European Journal of Pharmacology (v.578, #1)

Phosphorylation, desensitization and internalization of human α1B-adrenoceptors induced by insulin-like growth factor-I by Tzindilú Molina-Muñoz; M. Teresa Romero-Ávila; S. Eréndira Avendaño-Vázquez; J. Adolfo García-Sáinz (1-10).
The effect of insulin-like growth factor-I (IGF-I) on human α1B-adrenoceptor function, phosphorylation state and cellular location was studied. Rat-1 fibroblasts were transfected with a plasmid construction containing enhanced green fluorescent protein joined to the carboxyl terminus of the human α1B-adrenoceptor. Receptors were identified by radioligand binding and photoaffinity labeling, and were immunoprecipitated with an antiserum generated against the enhanced green fluorescent protein. The receptor was functional, as evidenced by noradrenaline action on intracellular calcium and inositol phosphate production. IGF-I had no significant effect by itself on these parameters but markedly reduced the effects of noradrenaline. IGF-I induced α1B-adrenoceptor phosphorylation, which was markedly reduced by the following agents: pertussis toxin, a metalloproteinase inhibitor, diphtheria toxin mutant CRM 197, an epidermal growth factor (EGF) receptor intrinsic kinase activity inhibitor, and by phosphoinositide 3-kinase and protein kinase C inhibitors. IGF-I action appears to involve activation of a pertussis toxin-sensitive G protein, shedding of heparin-binding EGF and autocrine activation of EGF receptors. G protein subunits and phosphotyrosine residues stimulate phosphoinositide 3-kinase activity leading to activation of protein kinase C, which in turn phosphorylates α1B-adrenoceptors. Confocal fluorescent microscopy showed that α1B-adrenoceptors fussed to the green fluorescent protein were located in plasma membrane and intracellular vesicles in the basal state. IGF-I induced receptor redistribution favoring the intracellular location; this effect was blocked by hypertonic sucrose and concanavalin A. Our data show that IGF-I induces α1B-adrenoceptor desensitization associated to receptor phosphorylation and internalization.
Keywords: α1B-adrenoceptor; IGF-I; Receptor phosphorylation; Receptor internalization; Desensitization;

Cadmium induces apoptotic cell death through p38 MAPK in brain microvessel endothelial cells by Yi-Sook Jung; Euy-Myoung Jeong; Eun Kyung Park; You-Mie Kim; Seonghyang Sohn; Soo Hwan Lee; Eun Joo Baik; Chang-Hyun Moon (11-18).
Cadmium (Cd), an ubiquitous heavy metal, is known to be accumulated outside of the blood–brain barrier. In this study, we investigated whether Cd has cytotoxicity in mouse brain microvascular endothelial cells (bEnd.3). Results from the cell viability assay showed that Cd caused a remarkable decrease in cell viability in a dose-dependent manner. The cell death induced by Cd appeared to involve apoptosis, based on our results from annexin V staining, electron microscopy and TUNEL staining. And the cell death induced by Cd was inhibited by caspase inhibitor ZVAD-fmk. To further investigate the mechanism of the Cd-induced cell death, we examined the effects of selective inhibitors for mitogen activated protein kinase (MAPK) pathways on the cell death. The Cd-induced cell death was significantly inhibited by p38 MAPK inhibitor SB202190, but not by either, c-Jun N-terminal kinase (JNK) inhibitor SP600125 or extracellular signal-regulated kinase (ERK) inhibitor U0126. Phosphorylations of p38 MAPK, JNK and ERK were stimulated by treatment with CdCl2. In summary, our results suggest that Cd can induce apoptotic cell death, at least in part, through the p38 MAPK pathway in brain microvascular endothelial cells.
Keywords: Cadmium; Brain microvascular endothelial cells; Apoptosis; p38 MAPK;

Single exposure to erythropoietin modulates Nerve Growth Factor expression in the spinal cord following traumatic injury: Comparison with methylprednisolone by Fabio Fumagalli; Laura Madaschi; Paola Brenna; Lucia Caffino; Giovanni Marfia; Anna Maria Di Giulio; Giorgio Racagni; Alfredo Gorio (19-27).
Acute lesions of the spinal cord lead to dramatic changes in neuronal function. In the present study, we examined the possible involvement of neurotrophic factors in the action of the drug of choice for the treatment of such an emergency, i.e. the glucocorticoid methylprednisolone is compared to erythropoietin, a cytokine recently shown to markedly shorten the time necessary for motor recovery following injury [Gorio, A., Gokmen, N., Erbayraktar, S., Yilmaz, O., Madaschi, L., Cichetti, C., Di Giulio, A.M., Vardar, E., Cerami, A., Brines, M., 2002. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc. Natl. Acad. Sci. 99, 9450–9455]. We found that methylprednisolone reduces the lesion-enhanced Nerve Growth Factor (NGF) mRNA levels 3 h after injury in the trauma epicenter and caudal section of the cord whereas erythropoietin reinforced the NGF gene expression. Three days after the occurrence of the lesion, erythropoietin, but not methylprednisolone, significantly up-regulated the NGF gene expression both caudally and rostrally to the lesion site, an effect that, based on the chemo-attractant properties of neurotrophin, might facilitate the growth of injured axons toward NGF-rich sites and contribute to the enhancement of the regenerative process. The differences between the effects of methylprednisolone and erythropoietin dissipate 7 days after the lesion when they both enhance NGF mRNA levels at the epicenter. These data show that methylprednisolone and erythropoietin display a different pattern of activation of the neurotrophin NGF which is strictly dependent on the portion of the cord examined and the time elapsed from the injury. Based on our results, we suggest that the higher increase of NGF expression mediated by erythropoietin soon after the injury might explain, at least in part, the improved recovery of motor functions produced by erythropoietin compared to methylprednisolone and saline.
Keywords: Regeneration; Neuroplasticity; Neurotrophic factor; Gene expression;

We examined the effect of ginsenoside Rg1 or Rb1, the active ingredients of ginseng, on the release of endogenous glutamate from glutamatergic nerve terminals purified from rat cerebral cortex. Result showed that the Ca2+-dependent release of glutamate evoked by 4-aminopyridine was facilitated by ginsenoside Rg1 or Rb1 in a concentration-dependent manner. Sequential experiments reveal that ginsenoside Rg1 or Rb1-mediated facilitation of glutamate release (i) results from an enhancement of vesicular exocytosis; (ii) is not due to an alternation of synaptosomal excitability; (iii) is associated with an increase in Ca2+ influx through presynaptic N- and P/Q-type voltage-dependent Ca2+ channels; (iv) appears to involve a protein kinase A pathway. These results conclude that ginsenoside Rg1 or Rb1 exerts their presynaptic facilitatory effect, likely through the activation of protein kinase A, which subsequently enhances Ca2+ entry to cause an increase in evoked glutamate release from rat cortical synaptosomes. This finding might provide important information regarding the action of ginseng in the central nervous system.
Keywords: Ginsenoside; Glutamate exocytosis; Voltage-dependent Ca2+ channel; Protein kinase A; Cerebrocortical synaptosomes;

Female rats are more sensitive than males to many behavioral effects of cannabinoids. The purpose of the present study was to determine if sex differences in the antinociceptive and motoric effects of Δ9-tetrahydrocannabinol (THC) are due to activational effects of gonadal steroid hormones. THC-induced antinociception (tail withdrawal, paw pressure tests) and motoric effects (horizontal locomotion, catalepsy) were compared in male and female gonadectomized rats that were chronically treated with hormone (testosterone in males, estradiol in females) vs. those that were gonadectomized and had no hormone replacement. THC's effects were also compared between gonadally intact females tested during vaginal estrus vs. diestrus. THC (5 and 10 mg/kg i.p.) produced very similar antinociceptive effects in no-hormone vs. testosterone-treated males, but significantly less locomotor suppression in testosterone-treated males than those with no hormone replacement. In gonadectomized females, estradiol enhanced THC's antinociceptive but not motoric effects. In gonadally intact, cycling females, 5 mg/kg THC produced slightly to significantly greater behavioral effects in estrous than in diestrous females. These results suggest that sex differences in THC-induced behavioral effects in the adult rat can be attributed to activational effects of testosterone in males and/or estradiol in females.
Keywords: Sex differences; Analgesia; Catalepsy; Locomotor activity; Cannabinoid; Estrogen; Testosterone;

The antidepressant effects of curcumin in the forced swimming test involve 5-HT1 and 5-HT2 receptors by Rui Wang; Ying Xu; Hong-Li Wu; Ying-Bo Li; Yu-Hua Li; Jia-Bin Guo; Xue-Jun Li (43-50).
Curcuma longa is a main constituent of many traditional Chinese medicines, such as Xiaoyao-san, used to manage mental disorders effectively. Curcumin is a major active component of C. longa and its antidepressant-like effect has been previously demonstrated in the forced swimming test. The purpose of this study was to explore the possible contribution of serotonin (5-HT) receptors in the behavioral effects induced by curcumin in this animal model of depression. 5-HT was depleted by the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA, 100 mg/kg, i.p.) prior to the administration of curcumin, and the consequent results showed that PCPA blocked the anti-immobility effect of curcumin in forced swimming test, suggesting the involvement of the serotonergic system. Moreover, pre-treatment of pindolol (10 mg/kg, i.p., a β-adrenoceptors blocker/5-HT1A/1B receptor antagonist), 4-(2′-methoxy-phenyl)-1-[2′-(n-2″-pyridinyl)-p-iodobenzamino-]ethyl-piperazine (p-MPPI, 1 mg/kg, s.c., a selective 5-HT1A receptor antagonist), or 1-(2-(1-pyrrolyl)-phenoxy)-3-isopropylamino-2-propanol (isamoltane, 2.5 mg/kg, i.p., a 5-HT1B receptor antagonist) was found to prevent the effect of curcumin (10 mg/kg) in forced swimming test. On the other hand, a sub-effective dose of curcumin (2.5 mg/kg, p.o.) produced a synergistic effect when given jointly with (+)-8-hydroxy-2-(di-n-propylamino)tetralin, (8-OH-DPAT, 1 mg/kg, i.p., a 5-HT1A receptor agonist), anpirtoline (0.25 mg/kg, i.p., a 5-HT1B receptor agonist) or ritanserin (4 mg/kg, i.p., a 5-HT2A/2C receptor antagonist), but not with ketanserin (5 mg/kg, i.p., a 5-HT2A/2C receptor antagonist with higher affinity to 5-HT2A receptor) or R(-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 1 mg/kg, i.p., a 5-HT2A receptor agonist). Taken together, these results indicate that the antidepressant-like effect of curcumin in the forced swimming test is related to serotonergic system and may be mediated by, at least in part, an interaction with 5-HT1A/1B and 5-HT2C receptors.
Keywords: Curcumin; Depression; Antidepressant; Forced swimming test; 5-HT1A/1B receptors; 5-HT2A/2C receptors;

Surgical flaps are commonly used in the reconstruction of tissue defects after tumour surgery and trauma. Flap failure continues to be a clinical problem and the underlying causes are not fully understood.In the present study a system that generates nitric oxide (NO) in a non-enzymatic fashion was created through the acidification with vitamin C of a cream containing increasing concentrations (0.125%, 0.25%, 0.5%, 1.25% and 2.5%) of nitrite (NO2 ). The cream was applied for 30 min to a modified epigastric island skin flap in the rat. Blood flow in the supplying artery was measured by transit-time ultrasound flowmetry throughout the experiment and superficial skin blood flow was measured by laser Doppler perfusion imaging before and after treatment. Mean arterial blood pressure was also monitored. NO and the gas nitrogen dioxide (NO2), which is formed when NO reacts with atmospheric oxygen, were measured above the cream using chemiluminescence.In flaps treated with the NO generating cream, a concentration-dependent increase in blood flow in the supplying artery and flap skin of up to 130% was observed. Cream base alone or cream base acidified with vitamin C had no effect on blood flow. Also, concentration-dependent formation of both NO and NO2 was seen.NO increases both supplying artery blood flow and superficial cutaneous blood flow in an epigastric island skin flap model in the rat indicating that NO is of importance in flap physiology and possibly also for flap survival.
Keywords: Surgical flaps; Nitric oxide; Laser Doppler; Transit-time ultrasound flowmetry;

