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Biochemical Pharmacology (v.75, #11)

Editorial Advisory Board (pp. iii).

Dopaminergic signaling in dendritic spines by Wei-Dong Yao; Roger D. Spealman; Jingping Zhang (pp. 2055-2069).

Dopamine regulates movement, motivation, reward, and learning and is implicated in numerous neuropsychiatric and neurological disorders. The action of dopamine is mediated by a family of seven-transmembrane G protein-coupled receptors encoded by at least five dopamine receptor genes (D1, D2, D3, D4, and D5), some of which are major molecular targets for diverse neuropsychiatric medications. Dopamine receptors are present throughout the soma and dendrites of the neuron, but accumulating ultrastructural and biochemical evidence indicates that they are concentrated in dendritic spines, where most of the glutamatergic synapses are established. By modulating local channels, receptors, and signaling modules in spines, this unique population of postsynaptic receptors is strategically positioned to control the excitability and synaptic properties of spines and mediate both the tonic and phasic aspects of dopaminergic signaling with remarkable precision and versatility. The molecular mechanisms that underlie the trafficking, targeting, anchorage, and signaling of dopamine receptors in spines are, however, largely unknown. The present commentary focuses on this important subpopulation of postsynaptic dopamine receptors with emphases on recent molecular, biochemical, pharmacological, ultrastructural, and physiological studies that provide new insights about their regulatory mechanisms and unique roles in dopamine signaling.

Keywords: Dopamine receptor; Synaptic plasticity; Extrasynaptic transmission; Dendritic spines; Postsynaptic density

Sleep homeostasis: A role for adenosine in humans? by Hans-Peter Landolt (pp. 2070-2079).

Sleep is not the mere absence of wakefulness, but an active state which is finely regulated. The homeostatic facet of sleep–wake regulation is keeping track of changes in ‘sleep propensity’ (or ‘sleep need’), which increases during wakefulness and decreases during sleep. Increased sleep propensity following extended prior wakefulness (sleep deprivation) is counteracted by prolonged sleep duration, but also by enhanced non-rapid-eye-movement (nonREM) sleep intensity as measured by electroencephalographic (EEG) slow-wave activity (SWA, power within ∼1–4Hz). This highly reliable regulatory feature of nonREM sleep may be the most important aspect of sleep in relation to its function. The neurochemical mechanisms underlying nonREM sleep homeostasis are poorly understood. Here we provide compelling and convergent evidence that adenosinergic neurotransmission plays a role in nonREM sleep homeostasis in humans. Specifically, a functional polymorphism in the adenosine metabolizing enzyme, adenosine deaminase, contributes to the high inter-individual variability in deep slow-wave sleep duration and intensity. Moreover, the adenosine receptor antagonist, caffeine, potently attenuates the EEG markers of nonREM sleep homeostasis during sleep, as well as during wakefulness. Finally, adenosinergic mechanisms modulate individual vulnerability to the detrimental effects of sleep deprivation on neurobehavioral performance, and EEG indices of disturbed sleep after caffeine consumption. While these convergent findings strongly support an important contribution of adenosine and adenosine receptors to nonREM sleep homeostasis, further research is needed to elucidate the underlying mechanisms that mediate the actions of adenosine on sleep and the sleep EEG.

Keywords: Sleep regulation; Electroencephalogram (EEG); Caffeine; A; ₁; receptor; A; _2A; receptor

The cytotoxicity of a Grb2-SH3 inhibitor in Bcr-Abl positive K562 cells by Yun-Bin Ye; Jian-Yin Lin; Qiang Chen; Fang Liu; Hui-Jing Chen; Jie-Yu Li; Wang-Qing Liu; Christiane Garbay; Michel Vidal (pp. 2080-2091).

