteoporosis than osteoarthritis. Osteoprotegerin secretion levels remained unchanged after each treatment. The osteoporotic group was more sensitive to treatment. Both raloxifene (34%) and estradiol (37%) increased osteoprotegerin mRNA expression, and alendronate (118%) and raloxifene (61%) increased the mRNA expression of RANKL. The RANKL/osteoprotegerin mRNA ratio was higher in osteoporotic than osteoarthritic patients. In the osteoporotic group, the RANKL/osteoprotegerin mRNA ratio was significantly increased after treatment with alendronate (112%) and after treatment with raloxifene (60%). These results indicate a direct action of alendronate and raloxifene on hOB cultures from osteoporotic patients, and the cited drugs are able to modulate the osteoprotegerin/RANKL system.
Keywords: Human osteoblast culture; Osteoporosis; Osteoprotegerin/RANKL; Alendronate and raloxifene;

Prolonged GIP receptor activation improves cognitive function, hippocampal synaptic plasticity and glucose homeostasis in high-fat fed mice by David W. Porter; Nigel Irwin; Peter R. Flatt; Christian Hölscher; Victor A. Gault (688-693).
Enzyme-resistant glucose-dependent insulinotropic polypeptide (GIP) agonists offer therapeutic potential for type 2 diabetes treatment. In addition, there is emerging evidence suggesting that GIP plays a direct role in modulating aspects of brain function. This study compared effects of dietary modification and/or twice-daily injection of the stable GIP agonist, (d-Ala2)GIP, on metabolic control, cognitive function and hippocampal synaptic plasticity in high-fat fed mice. Young Swiss mice were maintained on high-fat diet for 155 days, at which point half of the animals were switched to standard maintenance diet. Mice were subsequently injected with (d-Ala2)GIP (25 nmol/kg bodyweight; b.i.d.) or saline vehicle for 28 days. Both dietary intervention and (d-Ala2)GIP treatment were equally effective in restoring non-fasting glycaemic control (P  < 0.001) and improving (P  < 0.05 to P  < 0.001) glucose tolerance in high-fat fed mice. Switching to standard diet alone or in combination with (d-Ala2)GIP treatment returned body weights of high-fat fed mice to normal levels by day 28. However, body weights of high-fat fed mice treated with (d-Ala2)GIP were not significantly different from controls. (d-Ala2)GIP did not affect food intake or plasma insulin levels irrespective of diet. All mice treated with (d-Ala2)GIP exhibited a marked increase in recognition index (1.4-fold; P  < 0.05) highlighting improved cognitive function. Furthermore, switching to standard diet and/or (d-Ala2)GIP treatment rescued deleterious effects of high-fat feeding on long-term potentiation of synaptic neurotransmission. These results demonstrate that prolonged GIP activation is equally effective or superior to dietary intervention, in improving glucose intolerance and aspects of cognitive function and hippocampal synaptic plasticity in high-fat fed mice.
Keywords: Cognitive function; Glucose-dependent insulinotropic polypeptide (GIP); Glucose homeostasis; High-fat feeding; Long-term potentiation;

The soy isoflavone genistein reverses oxidative and inflammatory state, neuropathic pain, neurotrophic and vasculature deficits in diabetes mouse model by Anna Elisa Valsecchi; Silvia Franchi; Alberto Emilio Panerai; Alice Rossi; Paola Sacerdote; Mariapia Colleoni (694-702).
Treatment of diabetes complications remains a substantial challenge. The aim of this study was to explore the ability of the soy isoflavone genistein in attenuating the signs that follow diabetes onset: nociceptive hypersensitivity, oxidative and inflammatory state, nerve growth factor (NGF) decrease and vascular dysfunctions. Genistein (3 and 6 mg/kg) was administered to C57BL/6J streptozotocin diabetic mice from the 2nd till the 5th week after disease induction. The hind paw withdrawal threshold to mechanical stimulation (tactile allodynia) was evaluated by a von Frey filament. The oxidative stress was assessed measuring: reactive oxygen species by fluorimetric analysis, both the lipoperoxide content, as malondialdehyde, the antioxidant enzymatic activities spectrophotometrically and the glutathione content spectrofluorimetrically. Proinflammatory cytokines and NGF were measured in the sciatic nerve by enzyme-linked immunosorbent assay. Aortic inducible (iNOS) and endothelial nitric oxide synthase (eNOS) protein content was measured by western immunoblotting. Genistein relieved diabetic peripheral painful neuropathy, reverted the proinflammatory cytokine and reactive oxygen species overproduction, and restored the NGF content in diabetic sciatic nerve. Furthermore it restored the GSH content and the GSH and GSSG ratio, improved the antioxidant enzymes activities, decreased reactive oxygen species and lipoperoxide level in the brain and liver. Finally it restored the iNOS and eNOS content and the superoxide dismutase activity in thoracic aorta. Hyperglycaemia and weight decrease were not affected. Genistein is able to reverse a diabetes established condition of allodynia, oxidative stress and inflammation, ameliorates NGF content and the vascular dysfunction, thus suggesting its possible therapeutic use for diabetes complications.
Keywords: Cytokines; Diabetes; Genistein; Nerve growth factor; Neuropathy; Oxidative stress;

The DPP-4 inhibitor vildagliptin increases pancreatic beta cell mass in neonatal rats by Alokesh Duttaroy; Frank Voelker; Kimberley Merriam; Xia Zhang; Xianglin Ren; Kala Subramanian; Thomas E. Hughes; Bryan F. Burkey (703-707).
The present study addressed the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor vildagliptin ((1-[[(3-hydroxy-1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine), LAF237) on pancreatic beta cell mass in neonatal rats. Newborn rats were treated orally with vildagliptin (60 mg/kg) or vehicle once daily for 19 days starting from postnatal day 2. Pancreatic immunohistochemistry and morphometric analysis were performed to evaluate changes in beta cell mass, cell apoptosis (Apoptag stain) and replication (5′-Bromo-2′-deoxyuridine (BrdU)-incorporation) on days 7, 21, and 33. On day 7, an eight-fold increase in BrdU-positive pancreatic beta cells and a 71% decrease in Apoptag-positive cells were observed. On day 21, vildagliptin produced a two-fold increase in pancreatic beta cell mass compared to placebo (0.06 ± 0.01 mg vs 0.11 ± 0.02 mg, P < 0.05). Beta cell mass remained elevated (90%, 0.09 ± 0.02 mg vs 0.16 ± 0.03 mg, P < 0.05) on day 33, twelve days after discontinuing vildagliptin treatment. These data show that the DPP-4 inhibitor vildagliptin increased pancreatic beta cell mass through enhanced beta cell replication and reduced apoptosis. The increased beta cell mass was sustained for 12 days after vildagliptin washout. This study demonstrates that DPP-4 inhibitors can elicit beneficial effects on beta cell turnover that could help to prevent or retard the progression of type 2 diabetes.
Keywords: DPP-4; Vildagliptin; Beta cell mass; Apoptosis; Replication; Glucagon-like peptide-1; Pancreatic insulin content;