Ursodeoxycholic acid protects concanavalin A-induced mouse liver injury through inhibition of intrahepatic tumor necrosis factor-α and macrophage inflammatory protein-2 production by Kaoru Ishizaki; Tomomichi Iwaki; Shuji Kinoshita; Mamoru Koyama; Atsushi Fukunari; Hideki Tanaka; Makoto Tsurufuji; Kei Sakata; Yasuhiro Maeda; Teruaki Imada; Kenji Chiba (57-64).
Ursodeoxycholic acid (UDCA) is widely used for the therapy of liver dysfunction. In this study, we investigated the protective effect of UDCA in concanavalin A-induced mouse liver injury. The treatment with UDCA at oral doses of 50 and 150 mg/kg at 2 h before concanavalin A injection significantly reduced the elevated plasma levels of aminotransferases and the incidence of liver necrosis compared with concanavalin A-injected control group without affecting the concentrations of liver hydrophobic bile acids. UDCA significantly inhibited elevated levels of tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2), and interleukin 6 (IL-6) in blood of concanavalin A-injected mice. To clarify the influence of UDCA on production of cytokines, we examined intrahepatic mRNA expressions and the protein levels of TNF-α, MIP-2, interferon-γ (IFN-γ), IL-4, IL-6, and IL-10 at 1 h after concanavalin A injection. The treatment with UDCA significantly decreased the intrahepatic levels of TNF- α and MIP-2, whereas this compound showed no clear effect on IFN-γ, IL-4, IL-6, or IL-10. Furthermore, UDCA significantly decreased myeloperoxidase activity as well as MIP-2 level in the liver and histological examination of liver tissue revealed that intrasinusoidal accumulation of neutrophils was decreased markedly by UDCA. In addition, UDCA significantly inhibited the production of TNF-α and MIP-2 when cultured with nonparenchymal and lymph node cells. In conclusion, these findings suggest that UDCA protects concanavalin A-induced liver injury in mice by inhibiting intrahepatic productions of TNF-α and MIP-2, and the infiltration of neutrophils into the liver.
Keywords: Concanavalin A; Liver bile acid; Liver nonparenchymal cell; Lymph node cell; Macrophage inflammatory protein-2; Neutrophil; Tumor necrosis factor-α; Ursodeoxycholic acid;

Methacholine-induced pulmonary gas trapping in a mouse model of allergic asthma: Effect of inhaled budesonide and ciglitazone by Peter W. Stengel; Douglas J. Zeckner; Wen-Kai Kevin Guo; Jeffrey A. Wolos; David W. Snyder (65-75).
Previously, we found pulmonary gas trapping to be a rapid, simple and objective measure of methacholine-induced airway obstruction in naïve mice. In this study we extended that finding by using methacholine-induced pulmonary gas trapping to differentiate airway responses of ovalbumin-sensitized, ovalbumin-exposed (Positive Control) and ovalbumin-sensitized, sodium chloride-exposed (Negative Control) mice. Additionally, pulmonary gas trapping and enhanced pause were compared following methacholine exposure in sensitized and nonsensitized mice. Finally, we examined by nose-only inhalation the ability of the glucocorticosteroid budesonide and the peroxisome proliferator-activated receptor-γ agonist ciglitazone to modify methacholine-induced airway responses in ovalbumin-sensitized mice. Positive Controls exhibited a 7.8-fold increase in sensitivity and a 2.4-fold enhancement in the maximal airway obstruction to methacholine versus Negative Controls. Following methacholine, individual Positive and Negative Control mouse enhanced pause values overlapped in 9 of 9 studies, whereas individual Positive and Negative Control mouse excised lung gas volume values overlapped in only 1 of 9 studies, and log[excised lung gas volume] correlated (P  = 0.023) with in vivo log[enhanced pause] in nonsensitized mice. Finally, budesonide (100.0 or 1000.0 μg/kg) reduced methacholine-mediated airway responses and eosinophils and neutrophils, whereas ciglitazone (1000.0 μg/kg) had no effect on methacholine-induced pulmonary gas trapping, but reduced eosinophils. In conclusion, pulmonary gas trapping is a more reproducible measure of methacholine-mediated airway responses in ovalbumin-sensitized mice than enhanced pause. Also, excised lung gas volume changes can be used to monitor drug interventions like budesonide. Finally, this study highlights the importance of running a positive comparator when examining novel treatments like ciglitazone.
Keywords: Asthma; Airway inflammation; Airway obstruction; Airway resistance; Inhaled glucocorticosteroids; Peroxisome proliferator-activated receptor-γ agonist;

Preclinical pharmacology profile of CS-706, a novel cyclooxygenase-2 selective inhibitor, with potent antinociceptive and anti-inflammatory effects by Shigeru Ushiyama; Tomoko Yamada; Yukiko Murakami; Sei-ichiro Kumakura; Shin-ichi Inoue; Keisuke Suzuki; Akira Nakao; Akihiro Kawara; Tomio Kimura (76-86).
We report here the preclinical anti-inflammatory profile of CS-706 [2-(4-ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole], a novel cyclooxygenase-2 (COX-2) selective inhibitor. CS-706 selectively inhibited COX-2 in a human whole blood assay with an IC50 of 0.31 μM, compared with an IC50 of 2.2 μM for COX-1. The selectivity ratio of CS-706 was higher than those of the conventional non-steroidal anti-inflammatory drugs naproxen, indomethacin, and Diclofenac–Na, whereas it was lower than those of rofecoxib, valdecoxib and etoricoxib. It was similar to that of celecoxib. The pharmacokinetic profile of CS-706 showed rapid absorption and dose-proportional exposure after oral administration to rats. CS-706 inhibited prostaglandin E2 production in inflamed tissue induced by yeast-injection in rats with potency similar to that of indomethacin. However, it inhibited gastric mucosal prostaglandin E2 production in normal rats weakly compared with indomethacin. CS-706 ameliorated both yeast-induced inflammatory acute pain (ED50  = 0.0090 mg/kg) and adjuvant-induced chronic arthritic pain (ED50  = 0.30 mg/kg) in rats. CS-706 showed more potent antinociceptive activity than celecoxib and rofecoxib in these models. In an adjuvant-induced arthritic model in rats, CS-706 suppressed foot swelling prophylactically with an ID50 of 0.10 mg/kg/day, and decreased foot swelling in the established arthritis therapeutically in a dose range of 0.040 to 1.0 mg/kg/day. Single administration of up to 100 mg/kg of CS-706 induced no significant gastric lesions in rats. In conclusion, CS-706 is a COX-2-selective inhibitor with a potent antinociceptive and anti-inflammatory activity and a gastric safety profile.
Keywords: Cyclooxygenase-2; COX-2-selective inhibitor; CS-706 [2-(4-ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole]; Inflammation; Nociception; Non-steroidal anti-inflammatory drug;

Repeated instillations of Dermatophagoides farinae into the airways can induce Th2-dependent airway hyperresponsiveness, eosinophilia and remodeling in mice by Keiko Wakahara; Hiroyuki Tanaka; Go Takahashi; Mayumi Tamari; Reishi Nasu; Tatsuyuki Toyohara; Hirohisa Takano; Saburo Saito; Naoki Inagaki; Kaoru Shimokata; Hiroichi Nagai (87-96).
Dermatophagoides farinae are known to be a common environmental allergen causing allergic asthma; however, little is known about their pathophysiological effect via the allergenicities in vivo. Therefore, we first established a mouse model of asthma induced by repeated instillations of D. farinae. Second, to investigate whether the asthmatic responses are Th2-dependent, we examined the effect of the deficiency of interleukin-4 (IL-4) receptor α chain gene. Finally, we examined the effect of fluticasone propionate on this model. Mice were instilled with D. farinae without additional adjuvants into the trachea 8 times. After the final allergen instillation, the airway responsiveness to acetylcholine was measured, and bronchoalveolar lavage and histological examination were carried out. The instillation of the allergen-induced airway hyperresponsiveness, the accumulation of inflammatory cells and increases in the levels of Th2 cytokines and transforming growth factor-β1 production in the bronchoalveolar lavage fluid dose dependently. The number of goblet cells in the epithelium and the extent of the fibrotic area beneath the basement membrane were also increased in the morphometric study. In contrast, the defect of IL-4/IL-13 signaling through IL-4 receptor α chain completely abrogated all these responses. Furthermore, the simultaneous instillation of fluticasone propionate with the allergen showed significant inhibition or an inhibitory tendency of these changes. These findings demonstrate that the repetitive intratracheal instillations of D. farinae can induce airway remodeling through Th2-type inflammation, and that fluticasone propionate inhibits D. farinae-induced airway remodeling in mice, and this model would be useful for studying mechanisms involved in the development of allergic asthma.
Keywords: Airway; Asthma; Corticosteroid; Dermatophagoides farinae; Eosinophil; Goblet cell; IL-13; Mouse; Subepithelial fibrosis;

The purpose of this study is to clarify involvement ratios between central and peripheral cyclooxygenase (COX)-2 in rat inflammatory pain models, by evaluating celecoxib and [6-chloro-2-(4-chlorobenzoyl)-1H-indol-3-yl]acetic acid (CIAA) on carrageenan-induced mechanical and thermal hyperalgesia. Celecoxib and CIAA exhibited ID30 values with 1.5 and 7.7 mg/kg on mechanical hyperalgesia, respectively, and ID25 values with 0.54 and 36 mg/kg on thermal hyperalgesia, respectively. By solving quadratic functional analysis with prostaglandin E2 (PGE2) inhibitory activities, it was calculated that involvement ratios between central and peripheral COX-2 involvement were 0.47 and 0.53 on mechanical hyperalgesia, and 0.97 and 0.03 on thermal hyperalgesia, respectively. These data suggest that central and peripheral COX-2 are equally involved in mechanical hyperalgesia, while central COX-2 is predominantly involved in thermal hyperalgesia.
Keywords: COX-2; Hyperalgesia; Involvement ratio; CNS; Periphery;