Chronic myelogenous leukemia (CML) is characterized by the presence of Bcr-Abl oncoprotein. Gleevec has been designed to treat many CML patients by specifically targeting Bcr-Abl, but resistance to it is already apparent in many cases. In CML cells, Bcr-Abl activates several signaling pathways, including the Ras-dependent pathway, in which growth factor receptor binding 2 (Grb2) acts as an adaptor protein. A specific Grb2-SH3 inhibitor (denoted as peptidimer-c) that disrupts Grb2–Sos complex was designed and synthesized in our laboratory.In this study, we investigated the effect and the molecular mechanism of this inhibitor. Peptidimer-c was shown to bind to Grb2 in K562 cells, a cell line over-expressing Bcr-Abl oncoprotein. It caused cytotoxicity in the cells, and inhibited their ability of colony formation in the semi-solid medium. It was shown to induce apoptosis of K562 cells in a dose-dependent mode, the apoptotic effect of peptidimer-c being associated with caspase-3 activation. The effect of peptidimer-c on growth inhibition was also shown to be accompanied by S-phase arrest of cell cycle mediated by down-regulation of cyclin A and Cdk2, as well as phospho-Cdk2. The above results indicated that peptidimer-c may be another potential therapeutic agent for CML, which can induce S-phase arrest in the Bcr-Abl positive K562.

Keywords: Bcr-Abl; Chronic myelogenous leukemia; Grb2; SH3 domain; Cell cycle; Apoptosis

Treatment of lung cancer cells with cytotoxic levels of sodium selenite: Effects on the thioredoxin system by Markus Selenius; Aristi Potamitou Fernandes; Ola Brodin; Mikael Björnstedt; Anna-Klara Rundlöf (pp. 2092-2099).

Selenium at subtoxic doses has been shown to have tumor specific cytotoxic effects. In this work, viability measurements in different lung cancer cell lines showed that selenite was more effective compared to three different conventional cytotoxic drugs. In addition, the cell line most sensitive to selenite toxicity comprised the highest level of thioredoxin reductase 1 (TrxR1). The human selenoenzyme TrxR1 is a central enzyme for cell growth, differentiation, and the protection against oxidative stress. TrxR1, which in several studies has been shown to be up-regulated in various tumor cells, is also a target for many anticancer drugs. In this study, inhibition of TrxR resulted in enhanced selenite cytotoxicity, clearly connecting the thioredoxin system to the toxic effects mediated by selenite. The complex regulation of TrxR1, involving the expression of many different transcript forms of mRNA, was investigated by real-time qPCR in lung cancer cell lines following treatment with toxic doses (2.5–10μM) of sodium selenite. Selenium treatment resulted in increased expression of almost all TrxR1 mRNA variants with increasing concentrations of selenite. On the contrary, the TrxR protein level and activity, increased at low to moderate doses followed by a decrease at higher doses, indicating impairment of protein synthesis by selenite.

Keywords: Selenite; Thioredoxin reductase; Lung cancer; Cytotoxicity

Alterations in S-adenosylhomocysteine metabolism decrease O⁶-methylguanine DNA methyltransferase gene expression without affecting promoter methylation by Marina Hermes; Hermann Geisler; Hartmut Osswald; Renate Riehle; Doris Kloor (pp. 2100-2111).

The DNA repair enzyme O⁶-methylguanine DNA methyltransferase (MGMT) protects cells against the cytotoxic effects of alkylating agents. Therefore, modulation of MGMT expression in tumors is a possible strategy for improving the efficiency of cancer therapy. MGMT expression and activity is lost frequently in association with DNA hypermethylation of the MGMT promoter region. Since DNA and mRNA methylation are controlled by intracellular S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) levels, we hypothesized a role for AdoMet/AdoHcy ratio in the regulation of MGMT promoter methylation and mRNA expression.Our initial studies showed that AdoMet/AdoHcy ratios vary over a wide range (7.0–50) in different glioblastoma and hepatoma cell lines. The studied cell lines exhibit distinct MGMT promoter methylation patterns: MGMT promoter was completely unmethylated in LN-18 and Tu 132 cells, hypermethylated in LN-229, U87-MG, and Tu 113 cells, and partially methylated in HepG2 cells. Furthermore, MGMT promoter methylation patterns and global DNA methylation are not related to intracellular AdoMet/AdoHcy ratio under control conditions. To lower AdoMet/AdoHcy ratio to values <1 we used AdoHcy hydrolase inhibitor adenosine-2′,3′-dialdehyde (30μM) and found that neither short-term (24h) nor long-term changes (7 weeks) in AdoMet/AdoHcy ratio altered global or MGMT promoter methylation. However, experimentally elevated AdoHcy levels significantly decreased MGMT mRNA levels by >50% in all MGMT-expressing cell lines, which is most likely the result of impaired mRNA methylation. Thus, the present study suggests elevation of AdoHcy levels by AdoHcy hydrolase inhibition as a novel pharmacological approach to modulate MGMT expression and to increase the responsiveness to alkylating agents.

Keywords: Abbreviations; MGMT; O; ⁶; -methylguanine DNA methyltransferase; AdoMet; S; -adenosylmethionine; AdoHcy; S; -adenosylhomocysteine; DNMT; DNA methyltransferase; NCS; newborn calf serum; FCS; fetal calf serum; Adox; adenosine-2′,3′-dialdehyde; HPLC; high performance liquid chromatography; EC; energy charge; RT-PCR; reverse transcription-polymerase chain reaction O; ⁶; -Methylguanine DNA methyltransferase; Promoter methylation; S; -Adenosylhomocysteine hydrolase; DNA methylation; mRNA methylation

Inhibitory effect of acetyl-11-keto-β-boswellic acid on androgen receptor by interference of Sp1 binding activity in prostate cancer cells by Hui-Qing Yuan; Feng Kong; Xiao-Ling Wang; Charles Y.F. Young; Xiao-Yan Hu; Hong-Xiang Lou (pp. 2112-2121).

Androgen receptor (AR)-mediated signaling is crucial for the development and progression of prostate cancer (PCa). Naturally occurring phytochemicals that target the AR signaling offer significant protection against this disease. Acetyl-11-keto-β-boswellic acid (AKBA), a compound isolated from the gum-resin of Boswellia carterii, caused G1-phase cell cycle arrest with an induction of p21^WAF1/CIP1, and a reduction of cyclin D1 as well in prostate cancer cells. AKBA-mediated cellular proliferation inhibition was associated with a decrease of AR expression at mRNA and protein levels. Furthermore, the functional biomarkers used in evaluation of AR transactivity showed suppressions of prostate-specific antigen promoter-dependent and androgen responsive element-dependent luciferase activities. Additionally, down-regulation of an AR short promoter mainly containing a Sp1 binding site suggested the essential role of Sp1 for the reduction of AR expression in cells exposed to AKBA. Interruption effect of AKBA on Sp1 binding activity but not Sp1 protein levels was further confirmed by EMSA and transient transfection with a luciferase reporter driven by three copies of the Sp1 binding site of the AR promoter. Therefore, anti-AR properties ascribed to AKBA suggested that AKBA-containing drugs could be used for the development of novel therapeutic chemicals.

Keywords: Abbreviations; AR; androgen receptor; ARE; androgen responsive element; hK2; human glandular kallikrein; PSA; prostate-specific antigen; Sp1; promoter specificity protein 1Acetyl-11-keto-β-boswellic acid; Cell cycle arrest; Androgen receptor; Sp1; Prostate cancer cell lines

Deregulated expression of pro-survival and pro-apoptotic p53-dependent genes upon Elongator deficiency in colon cancer cells by Isabelle Cornez; Catherine Creppe; Magali Gillard; Benoît Hennuy; Jean-Paul Chapelle; Emmanuel Dejardin; Marie-Paule Merville; Pierre Close; Alain Chariot (pp. 2122-2134).

Elongator, a multi-subunit complex assembled by the IkappaB kinase-associated protein (IKAP)/hELP1 scaffold protein is involved in transcriptional elongation in the nucleus as well as in tRNA modifications in the cytoplasm. However, the biological processes regulated by Elongator in human cells only start to be elucidated. Here we demonstrate that IKAP/hELP1 depleted colon cancer-derived cells show enhanced basal expression of some but not all pro-apoptotic p53-dependent genes such as BAX. Moreover, Elongator deficiency causes increased basal and daunomycin-induced expression of the pro-survival serum- and glucocorticoid-induced protein kinase ( SGK) gene through a p53-dependent pathway. Thus, our data collectively demonstrate that Elongator deficiency triggers the activation of p53-dependent genes harbouring opposite functions with respect to apoptosis.

