Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.78, #6)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e4).

Inflammatory cytokines in vascular dysfunction and vascular disease by Alexander H. Sprague; Raouf A. Khalil (pp. 539-552).

The vascular inflammatory response involves complex interaction between inflammatory cells (neutrophils, lymphocytes, monocytes, macrophages), endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and extracellular matrix (ECM). Vascular injury is associated with increased expression of adhesion molecules by ECs and recruitment of inflammatory cells, growth factors, and cytokines, with consequent effects on ECs, VSMCs and ECM. Cytokines include tumor necrosis factors, interleukins, lymphokines, monokines, interferons, colony stimulating factors, and transforming growth factors. Cytokines are produced by macrophages, T-cells and monocytes, as well as platelets, ECs and VSMCs. Circulating cytokines interact with specific receptors on various cell types and activate JAK-STAT, NF-κB, and Smad signaling pathways leading to an inflammatory response involving cell adhesion, permeability and apoptosis. Cytokines also interact with mitochondria to increase the production of reactive oxygen species. Cytokine-induced activation of these pathways in ECs modifies the production/activity of vasodilatory mediators such as nitric oxide, prostacyclin, endothelium-derived hyperpolarizing factor, and bradykinin, as well as vasoconstrictive mediators such as endothelin and angiotensin II. Cytokines interact with VSMCs to activate Ca²⁺, protein kinase C, Rho-kinase, and MAPK pathways, which promote cell growth and migration, and VSM reactivity. Cytokines also interact with integrins and matrix metalloproteinases (MMPs) and modify ECM composition. Persistent increases in cytokines are associated with vascular dysfunction and vascular disease such as atherosclerosis, abdominal aortic aneurysm, varicose veins and hypertension. Genetic and pharmacological tools to decrease the production of cytokines or to diminish their effects using cytokine antagonists could provide new approaches in the management of inflammatory vascular disease.

Keywords: Abbreviations; AAA; abdominal aortic aneurysm; AngII; angiotensin II; BP; blood pressure; CSF; colony stimulating factors; CRP; C-reactive protein (CRP); ECM; extracellular matrix; EC; endothelial cell; EDHF; endothelium-derived hyperpolarizing factor; ET-1; endothelin; HTN; hypertension; IFN; inetrferon; IL; interleukin; MAPK; mitogen-activated protein kinase; MCP; monocyte chemoattractant protein; MMP; matrix metalloproteinase; NO; nitric oxide; NOS; NO synthase; O; ₂; ⁻; superoxide anion; PAI-1; plasminogen activator inhibitor-1; PGI; ₂; prostacyclin; PKC; protein kinase C; RANTES; regulated on activation, normal T-expressed and secreted; ROS; reactive oxygen species; TGF; transforming growth factor; TNF; tumor necrosis factor; VSMC; vascular smooth muscle cellInterleukins; Oxidative stress; Endothelium; Vascular smooth muscle; Matrix metalloproteinases; Aneurysm; Varicose veins; Hypertension

N-acylethanolamines, anandamide and food intake by Harald S. Hansen; Thi Ai Diep (pp. 553-560).

Oleoylethanolamide (OEA) formed locally in the intestine seems to regulate food intake via activation of PPARalpha, and dietary fat can decrease OEA levels in the intestine, an effect that may lead to over-consumption of food.Anandamide and the other N-acylethanolamines, e.g. oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and linoleoylethanolamide (LEA), may be formed by several enzymatic pathways from their precursors, which are the N-acylated ethanolamine phospholipids. The exact enzymatic pathways involved in their biosynthesis in specific tissues are not clarified. It has been suggested that endogenous anandamide could stimulate food intake by activation of cannabinoid receptors in the brain and/or in the intestinal tissue. On the other hand, endogenous OEA and PEA have been suggested to inhibit food intake by acting on receptors in the intestine. At present, there is no clear role for endogenous anandamide in controlling food intake via cannabinoid receptors, neither centrally nor in the gastrointestinal tract.However, OEA, PEA and perhaps also LEA may be involved in regulation of food intake by selective prolongation of feeding latency and post-meal interval. These N-acylethanolamines seem to be formed locally in the intestine, where they can activate PPARα located in close proximity to their site of synthesis. The rapid onset of OEA response and its reliance on an intact vagus nerve suggests that activation of PPARα does not result in formation of a transcription-dependent signal but must rely on an unidentified non-genomic signal that translates to activation of vagal afferents. Whether GPR119, TRPV1 and/or intestinal ceramide levels also contribute to the anorectic and weight-reducing effect of exogenous OEA is less clear.Prolonged intake of dietary fat (45energy%) may promote over-consumption of food by decreasing the endogenous levels of OEA, PEA and LEA in the intestine.

