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Biochemical Pharmacology (v.70, #8)

Editorial Advisory Board (pp. iii).

Brain cholinergic vulnerability: Relevance to behavior and disease by Michael McKinney; Mayo Clinic Jacksonville (pp. 1115-1124).

The major populations of cholinergic neurons in the brain include two “projection�? systems, located in the pontine reticular formation and in the basal forebrain. These two complexes comprise, in part, the anatomical substrates for the “ascending reticular activating system�? (ARAS). The pontine cholinergic system relays its rostral influences mainly through thalamic intralaminar nuclei, but it also connects to the basal forebrain and provides a minor innervation of cortex. The basal forebrain cholinergic complex (BFCC) projects directly to cortex and hippocampus, and has a minor connection with the thalamus. Recent data reveal that a parallel system of basal forebrain GABAergic projection neurons innervates cortex/hippocampus in a way that seems to complement the BFCC. Generally, the picture developed from more than 50 years of research is consistent with a “global�? influence of these two ascending cholinergic projections on cortical and hippocampal regions. Seemingly, the BFCC acts in tandem or in parallel with the pontine cholinergic projection to activate the electro-encephalogram, increase cerebral blood flow, regulate sleep–wake cycling, and modulate cognitive function. There are quite a number and variety of human brain conditions, notably including Alzheimer's disease, in which degeneration of basal forebrain cholinergic neurons has been documented. Whether the corticopetal GABA system is affected by disease has not been established. Studies of degeneration of the pontine projection are limited, but the available data suggest that it is relatively preserved in Alzheimer's disease. Hypotheses of BFCC degeneration include growth factor deprivation, intracellular calcium dysfunction, amyloid excess, inflammation, and mitochondrial abnormalities/oxidative stress. But, despite considerable research conducted over several decades, the exact mechanisms underlying brain cholinergic vulnerability in human disease remain unclear.

The novel lipophilic camptothecin analogue gimatecan is very active in vitro in human neuroblastoma: A comparative study with SN38 and topotecan by Angela Maria Di Francesco; Anna Shirley Riccardi; Giuseppe Barone; Sergio Rutella; Daniela Meco; Roberta Frapolli; Massimo Zucchetti; Maurizio D’Incalci; Claudio Pisano; Paolo Carminati; Riccardo Riccardi (pp. 1125-1136).

Neuroblastoma is one of the most common extracranial solid tumours in childhood with a poor prognosis in its advanced stage. Treatment failure is often associated to the occurrence of drug resistance. To date, treatment of paediatric neuroblastoma is still dismal, and therefore novel effective drugs are awaited. In recent years, an increasing interest has concentrated on camptothecin analogues. Topotecan and irinotecan, the only two clinically relevant camptothecin derivatives to date, have entered clinical trials in neuroblastoma but so far the results have been disappointing. Gimatecan (ST1481, LBQ707; 7- t-butoxyiminomethylcamptothecin), is a novel lipophilic camptothecin derivative that was selected from a series of lipophilic analogues rationally designed and synthesized in order to overcome some of the main drawbacks of conventional camptothecins, limiting their clinical efficacy. Gimatecan is endowed with potent antitumour activity, strong topoisomerase I inhibition, stable drug–target interactions and a better pharmacological profile. The present study deals with the comparative evaluation of cellular pharmacology features of gimatecan, topotecan and SN38 in neuroblastoma cell lines. We show that, despite the lowest intracellular accumulation, gimatecan was the most active among the camptothecin analogues studied. Our findings suggest that the high activity of gimatecan in neuroblastoma is related to the ability of this novel analogue to cause a very high number of DNA breaks as assessed by the Comet assay in both cellular or sub-cellular systems. We propose that DNA strand breaks efficiency as measured by the Comet assay might provide important information about the stability of the ternary complexes induced by camptothecin compounds.

Keywords: Gimatecan; Camptothecins; Topoisomerase I inhibitors; Neuroblastoma; Alkaline comet assay; DNA strand breaks

The influence of tumour microenvironmental factors on the efficacy of cisplatin and novel platinum(IV) complexes by H.R. Mellor; S. Snelling; M.D. Hall; S. Modok; M. Jaffar; T.W. Hambley; R. Callaghan (pp. 1137-1146).

