European Journal of Pharmacology (v.557, #1)

We investigated the mechanism of the transport of ribavirin (1-β-d-ribofuranosyl-1,2,4-trizole-3-carboxamide) into placental epithelial cells using human choriocarcinoma (BeWo) cells and Xenopus oocytes expressing human nucleoside transporters. In BeWo cells, when a relatively low concentration (123 nM) of ribavirin was used, both Na+-dependent uptake and -independent uptake of ribavirin were observed. On the other hand, when a higher concentration (100 μM) of ribavirin was used, Na+-independent uptake was observed, but there was only a slight Na+-dependent uptake. In Xenopus oocytes, influxes of ribavirin mediated by hCNT2 (concentrative nucleoside transporter 2), hCNT3 (concentrative nucleoside transporter 3), hENT1 (equilibrative nucleoside transporter 1) and hENT2 (equilibrative nucleoside transporter 2) were saturable, and apparent K m values were 18.0 μM, 14.2 μM, 3.46 mM and 3.71 mM, respectively. These data indicate that hCNT2 and hCNT3 have higher affinity for ribavirin than do hENT1 and hENT2. Moreover, analysis by RT-PCR showed that BeWo cells express mRNA of hCNT3, hENT1 and hENT2. These results suggest that ribavirin is taken up by BeWo cells via both the high-affinity Na+-dependent transporter hCNT3 and the low-affinity Na+-independent transporters hENT1 and hENT2.
Keywords: Ribavirin; Concentrative nucleoside transporter; Equilibrative nucleoside transporter; BeWo cells; (Xenopus laevis) oocytes;

Cytotoxic effects of intra and extracellular zinc chelation on human breast cancer cells by Mohammad Hashemi; Saeid Ghavami; Mehdi Eshraghi; Evan P. Booy; Marek Los (9-19).
Zinc is an essential trace element with cofactor functions in a large number of proteins of intermediary metabolism, hormone secretion pathways, immune defence mechanisms, and as a cofactor of transcription factors it is also involved in the control of gene expression. Our study demonstrates that the modulation of intra and extracellular zinc alone is sufficient to induce metabolic changes or even apoptosis in two model human breast cancer cell lines MCF-7 and MDA-MB468. Treatment of breast cancer cells with different concentrations of a cell membrane permeable zinc chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the membrane impermeable zinc chelator, diethylenetriaminepentacetic acid, (DTPA) resulted in a significant increase of cell death. Features of apoptosis, such as chromatin condensation and nuclear fragmentation accompanied the DTPA and TPEN-induced cell death. A significant increase in the activity of caspase-9 was observed in both cell lines; whereas, caspase-3 activity was only increased in MDA-MB468 cells since caspase-3 is not expressed in MCF-7 cells. Caspase-8 activation was negligible in both cell lines. Addition of Zn2+ or Cu2+ prevented DTPA and TPEN-induced cytotoxicity, indicating that both bivalent cations can be replaced functionally to a certain extent in our experimental system. Interestingly, addition of Ca2+, or Mg2+ had no effect. The antioxidant N-Acetyl–l-Cysteine inhibited the cytotoxic effect of DTPA and TPEN, indicating that oxidative stress is the likely mediator of Zn-deficiency-related cell death.
Keywords: Apoptosis; Breast cancer; Caspase; Cu/Zn-dismutase; Diethylenetriaminepentacetic acid; N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine;

Cyclosporine attenuates the adenylyl cyclase superactivation induced by chronic cannabinoid treatment by Hamid R. Banafshe; Mahmoud Ghazi-Khansari; Shahram Ejtemaei Mehr; Ahmad R. Dehpour (20-22).
Chronic cannabinoid treatment results in the development of tolerance. Adenylyl cyclase superactivation, induced by chronic cannabinoid agonist administration, is regarded as one of the molecular mechanisms leading to tolerance. In the present study, the effect of cyclosporine on adenylyl cyclase superactivation after chronic exposure to WIN 55,212-2, a cannabinoid receptor agonist, was studied. Chronic treatment (18 h) with WIN 55,212-2 induced a significant increase in cAMP levels in human astrocytoma cells (adenylyl cyclase superactivation). Acute treatment with cyclosporine (10 min) did not have any effect on WIN 55,212-2-induced adenylyl cyclase superactivation. But, chronic cyclosporine treatment (18 h), with concentration from 1 nM to 1 μM, attenuates the development of adenylyl cyclase superactivation after chronic WIN 55,212-2 treatment. Our findings show that cyclosporine attenuates chronic cannabinoid-mediated adenylyl cyclase superactivation.
Keywords: Cannabinoid; Adenylyl cyclase superactivation; Cyclosporine;

