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Biochemical Pharmacology (v.70, #11)
Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension
by Daisy A. Salamanca; Raouf A. Khalil (pp. 1537-1547).
Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca2+]i, Ca2+-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin–myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca2+]i and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca2+-dependent and Ca2+-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca2+ antagonist-resistant forms of hypertension.
Keywords: Abbreviations; AngII; angiotensin II; ATP; adenosine triphosphate; CPI-17; PKC-potentiated phosphatase inhibitor protein-17; kDa; CAM; calmodulin; DAG; diacylglycerol; ET-1; endothelin; IP; 3; inositol 1,4,5-trisphosphate; MAPK; mitogen-activated protein kinase; MARCKs; myristoylated alanine-rich C-kinase substrate; MEK; MAPK kinase; MLC; myosin light chain; NADP; nicotinamide adenine dinucleotide phosphate; O; 2; −; superoxide; PDBu; phorbol 12,13-dibutyrate; PIP; 2; phosphatidylinositol 4,5-bisphosphate; PLD; phospholipase D; PKC; protein kinase C; PMA; phorbol myristate acetate; RACKs; receptors for activated C-kinase; Rho-kinase; Rho-associated kinase; ROS; reactive oxygen species; SHR; spontaneously hypertensive rat; TPA; 12-; o; -tetradecanoylphorbol-13-acetate; VSM; vascular smooth muscle; WKY; Wistar–KyotoSignal transduction; Vascular smooth muscle; Calcium; Blood pressure
Transcriptional suppression of human microsomal triglyceride transfer protein by hypolipidemic insulin sensitizers
by Vered Sheena; Rachel Hertz; Ina Berman; Janna Nousbeck; Jacob Bar-Tana (pp. 1548-1559).
Microsomal triglyceride transfer protein (MTP) catalyzes the assembly and secretion of liver triglyceride-rich lipoproteins. The human MTP (hMTP) promoter activity is reported here to be suppressed by HNF-4α ligand antagonists (e.g., Medica analogs) or by PPARγ ligand agonists (e.g., thiazolidinediones), thus accounting for their hypolipidemic activity in humans. Suppression of liver hMTP by Medica analogs or by thiazolidinediones was mediated by the TAAA sequence that serves as non-canonical TATA box of the hMTP core promoter. MTP suppression was evident in the specific context of the wild type hMTP core promoter, but not in the context of the mutated rodent-conforming hMTP core promoter governed by a canonical TATA box conjoined with its proximal (−50/−38)DR-1 element. hMTP suppression by Medica analogs or thiazolidinediones mediated by hMTP TAAA was independent of HNF-4α or PPARγ. hMTP suppression by Medica analogs, but not by thiazolidinediones, was further complemented by inhibition of HNF-4α transcriptional activity transduced by the distal (−83/−70)DR-1 element of hMTP promoter. hMTP promoter activity was unaffected by PPARα activation. Furthermore, in contrast to hMTP, the promoter activity of the rodent-conforming hMTP was robustly activated by Wy-14,643-activated PPARα or by thiazolidinedione-activated PPARγ. Transcriptional activation by PPARα or PPARγ of the rodent-conforming, but not the wild type hMTP gene promoter, resulted from the species-specific context of the respective proximal DR-1 elements. Hence, suppression of hMTP transcription by hypolipidemic insulin sensitizers requires the specific context of hMTP core promoter. In light of the species-specific context of MTP core promoters, the rodent MTP promoter may not substitute for the human promoter when searching for hypolipidemic MTP suppressors.
Keywords: Abbreviations; AOX; peroxisomal acyl-CoA oxidase; Apo B; apolipoprotein B; CHIP; chromatin immunoprecipitation; Cl-DICA; α,α′-tetrachloro-tetradecanedioic acid; DR-1; direct repeat 1; hMTP/rMTP; human/rat MTP; HNF-1α; hepatocyte nuclear factor-1α; HNF-4α; hepatocyte nuclear factor-4α; Medica 16 (M16); β,β′-tetramethyl-hexadecanedioic acid; Medica 18 (M18); β,β′-tetramethyl-octadecanedioic acid; MTP; microsomal triglyceride transfer protein; P-DR1/D-DR1; proximal/distal DR-1; P-mut/D-mut; mutated P-DR1/D-DR1; PPARα; peroxisome proliferator activated receptor α; PPARγ; peroxisome proliferator activated receptor γ; PUFA; polyunsaturated fatty acids; TZD; thiazolidinedionesMTP; HNF-4α; PPARα; PPARγ; Thiazolidinediones; Medica
Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (−)-epigallocatechin-3-gallate
by Won-Ki Baek; Byeong-Churl Jang; Jun Hee Lim; Taeg-Kyu Kwon; Hyun-Young Lee; Chi-Heum Cho; Dae-Kwang Kim; Dong-Hoon Shin; Jong-Gu Park; Jeong-Geun Lim; Ji-Hyun Bae; Jae-Hoon Bae; Sun Kyun Yoo; Won-Kyun Park; Dae-Kyu Song (pp. 1560-1567).