The chemotherapeutic drug cisplatin is an important treatment for many types of solid tumours, in particular non-small cell lung cancer (NSCLC). Platinum(IV) complexes offer several advantages to cisplatin due to their requirement for reduction to the active platinum(II) form to elicit cytotoxicity. This should minimise non-specific effects and facilitate higher amounts of the active complexes reaching the target DNA. Hypoxia and a quiescent cell population are features of the tumour microenvironment known to lead to resistance to many chemotherapeutic agents. It is unclear how these microenvironmental factors will impact on the efficacy of novel platinum(IV) complexes. Consequently, the cytotoxicities of several platinum drugs were determined in monolayer and tumour spheroid cultures derived from NSCLC lines. Platinum(IV) reduction potential correlated well with cytotoxicity. The complex containing a chloro axial ligand demonstrated the greatest potency and the drug with the hydroxy ligand was the least effective. Although drug cytotoxicity was not enhanced under hypoxic conditions, both cisplatin and the platinum(IV) complexes retained full potency. In addition, all of the platinum drugs retained the ability to evoke apoptosis in quiescent cells. In summary, unlike many anticancer drugs, the platinum(IV) complexes retain cytotoxic potency under resistance-inducing tumour microenvironmental conditions and warrant further investigation as more selective alternatives to current platinum-based therapy for the treatment of solid tumours.

Keywords: Platinum(IV); Cisplatin; Hypoxia; Tumour microenvironment; Quiescent cells; Solid tumour models

Induction of apoptotic cell death by 2′-hydroxycinnamaldehyde is involved with ERK-dependent inactivation of NF-κB in TNF-α-treated SW620 colon cancer cells by Seung Ho Lee; Chung Woo Lee; Jae Woong Lee; Myoung Suk Choi; Dong Ju Son; Youn Bok Chung; Chong Kil Lee; Ki Wan Oh; Dong Chul Moon; Byoung Mog Kwon; Jin Tae Hong (pp. 1147-1157).

2′-Hydroxycinnamaldehyde (HCA) inhibits cell growth of several human cancer cells with unknown mechanisms. We investigated the inhibitory effect of HCA on TNF-α-induced cell growth and possible signal pathway in SW620 colon cancer cells. HCA inhibited TNF-α-induced SW620 colon cell growth in time- and dose-dependent manner through induction of apoptotic cell death. Parallel with inhibitory effect on cell growth, HCA dose dependency inhibited TNF-α-induced activation of NF-κB accompanied with inhibition of the translocation of p50. HCA also induced expression of caspase-3 and Bax, but decreased Bcl-2. HCA furthermore activated ERK pathway, and ERK inhibitor reversed inhibitory effect of HCA on cell growth and transcriptional activation of NF-κB. These results demonstrate that HCA inhibits cell growth through induction of apoptotic cell death by ERK pathway-dependent NF-κB inactivation.

Keywords: Abbreviations; HCA; 2′-hydroxycinnamaldehyde; NF-κB; nuclear transcription factor-κB; TNF-α; tumor necrosis factor-α; ERK; intracellular signal regulated kinase; MAPK; mitogen-activated protein kinase; IKK; IκB kinase; EMSA; electro mobility shift assay; JNK; c-jun NH; ₂; -terminal kinase2′-Hydroxycinnamaldehyde; NF-κB; ERK; Apoptotic cell death; Colon cancer

Primaquine synergises the activity of chloroquine against chloroquine-resistant P. falciparum by Patrick G. Bray; Samantha Deed; Emma Fox; Martha Kalkanidis; Mathirut Mungthin; Leslie W. Deady; Leann Tilley (pp. 1158-1166).

In recent years, resistance to the antimalarial drug, chloroquine, has become widespread. It is, therefore, imperative to find compounds that could replace chloroquine or work synergistically with this drug to overcome chloroquine resistance. We have examined the interaction between chloroquine, a 4-aminoquinoline, and a number of 8-aminoquinolines, including primaquine, a drug that is widely used to treat Plasmodium vivax infections. We find that primaquine is a potent synergiser of the activity of chloroquine against chloroquine-resistant Plasmodium falciparum. Analysis of matched transfectants expressing mutant and wild-type alleles of the P. falciparum chloroquine resistance transporter (PfCRT) indicate that primaquine exerts its activity by blocking PfCRT, and thus enhancing chloroquine accumulation. Our data suggest that a novel formulation of two antimalarial drugs already licensed for use in humans could be used to treat chloroquine-resistant parasites.