Keywords: Abbreviations; Acyl-S-CoA; acylated coenzyme A (thioester); CB1; cannabinoid receptor-1; FAAH; fatty acid amide hydrolase; FAAH-2; fatty acid amide hydrolase-2; FATP1; fatty acid transport protein 1; GDE1; glycerophospho diester phosphodiesterase-1; GP-NAE; glycerophospho-; N; -acyl ethanolamine; HF; high fat; HS-CoA; coenzyme A (free form); LEA; linoleoylethanolamide; NAAA; NAE-hydrolysing acid amidase; NAE; N; -acylethanolamine; NAPE; N; -acylated ethanolamine phospholipid; NAPE-PLD; NAPE-hydrolysing phospholipase D; N; -arachidonoyl-PE; N; -arachidonoyl ethanolamine phospholipids; OEA; oleoylethanolamide; PEA; palmitoylethanolamide; PPARα; peroxisome proliferators activated receptor α; PPARγ; peroxisome proliferators activated receptor γ; PPY; polypeptide Y; PTPN22; a non-receptor tyrosine phosphatase; SEA; stearoylethanolamide; TRPV1; transient receptor potential vanilloid receptor; WT; wild typeOleoylethanolamide; Palmitoylethanolamide; Satiety; Food intake; Dietary fat

Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs by Eva Lincová; Aleš Hampl; Zuzana Pernicová; Andrea Staršíchová; Pavel Krčmář; Miroslav Machala; Alois Kozubík; Karel Souček (pp. 561-572).

Antitumorigenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. However, the mechanisms driving these processes are not understood in all details. In our study, we observed significant differences in sensitivity of cancer epithelial cell lines to COX-independent antiproliferative effects of NSAIDs. The prostate cancer cell line LNCaP, lacking both critical enzymes in the negative control of PKB/Akt activation, PTEN and SHIP2, was the most sensitive to these effects, as assessed by analysing the cell cycle profile and expression of cell cycle regulating proteins. We found that p53 protein and its signalling pathway is not involved in early antiproliferative action of the selected NSAID—indomethacin. RNAi provided evidence for the involvement of p21^Cip1/Waf1, but not GDF-15, in antiproliferative effects of indomethacin in LNCaP cells. Interestingly, we also found that indomethacin activated PKB/Akt and induced nuclear localisation of p21^Cip1/Waf1 and Akt2 isoform. Our results are in agreement with other studies and suggest that maintaining of the p21^Cip1/Waf1 level and its intracellular localisation might be influenced by Akt2. Knock-down of SHIP2 by RNAi in PTEN negative prostate and colon cancer cell lines resulted in higher sensitivity to antiproliferative effects of indomethacin. Our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.

Keywords: Abbreviations; Cdk2; cyclin-dependent kinase-2; COX-1; cyclooxygenase-1; COX-2; cyclooxygenase-2; Egr-1; early growth response-1; ERK; extracellular signal-regulated protein kinase; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GDF-15; growth/differentiation factor-15; JNK; c-Jun N-terminal kinase; MAPK; mitogen activated protein kinase; NSAIDs; non-steroidal anti-inflammatory drugs; PDK1; phosphoinositide-dependent kinase-1; PGE; ₂; prostaglandin E; ₂; PI3K; phosphatidylinositol-3-kinase; PIP3; phosphatidylinositol-3,4,5-triphosphate; PKB; protein kinase B; POLR2A; polymerase (RNA) II (DNA directed) polypeptide A; PTEN; phosphatase and tensin homolog; SHIP; SH2-containing 5′-inositol phosphataseNSAIDs; Protein kinase B/Akt; Akt2 isoform; PTEN; SHIP2; Antiproliferative effects

Sensitization of human K562 leukemic cells to TRAIL-induced apoptosis by inhibiting the DNA-PKcs/Akt-mediated cell survival pathway by Mi-Ju Kim; Hak-Bong Kim; Jae-Ho Bae; Jae-Won Lee; Soo-Jung Park; Dong-Wan Kim; Sang-Ick Park; Chi-Dug Kang; Sun-Hee Kim (pp. 573-582).