(−)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic substance found in green tea, is well recognized to be beneficial for human health. However, it is still controversial as to what dose of this compound is indeed good for human health. Though some recent studies have interestingly reported various beneficial effects of EGCG in cell culture system, however, plasma levels of EGCG attainable by oral regular intake in humans are normally in nanomolar range. However, potential side effects of EGCG when administered parenterally at higher concentration have not been thoroughly tested. Here, we evaluated the effect of EGCG on ATP-sensitive potassium (KATP) channels expressed in Xenopus oocytes. EGCG inhibited the activity of the Kir6.2/SUR1 and Kir6.2ΔC36 channels with IC50 of 142±37 and 19.9±1.7μM, respectively. Inhibition of EGCG was also observed in Kir6.2/SUR2A or Kir6.2/SUR2B channels. Notably, (−)-epicatechin-3-gallate (ECG), another major polyphenolic substance in green tea, was found to reduce the channel activity with greater potency than EGCG. In contrast to EGCG and ECG, which have the gallic acid-ester moiety in their own structures, (−)-epigallocatechin and (−)-epicatechin exhibited very weak inhibition of the KATP channel. Collectively, these results suggest that the gallate-ester moiety of epicatechins may be critical for inhibiting the KATP channel activity via the pore-forming subunit Kir6.2 and this may be a possible mechanism by which green tea extracts or EGCG may cause unexpected side effects at micromolar plasma level.
Keywords: EGCG; ATP-sensitive potassium channel; Gallate-ester moiety; Kir6.2; Xenopus; oocyte; Pancreatic β-cell
EGFR blockade by cetuximab alone or as combination therapy for growth control of hepatocellular cancer
by Alexander Huether; Michael Höpfner; Viola Baradari; Detlef Schuppan; Hans Scherübl (pp. 1568-1578).
Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. In light of the very poor 5 year survival new therapeutic approaches are mandatory. Several reports indicate that the epidermal growth factor receptor (EGFR) is expressed frequently in HCC, most likely contributing to the aggressive growth characteristics of these tumors. Cetuximab, a chimeric monoclonal IgG1 antibody directed against the EGFR, potently suppresses the growth of various cancers but its effect on HCC remains to be explored. We therefore studied the antineoplastic potency of cetuximab in human HCC cells alone and in combination with growth factor tyrosine-kinase inhibition (TKI) or HMG-CoA-reductase inhibiton or conventional cytostatics.Cetuximab inhibited growth of p53 wild-type HepG2 hepatocellular cancer cells in a time- and dose-dependent manner. Cetuximab treatment resulted in arresting the cell cycle in the G1/G0-phase due to an increase of expression of the cyclin-dependent kinase inhibitors p21Waf1/CIP1 and p27Kip1 and a decrease in cyclin D1 expression. Additionally, we observed a moderate increase in apoptosis as demonstrated by caspase-3 activation. Combining cetuximab with TKIs (erlotinib or AG1024) or the HMG-CoA-reductase inhibitor fluvastatin or doxorubicin resulted in synergistic antiproliferative effects. In contrast, p53 mutated Huh-7 hepatocellular cancer cells proved to be less sensitive towards cetuximab, but when combined with TKIs or fluvastatin or doxorubicin a pronounced reduction of cell growth was observed. To conclude, our study may provide a rationale for future clinical investigations of cetuximab combination therapy for growth control of hepatocellular cancer.
Keywords: Abbreviations; AG1024; 3-bromo-5-; t; -butyl-4-hydroxy-benzylidenemalonitrile; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; erlotinib; (; N; -(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine); HCC; hepatocellular carcinoma; HMG-CoA-reductase; 3-hydroxy-3-methyl-glutaryl-coenzyme A-reductase; IGF-1R; insulin-like growth factor 1 receptor; Mab; monoclonal antibody; TK; tyrosine kinase; TKI; tyrosine kinase inhibitorEpidermal growth factor receptor; Insulin-like growth factor receptor; EGF; IGF; Statins; Chemoprevention; Erbituxâ„¢; Tarcevaâ„¢; Doxorubicin; Cisplatin
The ethyl pyruvate analogues, diethyl oxaloproprionate, 2-acetamidoacrylate, and methyl-2-acetamidoacrylate, exhibit anti-inflammatory properties in vivo and/or in vitro
by Penny L. Sappington; Ruy J. Cruz Jr.; Tomoyuki Harada; Runkuan Yang; Yusheng Han; Joshua A. Englert; Alfred A. Ajami; Meaghan E. Killeen; Russell L. Delude; Mitchell P. Fink (pp. 1579-1592).