Keywords: Abbreviations; CQ; chloroquine; DMSO; dimethyl sulfoxide; FP; ferriprotoporphyrin IX/haem; PfCRT; P. falciparum; chloroquine resistance transporter; PQ; primaquine; TQ; tafenoquineMalaria; 8-Aminoquinoline; Primaquine; Chloroquine; Drug resistance; Resistance reversing agent

Low-molecular-weight fucoidan enhances the proangiogenic phenotype of endothelial progenitor cells by Faouzia Zemani; Danielle Benisvy; Isabelle Galy-Fauroux; Anna Lokajczyk; Sylvia Colliec-Jouault; Georges Uzan; Anne Marie Fischer; Catherine Boisson-Vidal (pp. 1167-1175).

Endothelial progenitor cell (EPC) transplantation is a potential means of inducing neovascularization in vivo. However, the number of circulating EPC is relatively small, it may thus be necessary to enhance their proangiogenic properties ex vivo prior to injection in vivo. Fucoidan has previously been shown to potentiate in vitro tube formation by mature endothelial cells in the presence of basic fibroblast growth factor (FGF-2). We therefore examined whether fucoidan, alone or combined with FGF-2, could increase EPC proangiogenic potency in vitro. EPC exposure to 10μg/ml fucoidan induced a proangiogenic phenotype, including cell proliferation ( p<0.01) and migration ( p<0.01); moreover, differentiation into vascular cords occurred in the presence of FGF-2 ( p<0.01). This latter effect correlated with upregulation of the cell-surface #α6 integrin subunit of the laminin receptor ( p<0.05). Compared to untreated HUVEC, untreated EPC #α6 expression and adhesion to laminin were enhanced two-fold. Fucoidan treatment further enhanced HUVEC but not EPC adhesion to laminin. These results show that fucoidan enhances the proangiogenic properties of EPC and suggest that ex vivo fucoidan preconditioning of EPC might lead to increased neovascularization when injected into ischemic tissues.

Keywords: Abbreviations; EPC; endothelial progenitor cells; ECM; extracellular matrix; FGF-2; basic fibroblast growth factor; FACS; fluorescence-activated cell sorting; FCS; fetal calf serum; HUVEC; human umbilical vein endothelial cell; PBS; phosphate buffered saline; VEGF; vascular endothelial growth factor; KDR; vascular endothelial growth factor receptor-2; vWF; von Willebrand factorAlpha #6; Angiogenesis; Endothelial progenitor cells; FGF-2; Fucoidan

Double blockade of cell cycle progression by coptisine in vascular smooth muscle cells by H. Tanabe; H. Suzuki; H. Mizukami; M. Inoue (pp. 1176-1184).

Coptisine, an isoquinoline alkaloid isolated from rhizome of Coptis japonica, inhibits proliferation of vascular smooth muscle cells (VSMCs). The aim of this study was to evaluate the action of coptisine, along with berberine (a structurally similar isoquinoline alkaloid), on progression of the cell cycle in VSMCs. Coptisine displayed antiproliferative action against VSMCs by blocking the cell cycle at G₁ and G₂/M phases. The G₁ block was shown by inhibition of [³H]thymidine incorporation into VSMCs at coptisine concentrations higher than 15μM. The mechanism underlying the G₁ arrest involved a decrease in cyclin D1 protein, although cyclin E, A, and B were not affected by coptisine treatment. The selective reduction in cyclin D1 protein was mainly attributable to accelerated proteolysis via proteasome-dependent pathway, since it was inhibited by a proteasome inhibitor, N-carbobenzoxy-l-leucinyl-l-leucinyl-l-norleucinal (MG132) and further the mRNA level of cyclin D1, protein synthesis, and mitogen-activated protein kinase (MAPK) activity remained unaltered. The mechanism underlying the G₂/M arrest involved partial inhibition of tubulin polymerization, which was apparent at coptisine concentration of 3μM. Berberine arrested the cell cycle at G₁ phase via a mechanism identical with coptisine, but did not cause block at G₂/M phase. The results demonstrate that a small difference in the structure between isoquinoline alkaloids produces a big difference in activity, and that coptisine has a unique double action in arresting the cell cycle of VSMCs.

Keywords: Abbreviations; bFGF; basic fibroblast growth factor; CDK; cyclin dependent kinase; DEPC; diethyl pyrocarbonate; DMEM; Dulbecco's modified Eagle's medium; ERK; extracellular signal-regulated kinase; FCS; fetal calf serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; MAP; mitogen-activated protein; PDGF; platelet-derived growth factor; PVDF; polyvinylidene dufluoride; RNase; ribonuclease; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide electrophoresis; VSMC; vascular smooth muscle cellCoptisine; Berberine; Isoquinoline alkaloid; Vascular smooth muscle cell; Cell cycle; Selectivity

Effect of simvastatin on the oxidation of native and modified lipoproteins by Grazyna Sobal; Helmut Sinzinger (pp. 1185-1191).

Modified (oxidized) low-density lipoprotein (LDL) plays a significant role in atherosclerosis by accumulation in arteries. Also, glycated LDL, such as in diabetics, are increasing the risk for atherosclerosis, due to an increased oxidizability as compared to native LDL. For these reasons, the potential inhibition of such modifications is of clinical importance. We investigated the influence of simvastatin on oxidation of native and modified LDL as well as high-density lipoprotein (HDL), which plays a protective role in atherosclerosis. Quantitative assessment of the oxidation end-product malondialdehyde (MDA) revealed the highest inhibitory rate for HDL at concentrations of 1.6μg/ml and 0.8μg/ml by 30.3% and 20.4%, at 6h and 4h, respectively. At 24h, the inhibition was still persisting amounting to 27.9% and 20.3%, respectively.For native LDL, we found less inhibition of oxidation at a concentration of 1.6μg/ml amounting to 19.2% and 11.5%, for 4h and 6h, respectively. Similar effects were found at a concentration of 0.8μg/ml. For modified, glycated LDL, the most pronounced effect was found at a concentration of 1.6μg/ml amounting to 22.4% for the period of 2–24h of oxidation. For glycoxidated LDL, the inhibition of oxidation was less expressed amounting to 10.1% for the period of 2–6h at the same concentration.The influence of simvastatin on lag time (protection from oxidation) by diene conjugation was also investigated. At the highest concentration of simvastatin (1.6μg/ml), we found a prolongation of lag time from 73min to 99min for native LDL, for glycoxidated LDL 60min to 89min and for HDL 54min to 64min. For glycated LDL, only a small decrease of lag time (66min versus 71min) at same concentration was observed. For glycated and glycoxidated LDL, we found a moderate increase in relative electrophoretic mobility (REM) by 2.0 and 2.3, respectively, but no changes in the presence of simvastatin were observed. These data show that simvastatin besides its lipid-lowering action has also significant antioxidative properties.

Keywords: Abbreviations; Apo I; apolipoprotein I; BHT; butylated hydroxytoluene; CHD; coronary heart disease; CRP; C-reactive protein; EDTA; ethylenediaminetetraacetic acid; HDL; high-density lipoprotein; HMG-CoA; 3-hydroxy-3-methylglutaryl-coenzyme A; LDL; low-density lipoprotein; MDA; malondialdehyde; PBS; phosphate-buffered saline; REM; relative electrophoretic mobility; TBA; thiobarbituric acid; TCA; trichloroacetic acid; VLDL; very low-density lipoproteinLipoproteins; Modified lipoproteins; Glycation; Glycoxidation; Statins; Simvastatin; Atherosclerosis

Suppression of vascular cell adhesion molecule-1 expression by crocetin contributes to attenuation of atherosclerosis in hypercholesterolemic rabbits by Shuguo Zheng; Zhiyu Qian; Futian Tang; Liang Sheng (pp. 1192-1199).

To elucidate the molecular mechanism by which antioxidants alleviate atherosclerosis, we investigated the effect of crocetin, a naturally occurred carotinoid with potent antioxidant power, on vascular cell adhesion molecule-1 (VCAM-1) expression in atherosclerotic rabbits. Twenty-four male New Zealand White rabbits were allocated to three groups fed on standard diet (control group), high lipid diet (HLD group) or high lipid diet supplemented with crocetin (crocetin group), respectively. After 8 weeks of treatment, rabbits in HLD group developed severe hypercholesterolemia and atherosclerosis in aortas, together with a significantly up-regulated expression of both protein and mRNA for VCAM-1. In contrast, supplementation with crocetin resulted in markedly ameliorated atherosclerosis, coupled with a significantly decreased VCAM-1 expression, though plasma lipids level remained comparable to that of HLD group. Regression analysis revealed a positive correlation between VCAM-1 expression and the extent of atherosclerosis ( P<0.01). In addition, immunohistochemical analysis showed an increased activation of nuclear factor kappa B (NF-κB), a redox sensitive transcription factor essential for VCAM-1 expression, in aortas from rabbits fed on high lipid diet, which was evidently suppressed by crocetin supplementation. These findings suggest that the antiatherosclerotic effect of crocetin might be attributed, at least in part, to the suppressed expression of VCAM-1, which might result from reduced NF-κB activation. This study provides a further insight into the molecular mechanism by which antioxidants attenuate atherosclerosis and suggests a potential target for the treatment of atherosclerosis with antioxidants.

Keywords: Abbreviations; HDL; high density lipoprotein; HLD; high lipid diet; ICAM-1; intercellular adhesion molecule-1; IκB; inhibitory kappa B; LDL; low density lipoprotein; NF-κB; nuclear factor kappa B; Ox-LDL; oxidized LDL; ROS; reactive oxygen species; TC; total cholesterol; VCAM-1; vascular cell adhesion molecule-1Atherosclerosis; Cell adhesion molecule; Transcription factor; Reactive oxygen species; Antioxidants; Crocetin

Role of cytosolic phospholipase A₂ in the enhancement of α₂-adrenoceptor-mediated vasoconstriction by the thromboxane-mimetic U46619 in the porcine isolated ear artery: Comparison with vasopressin-enhanced responses by B. Bhattacharya; R. Williams; M.L. Latif; R.E. Roberts (pp. 1200-1210).

Pre-contraction with the thromboxane-mimetic U46619 enhances the subsequent α₂-adrenoceptor-mediated vasoconstriction in the porcine ear artery through an enhanced activation of ERK-MAP kinase. In this study we determined the role of cPLA₂ in this enhanced response, and determined whether vasopressin is also able to enhance α₂-adrenoceptor-mediated vasoconstriction through the same pathway. The cPLA₂ inhibitors AACOCF3 (50μM) and MAFP (50μM) both inhibited the U46619-enhanced α₂-adrenoceptor response, but had no effect on the direct α₂-adrenoceptor response. AACOCF3 also inhibited the enhanced ERK activation associated with the enhanced α₂-adrenoceptor-mediated vasoconstriction. Pre-contraction with arachidonic acid mimicked the effect of U46619 by enhancing the contractile response to the α₂-adrenoceptor agonist UK14304 (1μM) and enhancing the α₂-adrenoceptor-mediated ERK activation. Pre-contraction with vasopressin also enhanced the contractile response to UK14304, but neither PD98059 (50μM) nor AACOCF3 (50μM) had any effect this vasopressin-enhanced response, indicating that neither the ERK pathway, nor cPLA₂ are involved in vasopressin-enhanced responses. The α₂-adrenceptor-stimulated activation of ERK was also unaffected by pre-contraction with vasopressin. On the other hand, inhibition of PKCζ inhibited the enhanced α₂-adrenoceptor contraction after pre-contraction with both U46619 and vasopressin. This study demonstrates that α₂-adrenoceptor-mediated vasoconstriction can be enhanced through two different pathways—one dependent upon the enhanced activation of ERK-MAP kinase through activation of cPLA₂, and the other through a different, ERK/cPLA₂-independent pathway.

Keywords: Abbreviations; AEBSF; 4-(2-aminoethyl)benzenesulphonyl fluoride; ANOVA; analysis of the variance; E-64; trans-epoxysuccinyl-; l; -leucylamide-(4-guanidino) butane; ERK; extracellular signal-regulated kinase; HELSS; Haloenol lactone suicide substrate; MAP kinase; mitogen-activated protein kinase; MEK; mitogen-activated protein kinase kinase; PKC; protein kinase C; PLA; ₂; phospholipase A; ₂; TBS-T; tris-buffered saline containing 0.1% tween-20Porcine; Adrenergic agonists; Contractile function; Second messengers

6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-mediated JNK pathway in lipopolysaccharide-activated murine macrophages by Takuhiro Uto; Makoto Fujii; De-Xing Hou (pp. 1211-1221).

6-(Methylsulfinyl)hexyl isothiocyanate (6-MITC) is an active ingredient of Wasabi ( Wasabia japonica (Miq.) Matsumura), which is a very popular pungent spice in Japan. To clarify the cellular signaling mechanism underlying the anti-inflammatory action of 6-MITC, we investigated the effects of 6-MITC on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. 6-MITC showed a dose-dependent inhibition of LPS-induced nitric oxide (NO), iNOS mRNA and protein. LPS caused the c-Jun phosphorylation (a major component of AP-1) and IκB-α degradation. 6-MITC suppressed LPS-induced c-Jun phosphorylation, but did not inhibit IκB-α degradation. Cellular signaling analysis using MAPK-(U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) and Jak2-specific (AG490) inhibitors demonstrated that LPS stimulated iNOS expression via activating Jak2-mediated JNK, but not ERK and p38, pathway. 6-MITC suppressed iNOS expression through the inhibition of Jak2-mediated JNK signaling cascade with the attendant to AP-1 activation. In addition, the structure–activity study revealed that the inhibitory potency of methylsulfinyl isothiocyanates (MITCs) depended on the methyl chain length. These findings provide the molecular basis for the first time that 6-MITC is an effective agent to attenuate iNOS production.

Keywords: Abbreviations; AP-1; activator protein-1; ERK; extracellular signal-regulated kinase 1/2; iNOS; inducible nitric oxide synthase; Jak2; Janus kinase 2; JNK; c-Jun N-terminal kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; 6-MITC; 6-(methylsulfinyl)hexyl isothiocyanate; NF-κB; nuclear factor κB; NO; nitric oxide6-(Methylsulfinyl)hexyl isothiocyanate; Inducible nitric oxide synthase; Lipopolysaccharide; Janus kinase 2; c-Jun N-terminal kinase; Activator protein-1; Macrophage

In vivo treatment of acute Chlamydia pneumoniae infection with the flavonoids quercetin and luteolin and an alkyl gallate, octyl gallate, in a mouse model by Liisa T�rm�kangas; Pia Vuorela; Elise Saario; Maija Leinonen; Pekka Saikku; Heikki Vuorela (pp. 1222-1230).

Increasing evidence suggests that plant polyphenolic compounds may protect from cardiovascular diseases, which have been addressed to their antioxidative properties. In addition, these compounds have been shown to possess anti-inflammatory and anti-microbial potential. In the present study we tested the effects of two flavonoid compounds, quercetin and luteolin, and one alkyl gallate, octyl gallate, on the course of acute Chlamydia pneumoniae infection in vivo. C57BL/6J mice were treated with quercetin, luteolin or octyl gallate for 3 days prior to and 10 days after C. pneumoniae inoculation. Lung tissue was analysed for the presence of chlamydia by culture and quantitative PCR, and inflammatory responses were assessed. Luteolin was found histologically to suppress inflammation in lung tissue, the development of C. pneumoniae-specific antibodies and the presence of chlamydia in lung tissue. Octyl gallate had no significant effect on the course of infection, but quercetin increased both the inflammatory responses and the chlamydial load in the lungs. The infection and inflammation-enhancing effects of quercetin treatment may be attributable to the dose and the route of administration and should be reassessed in further studies with lower doses or with different metabolites of the compound. Contrariwise, the effects of luteolin treatment suggest this compound to have potential in decreasing the infection load and inflammatory reactions in vivo.

Keywords: Chlamydia pneumoniae; Lung infection; Mouse; Quercetin; Luteolin; Octyl gallate

The regulation of the expression of inducible nitric oxide synthase by Src-family tyrosine kinases mediated through MyD88-independent signaling pathways of Toll-like receptor 4 by Joo Y. Lee; Clifford A. Lowell; Danielle G. Lemay; Hyung S. Youn; Sang H. Rhee; Kyung H. Sohn; Byeong Jang; Jianping Ye; Jin H. Chung; Daniel H. Hwang (pp. 1231-1240).

Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to the expression of inflammatory gene products. Src-family tyrosine kinases (STKs) are known to be activated by LPS in monocytes/macrophages. Therefore, we determined the role of STKs in TLR4 signaling pathways and target gene expression in macrophages. The activation of NFκB, and p38 MAPK, and the expression of inducible nitric oxide synthase (iNOS) induced by LPS were not affected in macrophages deficient in three STKs (Lyn, Hck, and Fgr). These results suggest that the deletion of the three STKs among possibly nine STKs is not sufficient to abolish total activity of STKs possibly due to the functional redundancy of other STKs present in macrophages. However, two structurally unrelated pan-inhibitors of STKs, PP1 and SU6656, suppressed LPS-induced iNOS expression in MyD88-knockout as well as wild-type macrophages. The suppression of iNOS expression by the inhibitors was correlated with the downregulation of IFNβ (a MyD88-independent gene) expression and subsequent decrease in STAT1 phosphorylation. Moreover, PP1 suppressed the expression of IFNβ and iNOS induced by TRIF, a MyD88-independent adaptor of TLR4. PP1 suppressed STAT1 phosphorylation induced by LPS, but not by IFNβ suggesting that STKs are involved in the primary downstream signaling pathways of TLR4, but not the secondary signaling pathways downstream of IFNβ receptor. Together, these results demonstrate that STKs play a positive regulatory role in TLR4-mediated iNOS expression in a MyD88-independent (TRIF-dependent) manner. These results provide new insight in understanding the role of STKs in TLR4 signaling pathways and inflammatory target gene expression.

Keywords: Abbreviations; STKs; Src-family tyrosine kinases; TLR; Toll-like receptor; LPS; lipopolysaccharide; iNOS; inducible nitric oxide synthase; NFκB; nuclear factor κB; MyD88; myeloid differential factor 88; TRAF6; TNF receptor associated factor 6; TRIF; TIR domain-containing adapter inducing IFN-β; IFN-β; interferon β; IKK; IκB kinase; IRF-3; IFN-regulatory factor-3Lipopolysaccharide; Toll-like receptor; Src-family tyrosine kinases; Nitric oxide synthase; TRIF; IFN-beta

Calpain activation contributes to oxidative stress-induced pancreatic acinar cell injury by H. Weber; S. H�hns; F. L�then; L. Jonas; P. Schuff-Werner (pp. 1241-1252).

Oxygen radicals have been implicated as mediators in the pathogenesis of pancreatic acinar cell necrosis. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated whether the Ca²⁺-regulated cytosolic cysteine protease calpain is activated by oxidative stress and contributes to oxidant-induced acinar cell damage. Isolated rat pancreatic acinar cells were exposed to hydrogen peroxide (H₂O₂)-generated oxidative stress in the presence or absence of the Ca²⁺ chelator 1,2-bis-( o-aminophenoxy)-ethane- N, N, N′, N′-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and different calpain inhibitors including benzyloxycarbonyl-valyl-phenylalanine methyl ester. Calpain activation was studied by fluorescence spectrophotometry and immunoblotting. Cell injury was assessed by lactate dehydrogenase (LDH) release and characterization of the cellular ultrastructure including fluorescence-labeled actin filaments. Exposure of acinar cells to H₂O₂ provoked a time- and dose-dependent increase in calpain proteolytic activity involving the ubiquitous isoforms μ- and m-calpain. The activation of calpain reflected the time course of developing cytotoxicity as demonstrated by increased LDH release. Inhibition of oxidant-induced calpain activity by BAPTA-AM and various calpain inhibitors provoked a decline in oxidant-induced cell injury. In particular, changes in the actin filament organization characterized by an increase in the basolateral actin and by a detachment of actin from the cell membrane in the region of membrane blebs were clearly reduced. In summary, our findings suggest that acinar cell damage through oxidative stress requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease. The results support the hypothesis that calpain activation may play a role in the development of acute pancreatitis.

Keywords: Calpain; Calcium-mediated cell damage; Actin cytoskeleton; Phospholipase A; ₂; Necrosis

The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent by Barbara M.A. Lussenburg; Peter H.J. Keizers; Chris de Graaf; Mats Hidestrand; M. Ingelman-Sundberg; Nico P.E. Vermeulen; Jan N.M. Commandeur (pp. 1253-1261).

The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1′-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J 1998;331:783–92]. In the present study, the role of F483 in ligand binding and metabolism by CYP2D6 was examined experimentally using site-directed mutagenesis. