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Biochemical Pharmacology (v.85, #1)

Editorial Board (pp. iii).

Gene targeting in ischemic heart disease and failure: Translational and clinical studies by Shaina R. Eckhouse; Jeffrey A. Jones; Francis G. Spinale (pp. 1-11).

Gene delivery to the heart can include adenoviral vectors. Advancements and setbacks have been encountered in delivery, choice of vector, choice of target, and ultimately the effects on heart function.Alternative and innovative targeted strategies hold relevance in improving the current treatments for ischemic heart disease (IHD). One potential treatment modality, gene targeting, may provide a unique alternative to current IHD therapies. The principal function of gene targeting in IHD is to augment the expression of an endogenous gene through amplification of an exogenous gene, delivered by a plasmid or a viral vector to enhance myocardial perfusion, and limit the long-term sequelae. The initial clinical studies of gene targeting in IHD were focused upon induction of angiogenic factors and the outcomes were equivocal. Nevertheless, significant advancements have been made in viral vectors, mode of delivery, and potentially relevant targets for IHD. Several of these advancements, particularly with a focus on translational large animal studies, are the focus of this review. The development of novel vectors with prolonged transduction efficiency and minimal inflammation, coupled with hybrid perfusion-mapping delivery devices, and improving the safety of vector use and efficacy of gene systems are but a few of the exciting progresses that are likely to proceed to clinical studies in the near future.

Keywords: Gene therapy; Myocardial infarction; Ischemia; Plasmid; Adenovirus; Adeno-associated virus

Metabolomics and its potential in drug development by Diren Beyoğlu; Jeffrey R. Idle (pp. 12-20).

Metabolomics is the global and unbiased survey of the complement of small molecules (say, <1kDa) in a biofluid, tissue, organ or organism and measures the end-products of the cellular metabolism of both endogenous and exogenous substrates. Many drug candidates fail during Phase II and III clinical trials at an enormous cost to the pharmaceutical industry in terms of both time lost and of financial resources. The constantly evolving model of drug development now dictates that biomarkers should be employed in preclinical development for the early detection of likely-to-fail candidates. Biomarkers may also be useful in the preselection of patients and through the subclassification of diseases in clinical drug development. Here we show with examples how metabolomics can assist in the preclinical development phases of discovery, pharmacology, toxicology, and ADME. Although not yet established as a clinical trial patient prescreening procedure, metabolomics shows considerable promise in this regard. We can be certain that metabolomics will join genomics and transcriptomics in lubricating the wheels of clinical drug development in the near future.

Keywords: Abbreviations; MS; mass spectrometry; UPLC; ultraperformance liquid chromatography; UPLC–ESI-QTOFMS; UPLC–electrospray ionization-quadrupole time-of-flight MS; LCMS; liquid chromatography–mass spectrometry; GCMS; gas chromatography–mass spectrometry; NMR; nuclear magnetic resonance spectroscopy; ESI+; electrospray ionization in positive ion mode; ESI−; electrospray ionization in negative ion mode; MDA; multivariate data analysis; PCA; principal components analysis; PC; principal component; PLS-DA; projection to latent structures-discriminant analysis; OPLS-DA; orthogonal PLS-DA; NME; new molecular entity; POC; proof of concept; ADME; absorption, distribution, metabolism, and excretion; IVA; influenza A virus; HSV-1; Herpes simplex virus type-1; HCMV; human cytomegalovirus; PPARα; peroxisome proliferator-activated receptor alpha; PXR; pregnane X receptor; PCN; pregnenolone 16α-carbonitrile; CCl; ₄; carbon tetrachloride; ANIT; α-naphthylisothiocyanate; SOM; site of metabolismMetabolomics; Drug development; Ultraperformance liquid chromatography–electrospray ionization-quadrupole time-of-flight mass spectrometry; Pharmacology; Toxicology

Establishment of a robust hepatitis C virus replicon cell line over-expressing P-glycoprotein that facilitates analysis of P-gp drug transporter effects on inhibitor antiviral activity by Dennis Hernandez; Paul Falk; Fei Yu; Guangzhi Zhai; Yong Quan; Teresa Faria; Kai Cao; Paul Scola; Fiona McPhee (pp. 21-28).

P-glycoprotein (P-gp) is an active efflux pump affecting the pharmacokinetic (PK) profiles of drugs that are P-gp substrates. The Caco-2 bi-directional assay is widely used to identify drug-P-gp interactions in vitro. For molecules exhibiting non-classical drug properties however, ambiguous results limit its use in lead optimization. The goal of this study was to develop a robust cell-based assay system to directly measure the role of P-gp-driven efflux in reducing the potency of hepatitis C virus (HCV) replication inhibitors. Vinblastine (Vin) was employed to select for a Vin-resistant HCV replicon (313-11) from the parental cell line (377-2). The 313-11 cell line was >50-fold resistant to Vin and over-expressed P-gp, as determined by Western immunoblots. Increased expression of P-gp was mediated by up-regulation of the MDR1 transcript. The reduced potency of different classes of HCV replication inhibitors in the 313-11 P-gp cell line was restored in the presence of known P-gp inhibitors. Addition of the P-gp inhibitor, tariquidar, increased the uptake of a radiolabeled HCV replication inhibitor by 14-fold in the 313-11 replicon cell line. Finally, a positive correlation was demonstrated between potency in the 313-11 replicon and the bi-directional Caco-2 efflux ratio for a panel of HCV protease inhibitors. In conclusion, a robust P-gp HCV replicon cell-based assay has been developed to measure the effect of the P-gp efflux pump on the potency of different classes of HCV replication inhibitors. This system establishes a direct correlation between antiviral activity and the effect of P-gp efflux in a single cell line.

Keywords: P-glycoprotein; Hepatitis C virus; Efflux pump; Antiviral inhibitor; NS3 protease

Characterisation of the roles of ABCB1, ABCC1, ABCC2 and ABCG2 in the transport and pharmacokinetics of actinomycin D in vitro and in vivo by Christopher R. Hill; David Jamieson; Huw D. Thomas; Colin D.A. Brown; Alan V. Boddy; Gareth J. Veal (pp. 29-37).

Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI₅₀ was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7nM vs. 745nM, p<0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI₅₀ 25.7 and 40.4nM respectively, p<0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98nM vs. 0.1nM, p<0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b^−/− as compared to WT mice following administration of 0.5mg/kg actinomycin D (AUC0–6_h 242 vs. 152μg/Lh respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b^−/− and Abcc2^−/− mice, concentrations in the brain were significantly higher at 6h following drug administration in Abcb1a/1b^−/− mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.

Keywords: Abbreviations; ABC transporter; ATP-binding cassette transporter; Act D; actinomycin D; ANOVA; analysis of variance; GI; ₅₀; concentration at 50% growth inhibition; LC/MS; liquid chromatography/mass spectrometry; SEM; standard error of the mean; SNPs; single nucleotide polymorphismsActinomycin D; ABC transporters; Cancer

Molecular interaction of artemisinin with translationally controlled tumor protein (TCTP) of Plasmodium falciparum by Tolga Eichhorn; Dominic Winter; Berthold Büchele; Natalie Dirdjaja; Martin Frank; Wolf-Dieter Lehmann; Rolf Mertens; R. Luise Krauth-Siegel; Thomas Simmet; Joachim Granzin; Thomas Efferth (pp. 38-45).

Malaria causes millions of death cases per year. Since Plasmodium falciparum rapidly develops drug resistance, it is of high importance to investigate potential drug targets which may lead to novel rational therapy approaches. Here we report on the interaction of translationally controlled tumor protein of P. falciparum ( PfTCTP) with the anti-malarial drug artemisinin. Furthermore, we investigated the crystal structure of PfTCTP. Using mass spectrometry, bioinformatic approaches and surface plasmon resonance spectroscopy, we identified novel binding sites of artemisinin which are in direct neighborhood to amino acids 19–46, 108–134 and 140–163. The regions covered by these residues are known to be functionally important for TCTP function. We conclude that interaction of artemisinin with TCTP may be at least in part explain the antimalarial activity of artemisinin.

Keywords: Abbreviations; TCTP; translationally controlled tumor protein; MS; mass spectrometry; MS/MS; tandem mass spectrometry; m; /; z; mass; to charge ratio; LC; liquid chromatography; ESI; electron spray ionization; (Q)TOF; (quadrupole) time-of-flight; DTT; dithiothreitolArtemisinin; Translationally controlled tumor protein; Plasmodium falciparum

Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion by Samantha E. Griner; Jayashree P. Joshi; Rita Nahta (pp. 46-58).

Identification of novel molecular markers and therapeutic targets may improve survival rates for patients with ovarian cancer. In the current study, immunohistochemical (IHC) analysis of two human ovarian tumor tissue arrays showed high staining for GDF15 in a majority of tissues. Exogenous stimulation of ovarian cancer cell lines with recombinant human GDF15 (rhGDF15) or stable over-expression of a GDF15 expression plasmid promoted anchorage-independent growth, increased invasion, and up-regulation of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). MMP inhibition suppressed GDF15-mediated invasion. In addition, IHC analysis of human ovarian tumor tissue arrays indicated that GDF15 expression correlated significantly with high MMP2 and MMP9 expression. Exogenous and endogenous GDF15 over-expression stimulated phosphorylation of p38, Erk1/2, and Akt. Pharmacologic inhibition of p38, MEK, or PI3K suppressed GDF15-stimulated growth. Further, proliferation, growth, and invasion of GDF15 stable clones were blocked by rapamycin. IHC analysis demonstrated significant correlation between GDF15 expression and phosphorylation of mTOR. Finally, knockdown of endogenous GDF15 or neutralization of secreted GDF15 suppressed invasion and growth of a GDF15-over-expressing ovarian cancer cell line. These data indicate that GDF15 over-expression, which occurred in a majority of human ovarian cancers, promoted rapamycin-sensitive invasion and growth of ovarian cancer cells. Inhibition of mTOR may be an effective therapeutic strategy for ovarian cancers that over-express GDF15. Future studies should examine GDF15 as a novel molecular target for blocking ovarian cancer progression.

Keywords: GDF15; mTOR; PI3K; Ovarian cancer; Invasion; Rapamycin

Arsenic trioxide-induced hERG K⁺ channel deficiency can be rescued by matrine and oxymatrine through up-regulating transcription factor Sp1 expression by Ying Zhang; Zengxiang Dong; Liyan Jin; Kaiping Zhang; Xin Zhao; Jia Fu; Yan Gong; Mingming Sun; Baofeng Yang; Baoxin Li (pp. 59-68).

Matrine and oxymatrine may have a potential to rescue the hERG channel deficiency caused by As₂O₃, which include multi-pathway: promoting hERG channel activation and increasing hERG channel expression.The human ether-a-go-go-related gene (hERG) encodes the rapidly activating, delayed rectifier potassium channel (IKr) important for cardiac repolarization. Dysfunction of the hERG channel can cause Long QT Syndrome (LQTS). A wide variety of structurally diverse therapeutic compounds reduce the hERG current by acute direct inhibition of the hERG current or/and selective disruption of hERG protein expression. Arsenic trioxide (As₂O₃), which is used to treat acute promyelocytic leukemia, can cause LQTS type 2 (LQT2) by reducing the hERG current through the diversion of hERG trafficking to the cytoplasmic membrane. This cardiotoxicity limits its clinical applications. Our aim was to develop cardioprotective agents to decrease As₂O₃-induced cardiotoxicity. We reported that superfusion of hERG-expressing HEK293 (hERG-HEK) cells with matrine (1, 10μM) increased the hERG current by promoting hERG channel activation. Long-term treatment with 1μM matrine or oxymatrine increased expression of the hERG protein and rescued the hERG surface expression disrupted by As₂O_3. In addition, Matrine and oxymatrine significantly shortened action potential duration prolonged by As₂O₃ in guinea pig ventricular myocytes. These results were ascribed to the up-regulation of hERG at both mRNA and protein levels via an increase in the expression of transcription factor Sp1, an established transactivator of the hERG gene. Therefore, matrine and oxymatrine may have the potential to cure LQT2 as a potassium channel activator by promoting hERG channel activation and increasing hERG channel expression.

Keywords: As; ₂; O; ₃; Matrine; Oxymatrine; hERG; Sp1

Antiarrhythmic effects of (−)-epicatechin-3-gallate, a novel sodium channel agonist in cultured neonatal rat ventricular myocytes by Adonis Zhi-Yang Wu; Shih-Hurng Loh; Tzu-Hurng Cheng; Hsin-Hsiang Lu; Cheng-I. Lin (pp. 69-80).

(−)-Epicatechin-3-gallate (ECG), a polyphenol extracted from green tea, has been proposed as an effective compound for improving cardiac contractility. However, the therapeutic potential of ECG on the treatment of arrhythmia remains unknown. We investigated the direct actions of ECG on the modulation of ion currents and cardiac cell excitability in the primary culture of neonatal rat ventricular myocyte (NRVM), which is considered a hypertrophic model for analysis of myocardial arrhythmias. By using the whole-cell patch-clamp configurations, we found ECG enhanced the slowly inactivating component of voltage-gated Na⁺ currents ( I_Na) in a concentration-dependent manner (0.1–100μM) with an EC₅₀ value of 3.8μM. ECG not only shifted the current–voltage relationship of peak I_Na to the hyperpolarizing direction but also accelerated I_Na recovery kinetics. Working at a concentration level of I_Na enhancement, ECG has no notable effect on voltage-gated K⁺ currents and L-type Ca²⁺ currents. With culture time increment, the firing rate of spontaneous action potential (sAP) in NRVMs was gradually decreased until spontaneous early after-depolarization (EAD) was observed after about one week culture. ECG increased the firing rate of normal sAP about two-fold without waveform alteration. Interestingly, the bradycardia-dependent EAD could be significantly restored by ECG in fast firing rate to normal sAP waveform. The expression of dominant cardiac sodium channel subunit, Nav1.5, was consistently detected throughout the culture periods. Our results reveal how ECG, the novel I_Na agonist, may act as a promising candidate in clinical applications on cardiac arrhythmias.

Keywords: Arrhythmia; Electrophysiology; Na; ⁺; current; (−)-Epicatechin-3-gallate; Green tea

Chemical cholecystokinin receptor activation protects against obesity-diabetes in high fat fed mice and has sustainable beneficial effects in genetic ob/ob mice by Nigel Irwin; Ian A. Montgomery; R. Charlotte Moffett; Peter R. Flatt (pp. 81-91).

The current study has determined the ability of (pGlu-Gln)-CCK-8 to counter the development of diet-induced obesity-diabetes and examined persistence of beneficial metabolic effects in high fat and ob/ob mice, respectively. Twice daily injection of (pGlu-Gln)-CCK-8 in normal mice transferred to a high fat diet reduced energy intake ( p<0.001), body weight ( p<0.01), circulating insulin and LDL-cholesterol ( p<0.001) and improved insulin sensitivity ( p<0.001) as well as oral and intraperitoneal ( p<0.001) glucose tolerance. Energy intake, body weight, circulating insulin and glucose tolerance of (pGlu-Gln)-CCK-8 mice were similar to lean controls. In addition, (pGlu-Gln)-CCK-8 prevented the effect of high fat feeding on triacylglycerol accumulation in liver and muscle. Interestingly, (pGlu-Gln)-CCK-8 significantly ( p<0.001) elevated pancreatic glucagon content. Histological examination of the pancreata of (pGlu-Gln)-CCK-8 mice revealed no changes in islet number or size, but there was increased turnover of beta-cells with significantly ( p<0.001) increased numbers of peripherally located alpha-cells, co-expressing both glucagon and GLP-1. Beneficial metabolic effects were observed similarly in ob/ob mice treated twice daily with (pGlu-Gln)-CCK-8 for 18 days, including significantly reduced energy intake ( p<0.05), body weight ( p<0.05 to p<0.01), circulating glucose ( p<0.05 to p<0.01) and insulin ( p<0.05 to p<0.001) and improved glucose tolerance ( p<0.05) and insulin sensitivity ( p<0.001). Notably, these beneficial effects were still evident 18 days following cessation of treatment. These studies emphasize the potential of (pGlu-Gln)-CCK-8 for the treatment of obesity-diabetes.

Keywords: Abbreviations; CCK; cholecystokinin; GLP-1; glucagon-like peptide-1; HPLC; high performance liquid chromatography; MALDI-TOF; matrix-assisted laser desorption ionization-time of flight; MS; mass spectrometry; obese diabetic mice; (; ob/ob; ) mice; AUC; area under curve; TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP nick end labellingCholecystokinin (CCK); High fat; Obesity; Diabetes; ob/ob; mice; Insulin sensitivity; Glucose tolerance

Zymosan induces NADPH oxidase activation in human neutrophils by inducing the phosphorylation of p47phox and the activation of Rac2: Involvement of protein tyrosine kinases, PI3Kinase, PKC, ERK1/2 and p38MAPkinase by Karama Makni-Maalej; Mélanie Chiandotto; Margarita Hurtado-Nedelec; Samia Bedouhene; Marie-Anne Gougerot-Pocidalo; Pham My-Chan Dang; Jamel El-Benna (pp. 92-100).

Pathways involved in zymosan-induced NADPH oxidase (NOX2) activation in human neutrophils.Reactive oxygen species (ROS) production by the neutrophil NADPH oxidase plays a key role in host defense against pathogens, such as bacteria and fungi. Zymosan a cell-wall preparation from Saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan alone induced ROS production by human neutrophils. Zymosan-induced ROS production is higher than the formyl-methionyl-leucyl-phenylalanine (fMLF)- or the phorbol myristate acetate (PMA)-induced ROS production but is lower than the one induced by opsonized zymosan. Most of the zymosan-induced ROS production is intracellular. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on several sites which are Ser315, Ser328 and Ser345. Zymosan induced also the activation of the small G-protein Rac2. Phosphorylation of the p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor and by wortmannin a PI3Kinase inhibitor. GF109203X a PKC inhibitor inhibited phosphorylation of p47phox on Ser315 and Ser328. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway, respectively, inhibited phosphorylation of p47phox on Ser345. Zymosan-induced ROS production was completely inhibited by genistein and wortmannin and partially inhibited by SB203580, UO126 and GF109203X. These results show that zymosan alone is able to activate NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation and that a protein tyrosine kinase, PI3Kinase, p38MAPK, ERK1/2 and PKC are involved in this process. These pathways could be potential pharmacological targets to treat zymosan- and S. cerevisiae-induced inflammation.

Keywords: Neutrophils; NADPH oxidase; p47phox; Rac2; ROS; Signaling

Platelet-derived growth factor triggers PKA-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells by Florian Eisel; Meike Boosen; Martina Beck; Heinrich Heide; Ilka Wittig; Karl-Friedrich Beck; Josef Pfeilschifter (pp. 101-108).

Inflammatory glomerular kidney diseases are often accompanied with a massive production of reactive oxygen species (ROS) that affect the function of the glomerular filtration barrier and contribute to mesangiolysis via the induction of cell death in mesangial cells. Intriguingly, ROS also trigger fine-tuned signalling processes that affect gene expression and cell proliferation or migration. To define such redox-driven signalling devices, a proteomics approach was performed to identify the formation of protein complexes induced by ROS. To this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250μM). Thereafter cell lysates were subjected to diagonal 2D gel electrophoresis and putative redox-affected proteins were analysed by MS/MS analysis. Among others, the regulatory subunit of protein kinase A (PKA) could be identified that forms homodimers under oxidative conditions. To evaluate whether ROS dependent dimerization of PKA also occurs in a more physiological setting, rat mesangial cells were treated with platelet-derived growth factor-BB (PDGF-BB) to induce ROS formation. This regimen resulted in a redox dependent dimerization of the R-subunits of PKA. To demonstrate whether PDGF-BB induced ROS formation affects PKA dependent pathways, the effects of PDGF-BB on phosphorylation of serine 157 of vasodilator stimulated protein (VASP) a classical target of PKA were analysed. Interestingly PDGF-BB induced VASP phosphorylation in a ROS dependent manner but independent of changes in cAMP levels. Taken together, we demonstrate a redox-mediated activation of PKA by PDGF-BB thus highlighting a physiological role of ROS as regulator of PKA activity in rat mesangial cells.

Keywords: Redox signalling; Protein kinase A; Mesangial cells; Platelet-derived growth factor

Upregulation of phagocyte-like NADPH oxidase by cytokines in pancreatic beta-cells: Attenuation of oxidative and nitrosative stress by 2-bromopalmitate by Abiy M. Mohammed; Khadija Syeda; Timothy Hadden; Anjaneyulu Kowluru (pp. 109-114).

Phagocyte-like NADPH oxidase (Nox2) has been shown to play regulatory roles in the metabolic dysfunction of the islet β-cell under the duress of glucolipotoxic conditions and exposure to proinflammatory cytokines. However, the precise mechanisms underlying Nox2 activation by these stimuli remain less understood. To this end, we report a time-dependent phosphorylation of p47phox, a cytosolic subunit of Nox2, by cytomix (IL-1β+TNFα+IFNγ) in insulin-secreting INS-1 832/13 cells. Furthermore, cytomix induced the expression of gp91phox, a membrane component of Nox2. 2-Bromopalmitate (2-BP), a known inhibitor of protein palmitoylation, markedly attenuated cytokine-induced, Nox2-mediated reactive oxygen species (ROS) generation and inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) generation. However, 2-BP failed to exert any significant effects on cytomix-induced CHOP expression, a marker for endoplasmic reticulum stress. Together, our findings identify palmitoyltransferase as a target for inhibition of cytomix-induced oxidative (ROS generation) and nitrosative (NO generation) stress in the pancreatic β-cell.

Keywords: Abbreviations; 2-BP; 2-Bromopalmitate; BSA; Bovine serum albumin; CHAPS; 3-[3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CHOP; C/EBP homologous protein; DTT; Dithiothreitol; EDTA; Ethylenediaminetetraacetic acid; FBS; Fetal bovine serum; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; iNOS; inducible nitric oxide synthase; NO; nitric oxide; Nox2; phagocyte-like NADPH oxidase; Na; ₃; VO; ₄; Sodium orthovandate; PIC; Protease inhibitor cocktail; PMSF; Phenylmethanesulfonylfluoride; RIPA; Radioimmuno-precipitation assay; and ROS; Reactive oxygen speciesPancreatic β-cell; NADPH-oxidase; 2-Bromopalmitate; Cytokines; Oxidative stress; Nitrosative stress

Interorgan metabolism of ornithine phenylacetate (OP)—A novel strategy for treatment of hyperammonemia by Sherry Dadsetan; Michael Sørensen; Lasse K. Bak; Hendrik Vilstrup; Peter Ott; Arne Schousboe; Rajiv Jalan; Susanne Keiding; Helle S. Waagepetersen (pp. 115-123).

Combined administration of ornithine and phenylacetate (OP) is proposed as a novel treatment of hyperammonemia and hepatic encephalopathy. Ornithine is believed to increase ammonia fixation into glutamine in muscle tissue and glutamine is subsequently thought to react with phenylacetate forming phenylacetylglutamine (PAGN) which is excreted in urine. The aim of the present study was to elucidate the interorgan metabolism of ornithine and ammonia in cirrhotic rats treated with OP in order to obtain an understanding of the underlying mechanisms of the beneficial effect of the treatment, which are largely unknown. Bile duct ligated cirrhotic rats and SHAM rats were treated with OP or saline for five days. [2,5-¹⁵N]Ornithine or¹⁵NH₄⁺ were administered intravenously and the incorporation of¹⁵N in amino acids as well as the content of the amino acids were subsequently determined in plasma, skeletal muscle, liver and kidney. In BDL rats, OP treatment reduced arterial ammonia concentration and increased that of glutamine 30min after the treatment but not after 15h. OP treatment did not increase¹⁵N labeling in glutamine from [2,5-¹⁵N]ornithine and¹⁵NH₄⁺ in skeletal muscle or liver. However, the extent of glutamine labeling from [2,5-¹⁵N]ornithine or¹⁵NH₄⁺ was similar in arterial blood and liver and higher than that in skeletal muscle. These findings suggest that the effect of OP was related to hepatic metabolism of ornithine. PAGN could not be detected in urine or blood in any of the rats which may explain why OP treatment only reduced arterial ammonia transiently.

Keywords: Abbreviations; HE; hepatic encephalopathy; PA; phenylacetate; PAGN; phenylacetylglutamine; OP; ornithine phenylacetate; BDL; bile duct ligation; SHAM; sham-operation; OAT; ornithine-ω-aminotransferase; GSADH; glutamate-γ-semialdehyde dehydrogenase; GS; glutamine synthetase; LOLA; l; -ornithine; l; -aspartateHepatic encephalopathy; Hyperammonemia; Bile duct ligated rats; Phenylacetylglutamine; Ornithine; Phenylacetate

Autophagy upregulation promotes survival and attenuates doxorubicin-induced cardiotoxicity by Balindiwe J.N. Sishi; Benjamin Loos; Jacques van Rooyen; Anna-Mart Engelbrecht (pp. 124-134).

This study evaluated whether the manipulation of autophagy could attenuate the cardiotoxic effects of doxorubicin (DXR) in vitro as well as in a tumour-bearing mouse model of acute doxorubicin-induced cardiotoxicity. We examined the effect of an increase or inhibition of autophagy in combination with DXR on apoptosis, reactive oxygen species (ROS) production and mitochondrial function. H9C2 rat cardiac myoblasts were pre-treated with bafilomycin A1 (autophagy inhibitor, 10nM) or rapamycin (autophagy inducer, 50μM) followed by DXR treatment (3μM). The augmentation of autophagy with rapamycin in the presence of DXR substantially ameliorated the detrimental effects induced by DXR. This combination treatment demonstrated improved cell viability, decreased apoptosis and ROS production and enhanced mitochondrial function. To corroborate these findings, GFP-LC3 mice were inoculated with a mouse breast cancer cell line (EO771). Following the appearance of tumours, animals were either treated with one injection of rapamycin (4mg/kg) followed by two injections of DXR (10mg/kg). Mice were then sacrificed and their hearts rapidly excised and utilized for biochemical and histological analyses. The combination treatment, rather than the combinants alone, conferred a cardioprotective effect. These hearts expressed down-regulation of the pro-apoptotic protein caspase-3 and cardiomyocyte cross-sectional area was preserved. These results strongly indicate that the co-treatment strategy with rapamycin can attenuate the cardiotoxic effects of DXR in a tumour-bearing mouse model.

Keywords: Autophagy; Rapamycin; Cardiotoxicity; Doxorubicin; Mitochondria; GFP-LC3

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Biochemical Pharmacology (v.85, #1)