Ethyl pyruvate (EP) is a simple aliphatic ester derived from the endogenous metabolite, pyruvic acid. EP has been shown to decrease the expression of various pro-inflammatory mediators, including nitric oxide (NO), tumor necrosis factor (TNF), cyclooxygenase-2, and interleukin (IL)-6, in a variety of in vitro and in vivo model systems. In an effort to better understand the chemical features that might explain the anti-inflammatory properties of EP, we screened 15 commercially available compounds for cytoprotective or anti-inflammatory effects using two in vitro assay systems: TNF and NO production by lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage-like cells and changes in the permeability of Caco-2 human enterocyte-like monolayers stimulated with a cocktail of pro-inflammatory cytokines called cytomix (1000U/ml IFN-γ plus 10ng/ml TNF-α plus 1ng/ml IL-1β). Two compounds, namely diethyl oxaloproprionate (DEOP) and 2-acetamidoacrylate (2AA), demonstrated consistent anti-inflammatory or cytoprotective pharmacological properties in this screening process. Treatment of mice with either of these compounds ameliorated LPS-induced ileal mucosal hyperpermeability to the fluorescent probe, fluorescein isothiocyanate-labeled dextran (average molecular mass 4kDa), and bacterial translocation to mesenteric lymph nodes. Treatment with either of these compounds also improved survival in mice challenged with a lethal dose of LPS. Finally, in a study that compared 2AA to its methyl ester, we showed that methyl-2-acetamidoacrylate is at least 100-fold more potent than the parent carboxylate as an inhibitor of LPS-induced NO production by RAW 264.7 cells. Collectively, these data are consistent with the view that anti-inflammatory activity is demonstrable for a number of compounds that either incorporate an olefinic linkage conjugated to a carbonyl moiety or are capable of undergoing tautomeric rearrangement to form such a structure. Moreover, our findings suggest that esters with these general characteristics, perhaps because of their greater lipophilicity or electrophilicity, are more potent anti-inflammatory agents than are the parent carboxylates.
Rat sinusoidal liver endothelial cells (SECs) produce pro-fibrotic factors in response to adducts formed from the metabolites of ethanol
by Geoffrey M. Thiele; Michael J. Duryee; Thomas L. Freeman; Michael F. Sorrell; Monte S. Willis; Dean J. Tuma; Lynell W. Klassen (pp. 1593-1600).
Previous studies with alcohol-associated malondialdehyde–acetaldehyde (MAA)-modified proteins have demonstrated an increase in the expression of adhesion molecules, and the secretion of pro-inflammatory cytokines/chemokines by rat sinusoidal liver endothelial cells (SECs). However, no studies have been initiated to examine the effects of MAA-modified proteins on the expression of the extracellular matrix (ECM) protein, fibronectin and its isoforms. For these studies, SECs were isolated from the liver of normal rats, and exposed to MAA-modified bovine serum albumin (MAA–Alb). At selected time points, the total plasma and cellular fibronectin were determined by Western blot. Injection of rat liver via the mesenteric vein with MAA–Alb was performed in an effort to evaluate the potential in vivo role of MAA-modified proteins in the development of fibrosis. Expression of both plasma and cellular fibronectin was significantly increased over controls in the MAA–Alb stimulated SECs (>3-fold). Importantly, the isotype of fibronectin secreted was determined to be of the EIIIA variant and not EIIIB. These data were confirmed using RT-PCR procedures on liver tissue from; isolated SECs, and from an in vivo animal model wherein MAA–Alb was administered via the mesenteric vein. Thus, these studies demonstrate that MAA-modified proteins initiate a pro-fibrogenic response by initiating the expression of the fibronectin EIIIA isoform by SECs.