Keywords: Abbreviations; B2M; beta-2-microglobulin; BARD1; BRCA1-associated RING domain 1; BAX; BCL2-associated X protein; BTG2; B-cell translocation gene 2; CDK9; cyclin-dependent kinase 9; DMEM; Dulbecco's modified Eagle's medium; DUSP4; dual-specificity phosphatase 4; Elp; Elongator protein; EMEM; Eagle's modified essential medium; FACT; facilitates chromatin transcription; GFP; green fluorescent protein; HA; haemagglutinin; hELP1; human ELP1; IKAP; IKK-associated protein; IKK; inhibitor of kappa light chain gene enhancer in B cells kinase complex; NR2F2; nuclear receptor subfamily 2, group F, member 2; p21; cyclin-dependent kinase inhibitor 1A; PARP; poly (ADP-ribose) polymerase 1; PKIB; protein kinase, cAMP-dependent catalytic, inhibitor beta; RhoE/Rnd3; Rho family GTPase 3; SESN2; sestrin 2; SGK; serum- and glucocorticoid-induced protein kinase; shRNA; short hairpin RNA; TFIIF; general transcription factor IIF; TFIIH; general transcription factor IIH; TNFRSF10D; tumor necrosis factor receptor superfamily, member 10DIKK-associated protein (IKAP/hELP1); Elongator; p53; Familial dysautonomia; Serum- and glucocorticoid-induced protein kinase (SGK); BCL2-associated X protein (BAX)

Antibacterial and anti-atrophic effects of a highly soluble, acid stable UDCA formula in Helicobacter pylori-induced gastritis by Tran Dang Hien Thao; Ho-Cheol Ryu; Seo-Hong Yoo; Dong-Kwon Rhee (pp. 2135-2146).

Helicobacter pylori is one of the main causes of atrophic gastritis and gastric carcinogenesis. Gastritis can also occur in the absence of H. pylori as a result of bile reflux suggesting the eradication of H. pylori by bile acids. However, the bile salts are unable to eradicate H. pylori due to their low solubility and instability at acidic pH. This study examined the effect of a highly soluble and acid stable ursodeoxycholic acid (UDCA) formula on H. pylori-induced atrophic gastritis. The H. pylori infection decreased the body weight, mitochondrial membrane potential and ATP level in vivo. Surprisingly, H. pylori-induced expression of malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, at both the protein and mRNA levels. However, the UDCA formula repressed MDH expression and increased the membrane potential thereby increasing the ATP level and body weight in vivo. Moreover, UDCA scavenged the reactive oxygen species (ROS), increased the membrane potential, and inhibited apoptosis in AGS cells exposed to H₂O₂ in vitro through the mitochondria-mediated pathway. Taken together, UDCA decreases the MDH and ROS levels, which can prevent apoptosis in H. pylori-induced gastritis.

Keywords: Helicobacter Pylori; -induced atrophic gastritis; Ursodeoxycholic acid (UDCA); Mitochondria; Reactive oxygen species

Sarcoplasmic reticulum Ca²⁺ release channel ryanodine receptor (RyR₂) plays a crucial role in aconitine-induced arrhythmias by Min Fu; Ru-Xin Li; Li Fan; Guo-Wei He; Kent L. Thornburg; Zhao Wang (pp. 2147-2156).

The present study established a model of RyR₂ knockdown cardiomyocytes and elucidated the role of RyR₂ in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR₂ expression. Reduction of RyR₂ expression was documented by RT-PCR, western blot, and immunofluorescence. Ca²⁺ signals were investigated by measuring the relative intracellular Ca²⁺ concentration, spontaneous Ca²⁺ oscillations, caffeine-induced Ca²⁺ release, and L-type Ca²⁺ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]_i remained at the low level. Exposure to 3μM aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]_i and the level of caffeine-induced Ca²⁺ release were increased but the L-type Ca²⁺ currents were inhibited after application of 3μM aconitine for 5min. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]_i level; the level of caffeine-induced Ca²⁺ release was increased but L-type Ca²⁺ currents were inhibited. Alterations of RyR₂ are important consequences of aconitine-stimulation and activation of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca²⁺ leakage through the sarcoplasmic reticulum RyR₂ channel.