The mRNA levels of both DR4 and DR5 were significantly increased in K562 cells transfected with DNA-PKcs siRNA compared to the cells transfected with scrambled siRNA. We also found that suppression of DNA-PKcs using siRNA down-regulated c-FLIP and sensitized K562 cells to TRAIL-induced apoptosis through activation of caspase-8, -9 and -3.Moreover, the growth inhibitory effect of TRAIL in K562 cells was significantly increased after transfection with DNA-PKcs siRNA as compared with scrambled siRNA. This result was followed by increased susceptibility to TRAIL-induced apoptosis in K562 cells transfected with DNA-PKcs siRNA compared with that in the cells transfected with scrambled siRNA.Despite the fact that many cancer cells are sensitive to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, human K562 leukemic cells showed resistance to TRAIL-induced apoptosis. Interestingly, K562/R3 cells, a stable TRAIL-sensitive variant isolated from K562 cells, showed down-regulation of DNA-PK/Akt pathway and a high responsiveness to TRAIL-mediated growth inhibition and apoptosis. We revealed that siRNA-mediated suppression of DNA-PKcs led to decreased phosphorylation of Akt and Bad, a target molecule of Akt, and increased expression of DR4/DR5. Also, we found that suppression of DNA-PKcs using siRNA down-regulated c-FLIP and sensitized K562 cells to TRAIL-induced apoptosis through activation of caspase-8, -9 and -3. In addition, we revealed that treatment with DMNB, a specific inhibitor of DNA-PK, resulted in an increase of DR4/DR5 mRNA levels and their surface expression and a decrease of c-FLIP mRNA levels in K562 cells. DMNB potentiated TRAIL-induced cytotoxicity and apoptosis through inhibition of DNA-PK/Akt pathway and activation of caspase-8, -9 and -3 in K562 cells. This study is the first to show that a protective role of DNA-PK/Akt pathway against TRAIL-induced apoptosis and thus TRAIL in combination with agents that inhibit DNA-PK/Akt pathway would have clinical applicability in treating TRAIL-insensitive human leukemic cells. This model may provide a novel framework for overcoming TRAIL resistance of other cancer cells with agents that inhibit DNA-PK/Akt pathway.

Keywords: TRAIL; DNA-PKcs; Death receptors; Akt; Apoptosis; DMNB

Inhibition of ATP synthase by chlorinated adenosine analogue by Lisa S. Chen; Billie J. Nowak; Mary L. Ayres; Nancy L. Krett; Steven T. Rosen; Shuxing Zhang; Varsha Gandhi (pp. 583-591).

Phosphorylated metabolites of 8-chloroadenosine (8-Cl-Ado). Phosphorylation of 8-Cl-ADP by ATP synthase to form 8-Cl-ATP and subsequent inhibition of ATP synthase by 8-Cl-ATP product. Molecular modeling of 8-Cl-ADP in ATP synthase.8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F_O intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (α_dpβ_dp) and loose binding (α_tpβ_tp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the α_tpβ_tp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (α_dpβ_dp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF₃⁻. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.

Keywords: ATP synthase; 8-Chloroadenosine; Cellular bioenergy; Molecular modeling; Nucleoside analogue; Chemotherapeutics

The inhibition of TNF-α-induced NF-κB activation by marine natural products by Florence Folmer; Marcel Jaspars; Godofredo Solano; Silvia Cristofanon; Estelle Henry; Jioji Tabudravu; Kenny Black; David H. Green; Frithjof C. Küpper; William Aalbersberg; Klaus Feussner; Mario Dicato; Marc Diederich (pp. 592-606).

The deregulated activation of NF-κB is associated with cancer development and inflammatory diseases. With an aim to find new NF-κB inhibitors, we purified and characterized compounds from extracts of the Fijian sponge Rhabdastrella globostellata, the crinoid Comanthus parvicirrus, the soft corals Sarcophyton sp. nov. and Sinularia sp., and the gorgonian Subergorgia sp. after an initial screening of 266 extracts from different marine origins.Results obtained show that selected purified compounds had a cytotoxic effect on the human leukaemia cell line K562, inhibited both TNF-α-induced NF-κB-DNA binding as well as TNF-α-induced IκBα degradation and nuclear translocation of p50/p65. Furthermore, we observed the inhibition of NF-κB activation induced by an overexpression of IKKβ. Interestingly, natural products inhibited IKKβ kinase as well as the 26S proteasome proteolytic activity.

Keywords: NF-κB; Marine natural product; Anti-cancer drug discovery

Histamine H₄ receptor–RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H₄ receptor ligands by Erich H. Schneider; Roland Seifert (pp. 607-616).

When co-expressed with G_i-proteins in Sf9 cells, the histamine H₄ receptor shows only a low signal-to-background ratio. This ratio can be markedly enhanced by fusing the RGS protein GAIP to the receptor.The human histamine H₄ receptor (hH₄R), co-expressed with Gα_i2 and Gβ₁γ₂ in Sf9 cells, is highly constitutively active. In the steady-state GTPase assay, the full agonist histamine (HA) induces only a relatively small signal (∼20–30%), resulting in a low signal-to background ratio. In order to improve this system for ligand screening purposes, the effects of the regulators of G-protein signaling (RGS) RGS4 and RGS19 (GAIP) were investigated. RGS4 and GAIP were fused to the C-terminus of hH₄R or co-expressed with non-fused hH₄R, always combined with Gα_i2 and Gβ₁γ₂. The non-fused RGS proteins did not significantly increase the relative effect of HA. With the hH₄R–RGS4 fusion protein the absolute GTPase activities, but not the relative HA-induced signal were increased. Fusion of hH₄R with GAIP caused a selective increase of the HA signal, resulting in an enhanced signal-to-noise ratio. A detailed characterization of the hH₄R–GAIP fusion protein (co-expressed with Gα_i2 and Gβ₁γ₂) and a comparison with the data obtained for the non-fused hH₄R (co-expressed with Gα_i2 and Gβ₁γ₂) led to the following results: (i) the relative agonist- and inverse agonist-induced signals at hH₄R–GAIP are markedly increased. (ii) Compared to the wild-type hH₄R, standard ligands show unaltered potencies and efficacies at hH₄R–GAIP. (iii) Like hH₄R, hH₄R–GAIP shows high and NaCl-resistant constitutive activity. (iv) hH₄R–GAIP shows the same G-protein selectivity profile as the non-fused hH₄R. Collectively, hH₄R–GAIP provides a sensitive test system for the characterization of hH₄R ligands and can replace the non-fused hH₄R in steady-state GTPase assays.

Keywords: Histamine H; ₄; receptor; Fusion protein; RGS protein; Steady-state GTPase assay; Constitutive activity; G; _i; -proteins

Effects of CYP2D6 status on harmaline metabolism, pharmacokinetics and pharmacodynamics, and a pharmacogenetics-based pharmacokinetic model by Chao Wu; Xi-Ling Jiang; Hong-Wu Shen; Ai-Ming Yu (pp. 617-624).

Harmaline is a β-carboline alkaloid showing neuroprotective and neurotoxic properties. Our recent studies have revealed an important role for cytochrome P450 2D6 (CYP2D6) in harmaline O-demethylation. This study, therefore, aimed to delineate the effects of CYP2D6 phenotype/genotype on harmaline metabolism, pharmacokinetics (PK) and pharmacodynamics (PD), and to develop a pharmacogenetics mechanism-based compartmental PK model. In vitro kinetic studies on metabolite formation in human CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) hepatocytes indicated that harmaline O-demethylase activity ( V_max/ K_m) was about 9-fold higher in EM hepatocytes. Substrate depletion showed mono-exponential decay trait, and estimated in vitro harmaline clearance (CL_int, μL/min/10⁶cells) was significantly lower in PM hepatocytes (28.5) than EM hepatocytes (71.1). In vivo studies in CYP2D6-humanized and wild-type mouse models showed that wild-type mice were subjected to higher and longer exposure to harmaline (5 and 15mg/kg; i.v. and i.p.), and more severe hypothermic responses. The PK/PD data were nicely described by our pharmacogenetics-based PK model involving the clearance of drug by CYP2D6 (CL_CYP2D6) and other mechanisms (CL_other), and an indirect response PD model, respectively. Wild-type mice were also more sensitive to harmaline in marble-burying tests, as manifested by significantly lower ED₅₀ and steeper Hill slope. These findings suggest that distinct CYP2D6 status may cause considerable variations in harmaline metabolism, PK and PD. In addition, the pharmacogenetics-based PK model may be extended to define PK difference caused by other polymorphic drug-metabolizing enzyme in different populations.

Keywords: Abbreviations; CYP2D6; Cytochrome P450 2D6; PK; pharmacokinetics; PD; pharmacodynamics; EM; extensive metabolizer; PM; poor metabolizer; Tg-; CYP2D6; CYP2D6; -humanized; IDR; indirect response; HPLC; high performance liquid chromatography; LC-MS/MS; liquid chromatography tandem mass spectrometryCYP2D6; Pharmacogenetics; Harmaline; Pharmacokinetics; Transgenic mouse

Evaluation of a microfluidic based cell culture platform with primary human hepatocytes for the prediction of hepatic clearance in human by P. Chao; T. Maguire; E. Novik; K.-C. Cheng; M.L. Yarmush (pp. 625-632).

Integral to the discovery of new pharmaceutical entities is the ability to predict in vivo pharmacokinetic parameters from early stage in vitro data generated prior to the onset of clinical testing. Within the pharmaceutical industry, a whole host of assay methods and mathematical models exist to predict the in vivo pharmacokinetic parameters of drug candidates. One of the most important pharmacokinetic properties of new drug candidates predicted from these methods and models is the hepatic clearance. Current methods, while useful, are still limited in their predictive efficacy. In order to address this issue, we have established a novel microfluidic in vitro culture system, the patented HμREL^® device. The device comprises multiple compartments that are designed to be proportional to the physiological architectures and enhanced with the consideration of flow. Here we demonstrate the functionality of the liver-relevant chamber in the HμREL^® device, and the feasibility of utilizing our system for predicting hepatic clearance. Cryopreserved human hepatocytes from a single donor were seeded within the HμREL^® device to predict the in vivo hepatic clearance (CL_H) of six marketed model compounds (carbamazepine, caffeine, timolol, sildenafil, imipramine, and buspirone). The intrinsic clearance rates from static culture controls, as well as clearance rates from the HμREL^® device were subsequently compared to in vivo data available from the literature.

Keywords: Microfluidic; Human hepatocyte; Hepatic clearance

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site by Dana M. van Bemmel; Adam S. Brank; Ramon Eritja; Victor E. Marquez; Judith K. Christman (pp. 633-641).

Formation and reversal of 2-(1H)-pyrimidinone adduct with the activated thiol of a DNA C5-MTase.Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.

Keywords: Abbreviations; 2P; 2-(1H)-pyrimidinone (zebularine aglycon); AdoHcy; S; -adenosyl homocysteine; AdoMet; S; -adenosyl methionine; BME; beta-mercaptoethanol; bp; base pairs; Cyd; cytidine; C; cytosine; CDA; cytidine deaminase; DNA C5-MTase; DNA (cytosine C5)-methyltransferase; Dnmt1; DNA (cytosine C5)-methyltransferase-1; ds; double stranded; FCyt; 5-fluorocytosine; M.HhaI; HhaI methyltransferase; M.HgaI; HgaI methyltransferase; M.MspI; MspI methyltransferase; MR buffer; methylation reaction buffer; ODN; oligodeoxyribonucleotide; SDS; sodium dodecyl sulfate; ss; single stranded; ZCyt; 5-azacytosine; ZCyd; 5-azacytidine; ZdCyd; 5-aza-2′-deoxycytidine; DZCyt; 5,6-dihydro-5-azacytosineZebularine; DNA methylation; 2(1H)-Pyrimidinone riboside; DNA methyltransferase; 5-Aza-2′-deoxycytidine

Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid concentrations in rhesus monkeys by Cuyue Tang; Yuhsin Kuo; Nicole T. Pudvah; Joan D. Ellis; Maria S. Michener; Melissa Egbertson; Samuel L. Graham; Jacquelynn J. Cook; Jerome H. Hochman; Thomayant Prueksaritanont (pp. 642-647).

Brain penetration of drugs which are subject to P-glycoprotein (Pgp)-mediated efflux is attenuated, as manifested by the fact that the cerebrospinal fluid concentration ( C_CSF), a good surrogate of the unbound brain concentration ( C_ub), is lower than the unbound plasma concentration ( C_up) for Pgp substrates. In rodents, the attenuation magnitude of brain penetration by Pgp-mediated efflux has been estimated by correlating the ratio of CSF to plasma exposures ( C_CSF/ C_p) with the unbound fraction in plasma ( f_u) upon the incorporation of the in vivo or in vitro Pgp-mediated efflux ratios (ERs). In the present work, we investigated the impact of Pgp-mediated efflux on C_CSF in monkeys. Following intravenous administration to cisterna magna ported rhesus monkeys, the CSF and plasma concentrations were determined for 25 compounds from three discovery programs. We also evaluated their f_u in rhesus plasma and ER in human and African green monkey MDR-transfected LLC-PK1 cells. These compounds varied significantly in the f_u (0.025–0.73), and 24 out of 25 are considered Pgp substrates based on their appreciable directional transport (ER>2). The C_CSF/ C_p was significantly lower than the corresponding f_u (≥3-fold) for 16 compounds regardless of a significant correlation ( R²=0.59, p=4×10⁻⁵) when the C_CSF/ C_p was plotted against the f_u. When the f_u was normalized to the ER ( f_u/ER) the correlation was improved ( R²=0.75, p=8×10⁻⁸). More importantly, only one compound showed the C_CSF/ C_p that exceeded 3-fold of the normalized f_u. The results suggest that the impact of Pgp-mediated efflux in monkeys, similar to the case in rodents, is reasonably reflected by the gradient between the free concentrations in plasma and in CSF. Therefore, f_u and Pgp ER may serve as useful measurements in estimating in vivo C_CSF/ C_p ratios in monkeys, and potentially in humans.

Keywords: Abbreviations; Pgp; P-glycoprotein; MDR1; multidrug resistance protein; CNS; central nervous system; CSF; cerebral spinal fluid; ISF; interstitial fluid; BBB; blood–brain barrier; BCSFB; blood–CSF barrier; C; _CSF; compound concentration in CSF; C; _up; unbound compound concentration in plasma; C; _ub; unbound compound concentration in brain tissues; C; _p; total compound concentration in plasma; C; _b; total compound concentration in brain; ER; efflux ratio; f; _u; unbound fraction in plasma; AGM; African green monkey; CMP; cisterna magna portedP-glycoproteins; Cerebral spinal fluid; Blood–brain barrier; Rhesus monkeys

Ethanol selectively impairs clathrin-mediated internalization in polarized hepatic cells by David J. Fernandez; Benita L. McVicker; Dean J. Tuma; Pamela L. Tuma (pp. 648-655).

Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.

Keywords: Abbreviations; 5′NT; 5′nucleotidase; APN; aminopeptidase N; ASGP-R; asialoglycoprotein receptor; BC; bile canaliculus; CTxB; cholera toxin B subunit; DPP IV; dipeptidyl peptidase IV; GPI; glycophosphatidylinositol; HA; hemagglutinin; pIgA-R; polymeric IgA-receptor; Tf-R; transferrin receptor; TGN; trans; -Golgi networkWIF-B cells; Ethanol; Liver injury; Dynamin-2; Endocytosis; Clathrin

Sections

Personal tools

Biochemical Pharmacology (v.78, #6)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.78, #6)