Keywords: Abbreviations; Alb; bovine serum albumin; IL-1 β; interlukin-one beta; KCs; Kupffer cells; LPS; lipopolysaccharide; MAA; malondialdehyde–acetaldehyde-adduct; SEC; sinusoidal liver endothelial cells; TGF-β; transforming growth factor-betaAdducts; Aldehydes; Fibronectin; Extracellular matrix (ECM); Alcohol; Endothelial cells
Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors
by Katia Varani; Stefania Gessi; Stefania Merighi; Fabrizio Vincenzi; Elena Cattabriga; Annalisa Benini; Karl-Norbert Klotz; Pier Giovanni Baraldi; Mojgan Aghazadeh Tabrizi; Stephen Mac Lennan; Edward Leung; Pier Andrea Borea (pp. 1601-1612).
The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A2B adenosine receptors expressed in Chinese hamster ovary (hA2BCHO), in human embryonic kidney 293 (hA2BHEK-293) and at endogenous A2B receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A2B adenosine radioligand [3H]- N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1 H-purin-8-yl)-1-methyl-1 H-pyrazol-3-yloxy]-acetamide ([3H]-MRE 2029F20) revealed a single class of binding sites in hA2BCHO, hA2BHEK-293 and HMC-1 cells with KD (nM) of 1.65±0.18, 2.83±0.34, 2.62±0.27 and Bmax (fmol/mg protein) of 36±4, 475±50 and 128±15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA2BHEK-293 cells using [3H]-MRE 2029F20, showed a rank order of potency typical of the A2B receptors with K i values in the range 3.2–28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA2BCHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA2BCHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA2BCHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC50 value of 2.9±0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A2B receptors.
Keywords: Abbreviations; AS16; 2-(4-benzyloxy-phenyl)-; N; -[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yl]-acetamide; AS70; 8-[2-methyl-5-[2-oxo-2(4-phenyl-piperazin-1-yl)-ethoxy]-2H-pyrazol-3-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione; AS74; N; -benzo-[1,3]-dioxol-5-yl-2[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-isoxazol-3-yloxy]-acetamide; AS94; N; -[4-(ethoxycarbonyl)phenyl]2-[5-(2,6-dioxo-1,3dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yloxy]-acetamide; AS95; N-(4-carboxy-phenyl)-2-[5-(2,6-dioxo-1,3dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yloxy]-acetamide; AS96; 1,3-diallyl-8-[2-methyl-5-[2-oxo-2(4-phenyl-piperazin-1-yl)-ethoxy]-2H-pyrazol-3-yl]-3,7-dihydro-purine-2,6-dione; AS99; N; -[3,4] (dimethyl-phenyl)2-[5-(2,6-dioxo-1,3dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yloxy]-acetamide; AS100; N; -2,3,4 dichlorophenyl)-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yloxy]acetamide; AS101; N; -(3,4-dimethoxy-phenyl)-2-5-(2,6-di-oxo-1,3-dipropyl-2,3,6,7-tetra-hydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yloxy]-acetamide; cAMP; cyclic AMP; CGS 21680; 2-[; p; -(2-carboxyethyl)-phenetyl-amino]-5′-; N; -ethyl-carboxamidoadenosine; DPCPX; 1,3-dipropyl-8-cyclopentyl-xanthine; HE-NECA; 2-hexynyl-5′-; N; -ethylcarboxamidoadenosine; [; 3; H]-MRE 2029F20; [; 3; H]-; N; -benzo [1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yloxy]-acetamide; [; 3; H]-MRE 3008F20; [; 3; H]-5-; n; -(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; [; 3; H]-ZM 241385; [; 3; H]-4-(2-[7-amino-2-[furyl] [1,2,4] triazolo [2,3-a] [1,3,5]triazin-5-ylamino]ethyl] phenol; [; 3; H]-DPCPX; [; 3; H]-1,3-dipropyl-8-cyclopentyl-xanthine; hA; 2B; CHO; human A; 2B; adenosine receptor in Chinese hamster ovary; hA; 2B; HEK-293; human A; 2B; adenosine receptor in human embryonic kidney 293; HMC-1; human mast cells; MRE 3008F20; 5-; n; -(4-methoxy phenyl-carbamoyl) amino-8-propyl-2-(2-furyl) pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine; MRE 2029F20; N; -benzo[1,3]-dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1; H; -purin-8-yl)-1-methyl-1; H; -pyrazol-3-yloxy]-acetamide; NECA; 5′-; N; -ethylcarboxamidoadenosine; R-PIA; R(−)-; N; 6; (2-phenyl-isopropyl)-denosine; S-PIA; S(−)-; N; 6; (2-phenylisopropyl)-adenosine; SCH 58261; 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidineA; 2B; adenosine receptors; Inverse agonists; Cyclic AMP assays; hA; 2B; CHO cells; HEK-293 cells; HMC-1 cells
Inhibition of store-operated calcium entry-mediated superoxide generation by histamine trifluoromethyltoluide independent of histamine receptors
by Dong-Chan Kim; So-Young Lee; Dong-Jae Jun; Sun-Hee Kim; Jong-Hee Lee; Eun-Mi Hur; Nam-In Baek; Kyong-Tai Kim (pp. 1613-1622).
Store-operated calcium entry (SOCE) plays an important role in shaping the Ca2+ response of various tissues and cell types. In this report, we show that thapsigargin (TG)-induced SOCE was inhibited by the histamine receptor agonist, histamine-trifluoromethyltoluide (HTMT), in U937 and HL-60 human promyelocytes. Preincubation of HTMT resulted in a significant inhibition of subsequent TG-induced Ca2+ elevation without affecting Ca2+ release from intracellular stores. HTMT also inhibited TG-induced Ca2+ current and Ba2+/Mn2+ influx in a concentration-dependent manner. In contrast with HTMT, other H1 histamine receptor agonists, histamine, 2-methylhistamine and 2-thiazolylethylamine, did not affect TG-induced SOCE. In addition, HTMT also attenuated TG-induced cytosolic superoxide generation. Taken together, our data clearly suggest that the anti-inflammatory effect of HTMT may occur through direct inhibition of SOCE.
Keywords: Abbreviations; [Ca; 2+; ]; i; cytosolic calcium ion concentration; Fura-2/AM; fura-2 penta acetoxymethyl ester; H; 1; R; histamine 1 receptor; HTMT; histamine trifluoromethyltoluide; SOCE; store-operated Ca; 2+; entrySOCE; Calcium; HTMT; Superoxide; Inflammation
Role of the carboxy-terminal transactivation domain and active transcription in the ligand-induced and ligand-independent degradation of the mouse Ahb−1 receptor
by Richard S. Pollenz; Jesal Popat; Edward J. Dougherty (pp. 1623-1633).
To assess the importance of transactivation domains (TAD), DNA binding and transcription on the degradation of the AH receptor (AHR), Hepa-1 cells were pre-treated with actinomycin D (AD) or cycloheximide (CHX) and exposed to 2,3,7,8 tetrachlorodibenzo- p-dioxin (TCDD). AD or CHX did not affect nuclear localization or DNA binding of the AHR but inhibited ligand-induced degradation. In contrast, AD or CHX did not inhibit geldanamycin (GA) induced degradation of the AHR. To assess the role of the COOH-terminal TAD in AHR degradation, stop codons were placed at nucleotide 1501 and 1921 of the Ahb−1 AHR coding region to generate AHR500 and AHR640. Stable cell lines were generated and exposed to TCDD. Cells expressing AHR500 did not induce CYP1A1 protein, but exhibited significant degradation of AHR500. Cells expressing AHR640 induced CYP1A1 protein to 50% of the level of cells expressing wild type AHR and exhibited significant degradation of AHR640. Importantly, AD and CHX did not inhibit the TCDD-induced degradation of either AHR500 and AHR640 and these receptors showed a more rapid profile of ligand-induced degradation compared to cells expressing wild type AHR. TCDD exposure to Hepa-1 cells with reduced aryl hydrocarbon receptor nuclear translocator (ARNT), showed ligand-induced degradation of the AHR that was not blocked by AD. However, AD inhibited TCDD-induced degradation when ARNT expression was restored. These results show that multiple mechanisms exist for the ligand and GA-induced degradation of the AHR and suggest that ligand-induced degradation can switch between two mechanisms depending on the presence of a functional TAD and the binding to DNA.
Keywords: Abbreviations; AD; actinomycin D; AHR; aryl hydrocarbon receptor; ARNT; Ah receptor nuclear translocator; CHX; cycloheximide; ER; estrogen receptor; GA; geldanamycin; GAR-HRP; goat anti-rabbit antibodies conjugated to horseradish peroxidase; GAR-RHO; goat anti-rabbit antibodies conjugated to rhodamine; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; XRE; xenobiotic response elementAryl hydrocarbon receptor; TCDD; Geldanamycin; Protein degradation; Cycloheximide
Effect of magnesium sulfate on the calcium-stimulated adenosine triphosphatase activity and lipid peroxidation of red blood cell membranes from preeclamptic women
by Cilia Abad; Alejandro Teppa-Garrán; Teresa Proverbio; Sandy Piñero; Fulgencio Proverbio; Reinaldo Marín (pp. 1634-1641).
The effect of the treatment with magnesium sulfate (MgSO4) on Ca-ATPase activity and level of lipid peroxidation of red blood cells from preeclamptic pregnant women was examined because it is known that these parameters are affected with preeclampsia. Red cell ghosts from 11 normotensive and 11 preeclamptic pregnant women, before and after treatment with MgSO4, were assayed for Ca-ATPase activity and level of lipid peroxidation, determined as TBARS or conjugated dienes. It was found that the Ca-ATPase activity is significantly lower and the level of lipid peroxidation is significantly higher in the preeclamptic women with no treatment, as compared to normotensive pregnant women. Both parameters return to normal values after the MgSO4 therapy. These results can be mimicked by in vitro preincubation with MgSO4 of intact red blood cells from preeclamptic pregnant women, without any treatment. Our data indicate that MgSO4 treatment of preeclamptic pregnant women modifies both the Ca-ATPase activity and the level of lipid peroxidation of their red blood cell membranes, reaching values similar to those of normotensive pregnant women. The diminution of the level of lipid peroxidation by MgSO4, can account for the increase in Ca-ATPase activity.
Keywords: Abbreviations; Ca-ATPase; calcium-stimulated adenosine triphosphatase; MDA; malondialdehyde; Na,K-ATPase; sodium and potassium-stimulated adenosine triphosphatase; TBARS; thiobarbituric acid-reactive substancesMagnesium sulfate; Preeclampsia; Inside-out vesicles; Ca-ATPase; Lipid peroxidation; Human red blood cells
Novel potential neuroprotective agents with both iron chelating and amino acid-based derivatives targeting central nervous system neurons
by Hailin Zheng; Moussa B.H. Youdim; Lev M. Weiner; Mati Fridkin (pp. 1642-1652).
Antioxidants and iron chelating molecules are known as neuroprotective agents in animal models of neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this study, we designed and synthesized a novel bifunctional molecule (M10) with radical scavenging and iron chelating ability on an amino acid carrier likely to be a substrate for system L, thus targeting the compound to the central nervous system (CNS). M10 had a moderate iron affinity in HEPES buffer (pH 7.4) with log K3=12.25±0.55 but exhibited highly inhibitory action against iron-induced lipid peroxidation, with an IC50 value (12μM) comparable to that of desferal (DFO). EPR studies indicated that M10 was a highly potentOH scavenger with an IC50 of about 0.3 molar ratio of M10 to H2O2. In PC12 cell culture, M10 was at least as potent as the anti-Parkinson drug rasagiline in protecting against cell death induced by serum-deprivation and by 6-hydroxydopamine (6-OHDA). These results suggest that M10 deserves further investigation as a potential agent for the treatment of neurodegenerative disorders such as AD and PD.
Keywords: Antioxidant; Iron chelator; Lipid peroxidation; Neurodegenerative diseases; Neuroprotection; Oxidative stress
Fibrates inhibit aldose reductase activity in the forward and reverse reactions
by Ganesaratnam K. Balendiran; Balakrishnan Rajkumar (pp. 1653-1663).
Fibrates such as bezafibrate, gemfibrozil, clofibric acid, ciprofibrate and fenofibrate, are ligands for peroxisome proliferator-activated receptor α (PPARα), and are used as therapeutic agents in the treatment of hyperlipidemia. Synthesis and accumulation of sorbitol in cells due to aldose reductase (AR) activity is implicated in secondary diabetic complications. In pursuit of finding a lead compound identification to design an effective AR inhibitor employing fragment-based design-like approach, we found that this class of compounds and their nearest neighbors could inhibit AR. Bezafibrate and gemfibrozil displayed a mixed non-competitive inhibition pattern in the glyceraldehyde reduction activity and pure non-competitive inhibition pattern in the benzyl alcohol oxidation activity of AR. Clofibric acid, ciprofibrate and fenofibrate showed pure non-competitive inhibition patterns in the forward reaction. In the reverse reaction, clofibric acid displayed a non-competitive inhibition pattern while ciprofibrate and fenofibrate displayed competitive inhibition patterns. This finding reveals for the first time a novel attribute of the fibrates in the regulation of AR activity and may be useful as lead compounds to control the function of AR in the progression and treatment of secondary diabetic complications in addition to other clinical conditions. Alternatively, these findings demonstrate that AR plays a significant role in the fibrate metabolism under various scenarios.
Keywords: Aldose reductase inhibitors; Oxidoreductase; Fibrates; Polyol pathway; Hyperglycemia; Diabetes complication
Effects of nicotine-specific antibodies, Nic311 and Nic-IgG, on the transfer of nicotine across the human placenta
by Ilona A. Nekhayeva; Tatiana N. Nanovskaya; Paul R. Pentel; Dan E. Keyler; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 1664-1672).
The adverse effects of smoking during pregnancy on fetal development are, in part, due to nicotine. These effects may be due to the actions of nicotine in fetal circulation or on placental functions. In pregnant rats, vaccination with a nicotine immunogen reduces the transfer of nicotine from the maternal to fetal circulation. However, extrapolation of these results to pregnant women might not be valid due to the well-recognized differences between human and rat placentas. In the current investigation, the effects of nicotine-specific antibodies on the transfer of nicotine from the maternal to fetal circuit of the dually perfused human placental lobule were determined. Two types of nicotine-specific antibodies were investigated; nicotine-specific mouse monoclonal antibody (Nic311, Kd for nicotine 60nM) and IgG from rabbits vaccinated with a nicotine immunogen (Nic-IgG, Kd 1.6nM). Transfer of the antibodies from maternal to fetal circuits was negligible. Both rabbit Nic-IgG and, to a lesser extent, mouse monoclonal Nic311 significantly reduced nicotine transfer from the maternal to fetal circuit as well as the retention of the drug by placental tissue. These effects were mediated by a substantial increase in the protein binding of nicotine and a reduction in the unbound nicotine concentration. Therefore, the data cited in this report suggest that the use of nicotine-specific antibodies might reduce fetal exposure to the drug, and that antibody affinity for nicotine is a key determinant of the extent of nicotine transfer.
Keywords: Nicotine; Nicotine-specific antibodies; Human placenta; Pregnancy; Transplacental transfer
Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma
by Bin Li; Meghan Sedlacek; Indumathi Manoharan; Rathnam Boopathy; Ellen G. Duysen; Patrick Masson; Oksana Lockridge (pp. 1673-1684).
The goal of this work was to identify the esterases in human plasma and to clarify common misconceptions. The method for identifying esterases was nondenaturing gradient gel electrophoresis stained for esterase activity. We report that human plasma contains four esterases: butyrylcholinesterase (EC 3.1.1.8), paraoxonase (EC 3.1.8.1), acetylcholinesterase (EC 3.1.1.7), and albumin. Butyrylcholinesterase (BChE), paraoxonase (PON1), and albumin are in high enough concentrations to contribute significantly to ester hydrolysis. However, only trace amounts of acetylcholinesterase (AChE) are present. Monomeric AChE is seen in wild-type as well as in silent BChE plasma. Albumin has esterase activity with alpha- and beta-naphthylacetate as well as with p-nitrophenyl acetate. Misconception #1 is that human plasma contains carboxylesterase. We demonstrate that human plasma contains no carboxylesterase (EC 3.1.1.1), in contrast to mouse, rat, rabbit, horse, cat, and tiger that have high amounts of plasma carboxylesterase. Misconception #2 is that lab animals have BChE but no AChE in their plasma. We demonstrate that mice, unlike humans, have substantial amounts of soluble AChE as well as BChE in their plasma. Plasma from AChE and BChE knockout mice allowed identification of AChE and BChE bands without the use of inhibitors. Human BChE is irreversibly inhibited by diisopropylfluorophosphate, echothiophate, and paraoxon, but mouse BChE spontaneously reactivates. Since human plasma contains no carboxylesterase, only BChE, PON1, and albumin esterases need to be considered when evaluating hydrolysis of an ester drug in human plasma.
Keywords: Butyrylcholinesterase knockout mice; Acetylcholinesterase; Carboxylesterase; Paraoxonase; Human plasma; Albumin; Cocaine esterase; Aspirin; Irinotecan; Procaine; Succinylcholine; Mivacurium
Inhibition of insulin/IGF-1 receptor signaling enhances bile acid toxicity in primary hepatocytes
by Paul Dent; Song Iy Han; Clint Mitchell; Elaine Studer; Adly Yacoub; Jennifer Grandis; Steven Grant; Geoffrey W. Krystal; Philip B. Hylemon (pp. 1685-1696).
Modulation of ERBB and insulin-like growth factor 1 (IGF-1) receptor function is recognized as a potential mechanism to inhibit tumor growth. We and others have shown that inhibition of ERBB1 can enhance bile acid toxicity. Herein, we extend our analyses to examine the impact of insulin/IGF-1 receptor inhibition on primary hepatocyte survival when exposed to the secondary bile acid deoxycholic acid (DCA) and compare the impact inhibition of this receptor has on bile acid toxicity effects to that of ERBB1/MEK1/2 inhibition. The insulin/IGF-1 receptor inhibitor NVP-ADW742 at concentrations which inhibit both the insulin and IGF-1 receptors had a modest negative impact on hepatocyte viability, and strongly potentiated DCA-induced apoptotic cell death. Identical data were obtained expressing a dominant negative IGF-1 receptor in hepatocytes; a receptor which acts to inhibit both the IGF-1 receptor and the insulin receptor in trans. Inhibition of ERBB1 function using Iressa (gefitinib) or the tyrphostin AG1478 had more modest effects at enhancing DCA lethality than inhibition of the insulin/IGF-1 receptor function. In contrast, over-expression of a dominant negative ERBB1 protein had pleiotropic effects on multiple signaling pathways in an apparently non-specific manner. These findings suggest that novel therapeutic kinase inhibitors, targeted against growth factor receptors, have the potential to promote bile acid toxicity in hepatocyte when bile flow may be impaired.
Keywords: Abbreviations; AG; AG1478; ASM; acidic sphingomyelinase; ca; constitutively active; CD533; dominant negative ERBB1; DCA; deoxycholic acid; dn; dominant negative; EGF; epidermal growth factor; ERK; extracellular regulated kinase; FADD; FAS associating death domain protein; IGF; insulin-like growth factor; JNK; c-Jun NH; 2; -terminal kinase; JNK I; JNK1/2 inhibitory peptide derived from JIP1; LY; LY294002; MAPK; mitogen activated protein kinase; MEK; mitogen activated extracellular regulated kinase; NVP; NVP-ADW742; P; phospho-; PD; PD184352; R; receptor; WT; wild type; −/−; null/gene deletedBile acid; Kinase; Receptor; Apoptosis; Dominant negative; Insulin
The effect of inhibition of Ca2+-independent phospholipase A2 on chemotherapeutic-induced death and phospholipid profiles in renal cells
by Ling Zhang; Brianna L. Peterson; Brian S. Cummings (pp. 1697-1706).
We demonstrate that cells derived from primary cultures of rabbit proximal tubules (RPTC), human embryonic kidney (HEK293) and human kidney carcinomas (Caki-1) express microsomal Ca2+-independent phospholipase A2 (iPLA2γ) and cytosolic Ca2+-independent phospholipase A2 (iPLA2β). Inhibition of iPLA2 activity in these cells using the iPLA2 inhibitor bromoenol lactone (BEL) (0–5.0μM) for 24h did not induce cell death as determined by annexin V and propidium iodide (PI) staining. However, BEL treatment prior to cisplatin (50μM) or vincristine (2μM) exposure reduced apoptosis 30–50% in all cells tested (RPTC, HEK293 and Caki-1 cells). To identify the phospholipids altered during cell death electrospray ionization-mass spectrometry and lipidomic analysis of HEK293 and Caki-1 cells was performed. Cisplatin treatment reduced 14:0–16:0 and 16:0–16:0 phosphatidylcholine (PtdCho) 50% and 35%, respectively, in both cell lines, 16:0–18:2 PtdCho in Caki-1 cells and increased 16:1–22:6 plasmenylcholine (PlsCho). BEL treatment prior to cisplatin exposure further decreased 14:0–16:0 PtdCho, 16:0–16:1 PlsCho and 16:0–18:1 PlsCho in HEK293 cells, and inhibited cisplatin-induced increases in 16:1–22:6 PlsCho in Caki-1 cells. Treatment of cells with BEL prior to cisplatin exposure also increased the levels of several arachidonic containing phospholipids including 16:0–20:4, 18:1–20:4, and 18:0–20:4 PtdCho, compared to cisplatin only treated cells. These data demonstrate that inhibition of iPLA2 protects against chemotherapeutic-induced cell death in multiple human renal cell models, identifies specific phospholipids whose levels are altered during cell death, and demonstrates that alterations in these phospholipids correlate to the protection against cell death in the presence of iPLA2 inhibitors.
Keywords: Abbreviations; PLA; 2; phospholipase A; 2; sPLA; 2; secretory phospholipase A; 2; cPLA; 2; cytosolic phospholipase A; 2; iPLA; 2; Ca; 2+; -independent phospholipase A; 2; BEL; bromoenol lactone; RPTC; rabbit renal proximal tubule cell(s); PtdCho; phosphatidylcholine; PlsCho; plasmenylcholinePhospholipase A; 2; Apoptosis; Phospholipids; Electron ionization-mass spectrometry
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