Keywords: Abbreviations; RyR; ₂; type 2-ryanodine receptor; KD; knockdown; e–c coupling; excitation–contraction coupling; ACO; aconitine; SR; sarcoplasmic reticulum; siRNA; small interfering RNARyR; ₂; Knockdown; Aconitine; Arrhythmia; Excitation–contraction coupling

STIM1 regulates acidic Ca²⁺ store refilling by interaction with SERCA3 in human platelets by Jose J. López; Isaac Jardín; Regis Bobe; Jose A. Pariente; Jocelyne Enouf; Gines M. Salido; Juan A. Rosado (pp. 2157-2164).

Ca²⁺ mobilization regulates a wide variety of cellular functions. Platelets posses agonist-releasable Ca²⁺ stores in acidic organelles where sarcoendoplasmic reticulum Ca²⁺-ATPase-3 (SERCA) pump is involved in store refilling. Stromal interaction molecule 1 (STIM1), which has been presented as a central regulator of platelet function, is a Ca²⁺ sensor of the intracellular Ca²⁺ stores. Here we present that STIM1 is required for acidic store refilling. Electrotransjection of cells with anti-STIM1 (Y²³¹–K²⁴³) antibody, directed towards a cytoplasmic sequence of STIM1, significantly reduced acidic store refilling, which was tested by remobilizing Ca²⁺ from the acidic stores using 2,5-di-( t-butyl)-1,4-hydroquinone (TBHQ) after a brief refilling period that followed thrombin stimulation. Platelet treatment with thrombin or thapsigargin in combination with ionomycin, to induce extensive Ca²⁺ store depletion, resulted in a transient increase in the interaction between STIM1 and SERCA3, reaching a maximum 30s after stimulation. The coupling between STIM1 and SERCA3 was abolished by electrotransjection with anti-STIM1 antibody. The interaction between STIM1 and SERCA3 induced by thrombin or by treatment with thapsigargin plus ionomycin is reduced in platelets from type 2 diabetic patients, as well as Ca²⁺ reuptake into the acidic Ca²⁺ stores. These findings provide evidence for a role of STIM1 in acidic store refilling in platelets probably acting as a Ca²⁺ sensor and regulating the activity of SERCA3. This action is impaired in platelets from type 2 diabetics, which might lead to the enhanced cytosolic Ca²⁺ concentration observed and, therefore, in platelet hyperactivity.

Keywords: STIM1; SERCA3; Ca; ²⁺; reuptake; Platelets; Thrombin; Diabetes mellitus

A cyclopentanediol analogue selectively suppresses the conserved innate immunity pathways, Drosophila IMD and TNF-α pathways by Mizuki Sekiya; Kazunori Ueda; Kaori Okazaki; Haruhisa Kikuchi; Shoichiro Kurata; Yoshiteru Oshima (pp. 2165-2174).

Innate immunity comprises evolutionarily conserved self-defense mechanisms against microbial infections. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Using Drosophila ex vivo culture systems, we isolated a cyclopentanediol analogue from Aspergillus sp. as an immunosuppressive substance. This compound selectively suppressed activation of the IMD pathway in Drosophila in vivo and the target molecules of the compound lie between the Imd adaptor protein and dTAK1 kinase in the IMD pathway. In human cells, the compound suppressed TNF-α, but not IL-1β, stimulation-induced activation of NF-κB, suggesting that its target molecules are upstream of TAK1 in mammalian innate immunity.

Keywords: Abbreviations; Dif; dorsal related immune factor; Dpt; Diptericin; Drs; Drosomycin; HREIMS; high resolution electron ionization mass spectrometry; HUVECs; human umbilical vein endothelial cells; IκB; inhibitor of κB; IKK; IκB kinase; IL; interleukin; IMD; immune deficiency; LPS; lipopolysaccharide; MCP; monocyte chemoattractant protein; NF-κB; nuclear factor κB; PGRP; peptidoglycan recognition protein; TAB; TAK1-binding protein; TAK; TGF-β activated kinase; TGF; transforming growth factor; TLR; Toll-like receptor; TNF; tumor necrosis factor; TRAF; TNF receptor associated factorCyclopentanediol analogue; Drosophila; Immune suppressor; Innate immunity; NF-κB; TAK1 kinase

Bavachalcone inhibits osteoclast differentiation through suppression of NFATc1 induction by RANKL by Cheol Kyu Park; Youngkyun Lee; Eun-Ju Chang; Ming Hong Lee; Jeong Hoon Yoon; Jae-Ha Ryu; Hong-Hee Kim (pp. 2175-2182).

Osteoclasts are cells that have a specialized role for bone resorption and are responsible for many bone diseases such as osteoporosis. As herbal products are invaluable sources in discovery of compounds for new therapies, we sought to identify compounds efficacious in suppressing osteoclastogenesis from medicinal plants that have been implicated for treatment of osteoporotic conditions. Bavachalcone was isolated from Psoralea corylifolia, and its effects on osteoclast differentiation were evaluated with primary cultures of osteoclast precursor cells. In addition, the molecular mechanism of action was investigated. Bavachalcone inhibited osteoclast formation from precursor cells with the IC₅₀ of ∼1.5μgml⁻¹. The activation of MEK, ERK, and Akt by receptor activator of nuclear factor kappaB ligand (RANKL), the osteoclast differentiation factor, was prominently reduced in the presence of bavachalcone. The induction of c-Fos and NFATc1, key transcription factors for osteoclastogenesis, by RANKL was also suppressed by bavachalcone. In conclusion, bavachalcone inhibits osteoclastogenesis by interfering with the ERK and Akt signaling pathways and the induction of c-Fos and NFATc1 during differentiation. Our results suggest that bavachalcone may be useful as a therapeutic drug for bone resorption-associated diseases.

Keywords: Bavachalcone; Osteoclast differentiation; NFATc1; c-Fos; RANKL

Geldanamycin inhibits tyrosine phosphorylation-dependent NF-κB activation by Julie Crèvecoeur; Marie-Paule Merville; Jacques Piette; Geoffrey Gloire (pp. 2183-2191).

Hsp90 is a protein chaperone regulating the stability and activity of many signalling molecules. The requirement of Hsp90 activity in the NF-κB pathway has been recently reported by several authors using the Hsp90 ATPase inhibitor geldanamycin (GA), an anti-tumor drug. Hsp90 inhibition blocks the synthesis and activation of the IKK complex, the major kinases complex responsible for IκBα phosphorylation on serine 32 and 36, a key step for its degradation and the nuclear translocation of NF-κB. However, the effect of GA on other IκBα kinases, including tyrosine kinases, is unknown. In the present study, we investigated the effect of GA on NF-κB activation induced by sodium pervanadate (PV), a tyrosine phosphatase inhibitor triggering c-Src-mediated tyrosine phosphorylation of IκBα. We report for the first time that GA inhibits PV-induced IκBα tyrosine phosphorylation and degradation. Using an in vitro kinase assay, we demonstrated that GA inhibits the activity of c-Src as an IκBα tyrosine kinase, but not its cellular expression. As a result, GA blocked PV-induced NF-κB DNA-binding activity on an exogenous κB element and on the endogenous iκbα promoter, thereby inhibiting iκbα transcription. Finally, we demonstrated that, despite NF-κB inhibition, pre-treatment with GA does not potentiate PV-induced apoptosis. We conclude that c-Src requires Hsp90 for its tyrosine kinase activity, and its inhibition by GA blocks c-Src-dependent signalling pathways, such as NF-κB activation induced by sodium pervanadate. The effect of GA on PV-induced apoptosis is discussed in the light of recent publications in the literature.

Keywords: Abbreviations; Hsp90; heat shock protein 90; NF-κB; nuclear factor-κB; IκB; inhibitor of κB; IKK; IκB kinase; NEMO; NF-κB essential modulator; TNFα; tumor necrosis factor α; IL-1β; interleukin-1β; ELKS; glutamic acid (E), leucine (L), lysine (K), and serine (S); PV; sodium pervanadateGeldanamycin; Hsp90; c-Src; NF-κB; IκBα; Pervanadate

Antagonist-radioligand binding to D_2L-receptors in intact cells by Ann Packeu; Jean-Paul De Backer; Isabelle Van Liefde; Patrick M.L. Vanderheyden; Georges Vauquelin (pp. 2192-2203).

D₂-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D₂-receptor antagonists [³H]-raclopride, a hydrophilic benzamide, and [³H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [³H]-raclopride and [³H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D_2L-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [³H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [³H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the membrane.

Keywords: Abbreviations; CHO-K1; Chinese Hamster Ovary cells; CHO-AEQ; Chinese Hamster Ovary cells stably transfected with the cDNA for apo-aquorin of; Aquoria victoria; , as well as the GTP-binding protein G; _α16; CHO-D; _2L; CHO-AEQ cells stably transfected with the cDNA of the human D2DR (transcript variant 1)Dopamine receptor; Radioligand binding; Intact cells; Raclopride; Spiperone

Retinoids induce cytochrome P450 3A4 through RXR/VDR-mediated pathway by Kun Wang; Shiyong Chen; Wen Xie; Yu-Jui Yvonne Wan (pp. 2204-2213).

A panel of retinoids and carotenoids was screened as potential inducers of CYP3A4 through the RXR/VDR-mediated signaling pathway. Transient transfection assays revealed that 3 out of 12 retinoids screened transactivated RXRα/VDR and induced CYP3A4 reporter activity. These three retinoids are the active metabolites of retinoids, 9- cis-retinal, 9- cis-retinoic acid (9- cis-RA), and all- trans-retinoic acid (all- trans-RA). 9- cis-RA and all- trans-RA preferentially transactivated the RXR/VDR heterodimers and RXR homodimers. Retinoids and VDR agonist 1α, 25-dihydroxyvitamin D₃, but not PXR or CAR activator, could induce Cyp3a11 mRNA level in hepatocytes derived from PXR/CAR-double null mouse. Moreover, retinoids induced CYP3A4 enzyme activity in HepG2 human hepatoma and Caco-2 human colorectal adenocarcinoma cells. A direct role of retinoid-mediated CYP3A4 induction through RXRα/VDR was proved by the results that 9- cis-retinal, 9- cis-RA, and all- trans-RA recruited RXRα and VDR to CYP3A4 regulatory region pER6 (proximal everted repeat with a 6-nucleotide spacer) and dXREM (distal xenobiotic-responsive enhancer module). Thus, using various approaches, we have unequivocally demonstrated that retinoids transactivate RXR/VDR heterodimers and RXR homodimers and induce CYP3A expression at mRNA as well as enzyme activity levels in both liver and intestinal cells. It is possible that retinoids might alter endobiotic metabolism through CYP3A4 induction in vivo.

Keywords: Abbreviations; CYP; cytochrome P450; RA; retinoic acid; all-; trans; -RP; all-trans-retinol palmitate; RBP; retinol-binding protein; ADHs; alcohol dehydrogenase/reductases; ALHDs; aldehyde dehydrogenase; ER; everted repeat (prefixes “p” and “d” indicate proximal and distal, respectively); DR; direct repeat; NR; nuclear receptor; dXREM; distal xenobiotic-responsive enhancer module; D; ₃; or 1α,25-(OH); ₂; D; ₃; 1α, 25-dihydroxyvitamin D; ₃; VDRE; vitamin D-responsive element; RT; reverse transcription; Gadph; glyceraldehyde-3-phosphate dehydrogenase; PCN; pregnenalone 16α-carbonitrile; TCPOBOP; 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; CITCO; 6-(4-chlorophenyl)imidazo[2,1-; b; ][1,3]thiazole-5-carbaldehyde; O; -(3,4-dichlorobenzyl)oxime; TTNBP; 4-(; E; -2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acidVDR; RXR; Retinoid; CYP3A4

Sulforaphane protects kidneys against ischemia-reperfusion injury through induction of the Nrf2-dependent phase 2 enzyme by Ha-Yong Yoon; Nam-In Kang; Hern-Ku Lee; Kyu Yun Jang; Jin-Woo Park; Byung-Hyun Park (pp. 2214-2223).

Reactive oxygen species are important mediators that exert a toxic effect during ischemia-reperfusion injury of various organs. Sulforaphane, which is a naturally occurring isothiocyanate that is present in cruciferous vegetables such as broccoli, is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. Phase 2 enzymes such as heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, glutathione reductase, and glutathione peroxidase participate in adaptive and protective responses to oxidative stress and various inflammatory stimuli. Therefore, we evaluated the preactivation of Nrf2 by sulforaphane to determine if it could inhibit ischemia-reperfusion-induced kidney damage. Treatment of HK2 renal tubular epithelial cells with sulforaphane effectively protected cells against cytotoxicity induced by hypoxia-reoxygenation, and sulforaphane dramatically induced phase 2 enzymes by decreasing the Keap1 protein levels and increasing Nrf2 nuclear translocation. Additionally, a second set of experiments using a renal ischemia-reperfusion model produced results that were essentially the same as those observed when HK2 cells were used; namely, that sulforaphane induced Nrf2-dependent phase 2 enzymes and thereby improved ischemia-reperfusion-induced changes in the lipid hydroperoxides, glutathione, creatinine clearance, kidney weight, and histologic abnormalities. Collectively, these results suggest that sulforaphane can be used as an effective adjunct for the prevention of renal oxidative insults during ischemia-reperfusion injury.

Keywords: Abbreviations; ROS; reactive oxygen species; HO-1; heme oxygenase 1; NQO1; NAD(P)H quinone oxidoreductase 1; GR; glutathione reductase; GPx; glutathione peroxidase; Nrf2; NF-E2-related factor-2; Keap1; Kelch-like ECH-associated protein 1; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GSH; reduced form of glutathioneSulforaphane; Kidney; Ischemia-reperfusion; Nrf2; Phase 2 enzymes; ROS

Short-term regulation of the Cl⁻/HCO₃⁻ exchanger in immortalized SHR proximal tubular epithelial cells by Sónia Simão; Rui Pedrosa; Ulrich Hopfer; David B. Mount; Pedro A. Jose; Patrício Soares-da-Silva (pp. 2224-2233).

The present study evaluated the activity of Cl⁻/HCO₃⁻ exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar–Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO₃⁻ was identical in WKY and SHR PTE cells, but V_max values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly ( P<0.05) increased the Cl⁻/HCO₃⁻ exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl⁻/HCO₃⁻ exchanger. The Cl⁻/HCO₃⁻ exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.

Keywords: Cl; ⁻; /HCO; ₃; ⁻; exchanger; PKA; PKC; p38 MAPK; Hypertension; SHR; WKY

Inhibition of caspase-3 activity and activation by protein glutathionylation by Zhishan Huang; John T. Pinto; Haiteng Deng; John P. Richie Jr. (pp. 2234-2244).

Protein glutathionylation is a post-translational modification that may account for a broad mechanism of redox signaling. The caspase family of cysteine proteases represents a potential target for regulation by glutathionylation. To examine this, caspase proteins, derived from HL-60 cells after activation with actinomycin D, were incubated with GSSG. Total protein glutathionylation was enhanced and caspase-3 activity was inhibited in a dose- and time-dependent manner by GSSG. Caspase inhibition was reversible by thiol-specific reducing reagents. Proteolytic activation of caspases was also affected, as the activation of procaspase-3 and procaspase-9 in HL-60 cell extracts induced by cytochrome c and dATP was inhibited by pre-incubation with GSSG. When biotin-labeled GSSG was incubated with recombinant caspase-3, biotin label was found associated with both p12 and p17 subunits of active caspase-3 by non-reducing SDS-PAGE. Caspase-3 glutathionylation was confirmed by matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis of GSSG-treated recombinant caspase-3. Specific sites of glutathionylation were identified as Cys¹³⁵ of the p17 protein (the active site) and Cys⁴⁵ of the p12 protein. These results indicate that glutathionylation of caspase can occur at physiologically relevant concentrations of GSSG and results in the inhibition of caspase activation and activity.

Keywords: Abbreviations; dATP; 2′-deoxyadenosine 5′-triphosphate; DTNB; 5,5′-dithiobis (2-nitrobenzoic acid); DTT; dithiothreitol; HRP; horseradish peroxidase; MALDI; matrix assisted laser desorption ionization; MPA; metaphosphoric acid; PBS; phosphate-buffered saline; PKC; protein kinase C; TBS; Tris-buffered saline; TOF; time of flightGlutathionylation; Glutathione; Caspase; Thionylation; Oxidative stress; Apoptosis

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Biochemical Pharmacology (v.75, #11)

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Biochemical Pharmacology (v.75, #11)