Cilostazol, a phosphodiesterase 3 inhibitor, protects mice against acute and late ischemic brain injuries by Yi-Lu Ye; Wen-Zhen Shi; Wei-Ping Zhang; Meng-Ling Wang; Yu Zhou; San-Hua Fang; Li-Ying Liu; Qi Zhang; Yue-Ping Yu; Er-Qing Wei (23-31).
Cilostazol, a selective inhibitor of phosphodiesterase 3, exerts neuroprotective effects on acute brain injury after cerebral ischemia in rats. However, it is unknown whether cilostazol affects the subacute or chronic ischemic injury. In the present study, we evaluated the dose- and time-dependent effects of cilostazol on acute ischemic brain injury and the long-lasting effect on the late (subacute/chronic) injury in mice with focal cerebral ischemia induced by transient middle cerebral artery occlusion. We found that pre-treatment of cilostazol (injected i.p. at 30 min before ischemia) significantly ameliorated the acute injury 24 h after ischemia, and the effective doses were 3–10 mg/kg. The post-treatment of cilostazol (10 mg/kg) was effective on the acute injury when it was injected 1 and 2 h after ischemia. In addition, for the late injury, post-treatment of cilostazol (10 mg/kg, i.p., for 7 consecutive days after ischemia) attenuated neurological dysfunctions, brain atrophy and infarct volume. It also inhibited astrocyte proliferation/glial scar formation and accelerated the angiogenesis in the ischemic boundary zone 7 and 28 days after ischemia. Thus, we conclude that cilostazol protects against not only the acute injury, but also the late injury in mice with focal cerebral ischemia; especially it can modify brain remodeling, astrogliosis and angiogenesis.
Keywords: Cilostazol; Cerebral ischemia; Neuroprotection; Blood–brain barrier; Astrocyte proliferation; Angiogenesis;

Nociceptin/orphanin FQ (N/OFQ), a 17-amino-acid peptide, is an endogenous agonist whose receptor is similar in sequence to mu, delta and kappa opioid receptors. It has been reported that N/OFQ can block antinociceptive effects induced by opioid receptor agonists in the radiant heat tail-flick test and warm water tail-withdrawal test. The present study was designed to see the effect of N/OFQ on antinociception induced by opioid receptor agonists in the cold water tail-flick (CWT) test, which measures a different type of pain. In adult male Sprague–Dawley (S–D) rats given subcutaneous (s.c.) injections of saline or morphine (8 mg/kg), intracerebroventricular (i.c.v.) injection of N/OFQ (18 μg) 15 min later produced a significant reversal of morphine antinociception (P  < 0.01, ANOVA followed by Duncan's test), compared to the corresponding saline control group. Saline (t  = + 15 min, i.c.v.) had no effect on s.c. morphine antinociception (P  > 0.01), compared to the corresponding saline control group. When the kappa opioid receptor agonist spiradoline (80 mg/kg, s.c.) was used instead of morphine, similar results were observed. In another series of experiments, it was found that i.c.v. injection of N/OFQ (18 μg) reversed the antinociception induced by i.c.v. injection of the selective mu opioid agonist PL017 (2 μg), delta opioid agonist DPDPE (50 ng) and kappa opioid agonist dynorphin (21.5 μg), respectively. These results indicate that N/OFQ may be an endogenous anti-opioid peptide in the brain of rats in the CWT test.
Keywords: Nociceptin/orphanin FQ; Opioid agonists; Antinociception; Cold water tail-flick test; (Rats);

Subchronic nicotine exposure in adolescence induces long-term effects on hippocampal and striatal cannabinoid-CB1 and mu-opioid receptors in rats by Eva M. Marco; Oleg Granstrem; Enrique Moreno; Ricardo Llorente; Walter Adriani; Giovanni Laviola; Maria-Paz Viveros (37-43).
There is evidence for the existence of functional interactions between nicotine and cannabinoids and opioid compounds in adult experimental animals. However, there is scarce information about these relationships in young animals. In the present study we evaluated short and long-term effects of a subchronic nicotine treatment [0.4 mg/kg daily i.p. injections from postnatal day (PND) 34 to PND 43], upon hippocampal and striatal cannabinoid-CB1 and mu-opioid receptors in Wistar rats of both genders. Rats were sacrificed 2 h after the last nicotine injection (short-term effects, PND 43) or one month later (long-term effects, PND 75). Hippocampal and striatal cannabinoid CB1 and mu-opioid receptors were quantified by Western blotting. The subchronic nicotine treatment induced a region-dependent long-lasting effect in cannabinoid CB1 receptor: a significant increase in hippocampal cannabinoid CB1 receptors and a significant decrease in striatal cannabinoid CB1 receptors, with these effects being similar in males and females. With respect to mu-opioid receptors, subchronic nicotine induced a significant down-regulation in hippocampal and striatal mu-opioid receptors in the long-term, and within the striatum the effects were more marked in adult males than in females. The present results indicate that juvenile nicotine taking may have implications for the endocannabinoid and endogenous opioid function and for the behaviors served by those systems, this includes possible modification of the response of adults to different psychotropic drugs, i.e. cannabis and morphine/heroin when taken later in life.
Keywords: Nicotine; Adolescent rats; Cannabinoid CB1 receptor; mu-opioid receptor; Hippocampus; Striatum;

Assessment of anandamide's pharmacological effects in mice deficient of both fatty acid amide hydrolase and cannabinoid CB1 receptors by Laura E. Wise; Christopher C. Shelton; Benjamin F. Cravatt; Billy R. Martin; Aron H. Lichtman (44-48).
In the present study, we investigated whether anandamide produces its behavioral effects through a cannabinoid CB1 receptor mechanism of action. The behavioral effects of anandamide were evaluated in mice that lacked both fatty acid amide hydrolase (FAAH) and cannabinoid CB1 receptors (DKO) as compared to FAAH (−/−), cannabinoid CB1 (−/−), and wild type mice. Anandamide produced analgesia, catalepsy, and hypothermia in FAAH (−/−) mice, but failed to elicit any of these effects in the other three genotypes. In contrast, anandamide decreased locomotor behavior regardless of genotype, suggesting the involvement of multiple mechanisms of action, including its products of degradation. These findings indicate that the cannabinoid CB1 receptor is the predominant target mediating anandamide's behavioral effects.
Keywords: Cannabinoid CB1 receptor; FAAH [Fatty acid amide hydrolase]; N-arachidonoyl ethanolamine (anandamide); Pain; Analgesia; Marijuana;

Participation of CGRP and prostanoids in the sex-linked differences of vascular anandamide effects in mesenteric beds of Sprague-Dawley rats by Roxana N. Peroni; Maria L. Orliac; Tamara Abramoff; Maria L. Ribeiro; Ana M. Franchi; Edda Adler-Graschinsky (49-57).
The in vitro exposure to anandamide elicits greater relaxations in mesenteric beds isolated from female compared to male rats. The present work shows that in mesenteric beds precontracted with noradrenaline the removal of endothelium increased the relaxation caused by anandamide in male and ovariectomized female but not in sham-operated female rats. The nitric oxide synthase inhibition with 100 μM N ω-nitro-l-arginine methyl ester hydrochloride (l-NAME) and the sensory in vivo denervation through neonatal administration of capsaicin also reduced anandamide-induced relaxations but these effects had the same extent in male and in female mesenteries. The content of calcitonin gene related peptide (CGRP) in mesenteric beds, that was higher in intact female than in male rats, was reduced by ovariectomy and restored to control values 21 days after a 3 weekly i.m. administration of 450 μg/kg 17β-oestradiol. This latter treatment also increased CGRP content in mesenteries from males up to the same levels observed in females. The basal release of CGRP in mesenteric beds was equivalent in either sex, but the exposure to anandamide increased CGRP release solely in female mesenteries. The ratio prostacyclin/thromboxane A2 was selectively reduced in mesenteries from male rats after exposure to anandamide, due to the decrease of the tissue levels of prostacyclin. Moreover, the cyclooxygenase-2 inhibitor 0.1 μM N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulphonamide (NS-398) diminished the relaxations caused by anandamide solely in female rats. It is proposed that relaxing factors such as CGRP and prostacyclin contribute to the higher relaxations caused by anandamide in the vasculature of female rats.
Keywords: Anandamide; CGRP; Prostacyclin; 17β-oestradiol; Cyclooxygenase-2; Vasorelaxation;

Anticlastogenic activity of morin against whole body gamma irradiation in Swiss albino mice by Vipan Kumar Parihar; Koiram Rajanna Prabhakar; Veeresh Prabhakar Veerapur; Kavirayani Indira Priyadarsini; Mazhuvancherry Kesavan Unnikrishnan; Chamallamudi Mallikajuna Rao (58-65).
Anticlastogenic activity of morin was explored against whole body gamma radiation, at a dose rate of 1.66 Gy/min in Swiss albino mice pretreated intraperitoneal or orally. Pretreatment with morin 10, 25, 50, 75, 100, 125, and 150 mg/kg, i.p. delayed and reduced percentage mortality and increased mean survival times in mice irradiated with 10 Gy gamma radiation. Intraperitoneal route was found superior to oral route. An i.p. dose of 100 mg/kg was found to be the most effective dose in preventing radiation-induced weight loss, increasing the mean survival times and reducing percentage mortality. Morin (100 mg/kg) pretreatment effectively maintained spleen index (spleen weight / body weight × 100) and stimulated endogenous spleen colony forming units. Pretreatment with morin (100 mg/kg) significantly reduced dead, inflammatory, and mitotic cells in irradiated mice jejunum along with a significant increase in goblet cells and rapidly multiplying crypt cells. Morin (100 mg/kg) also maintained the villus height close to normal, prevented mucosal erosion and basement membrane damage in irradiated jejunum. Nuclear enlargement in epithelial cells of jejunum was lower in morin treated mice compared to radiation control. Morin (100 mg/kg) also significantly elevated the endogenous antioxidant enzymes viz. glutathione S transferase (GST), superoxide dismutase (SOD) and reduced glutathione (GSH), in normal mice at 2, 4 and 8 h post treatment. Drastic decrease in endogenous enzymes (GSH, GST, catalase and SOD) and total thiols was observed in irradiated mice at 2, 4 and 8 h post irradiation, while pretreatment with morin (100 mg/kg) prevented this decrease. Morin (100 mg/kg) also elevated radiation LD50 from 9.2 to 10.1 Gy, indicating a dose modifying factor (DMF) of 1.11.
Keywords: Morin; Radioprotection; Jejunum; Spleen index; Glutathione; GST; SOD catalase; Total thiol; Dose modifying factor;

Inhaled anandamide reduces leukotriene D4-induced airway obstruction in guinea pigs by Peter W. Stengel; Sandra L. Cockerham; Steven A. Silbaugh (66-68).
In guinea pigs, we found that intravenous 5,8,11,14-eicosatetraenamide (N-2-hydroxyethyl), arachidonylethanolamide (anandamide), 0.3–10.0 mg/kg, did not inhibit leukotriene D4 (LTD4)-induced airway obstruction, while inhaled anandamide, 21.8 and 43.6 μg/kg (estimated inhaled doses), significantly reduced this airway obstructive effect by 24.8 ± 8.8 and 42.0 ± 11.2%, respectively. In contrast, aerosolized anandamide (43.6 μg/kg) was ineffective against histamine-induced bronchoconstriction. Thus, inhaled, but not intravenous anandamide selectively antagonizes the bronchospasm produced by LTD4.
Keywords: Asthma; Eicosanoid; Endocannabinoid;

Oxidative renal damage in pyelonephritic rats is ameliorated by montelukast, a selective leukotriene CysLT1 receptor antagonist by Halil Tuğtepe; Göksel Şener; Şule Çetinel; Ayliz Velioğlu-Öğünç; Berrak Ç. Yeğen (69-75).
Urinary tract infections may induce severe inflammation, transient impairment in renal function and scar formation, ranging in severity from acute symptomatic pyelonephritis to chronic pyelonephritis, which have a potential to lead to renal failure and death. The present study aimed to investigate the possible protective effect of montelukast, a selective antagonist of cysteinyl leukotriene receptor 1 (leukotriene CysLT1), against Escherichia coli-induced oxidative injury and scarring in renal tissue. Wistar rats were injected 0.1 ml of E. coli (ATCC 25922 1010 cfu/ml) or saline into left renal medullae. Six rats were assigned as the sham group and were given 0.1 ml 0.9% NaCl. Pyelonephritic rats were treated with either saline or montelukast immediately after surgery and at daily intervals. Twenty-four hours or one week after E. coli injection, rats were decapitated and the kidney samples were taken for histological examination or determination of renal malondialdehyde, glutathione (GSH) levels, myeloperoxidase (MPO) activity, and collagen contents. Formation of reactive oxygen species in renal tissue samples was monitored by using chemiluminescence technique with luminol and lucigenin probes. Creatinine, blood urea nitrogen and lactate dehydrogenase (LDH) activity were measured in the serum samples. E. coli inoculation caused significant increases in malondialdehyde level, MPO activity, chemiluminescence levels and collagen content, while GSH level was decreased in the renal tissues (p  < 0.05–0.001). On the other hand, serum TNF-alpha, LDH, blood urea nitrogen and serum creatinine levels were elevated in the pyelonephritic rats as compared to control group. Leukotriene CysLT1 receptor antagonist montelukast reversed all these biochemical indices, as well as histopathological alterations, that were induced by acute pyelonephritis. It seems likely that montelukast protects kidney tissue by inhibiting neutrophil infiltration, balancing oxidant–antioxidant status, and regulating the generation of inflammatory mediators suggesting a future role for leukotriene CysLT1 receptor antagonists in the treatment of pyelonephritis.
Keywords: Escherichia coli; Montelukast; Glutathione; Lipid peroxidation; Myeloperoxidase activity; TNF-α;

Anti-inflammatory mechanism of simvastatin in mouse allergic asthma model by Dae Yong Kim; Su Youn Ryu; Ji Eun Lim; Yun Song Lee; Jai Youl Ro (76-86).
Statins have anti-inflammatory property and immunomodulatory activity. In this study we aimed to investigate the inhibitory mechanism of simvastatin in allergic asthmatic symptoms in mice. BALB/c mice were sensitized and challenged by ovalbumin to induce asthma. Ovalbumin-specific serum IgE levels were measured by enzyme-linked immunosorbent assay (ELISA), and the recruitment of inflammatory cells into bronchoalveolar lavage fluid or lung tissues was measured by Diff-Quik staining and hematoxylin and eosin (H&E) staining, respectively, the expressions of CD40, CD40 ligand (CD40L), and vascular cell adhesion molecule-1 (VCAM-1) by immunohistochemistry, the mRNA and protein expressions of cytokines in lung tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) or ELISA, epithelial hyperplasia by periodic acid-Schiff (PAS) staining, activities of matrix metalloproteinases (MMPs) by zymography, the activities of small G proteins, mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB) in bronchoalveolar lavage cells and lung tissues by western blot and EMSA, respectively. Simvastatin reduced ovalbumin-specific IgE level, the number of total inflammatory cells, macrophages, neutrophils, and eosinophils into bronchoalveolar lavage fluid, the expressions of CD40, CD40L or VCAM-1, the mRNA and protein levels of interleukin (IL)-4, IL-13 and tumor necrosis factor (TNF)-α, the numbers of goblet cells, activities of MMPs, and further small G proteins, MAP kinases and NF-κB activities in bronchoalveolar lavage cells and lung tissues increased in ovalbumin-induced allergic asthma in mice. Our data suggest that simvastatin may be used as a therapeutic agent in asthma, based on reductions of various allergic responses via regulating small G proteins/MAP kinases/NF-κB in mouse allergic asthma.
Keywords: Allergic asthma; CD40 molecules; Adhesion molecules; Rho protein family; MAP kinases; NF-κB;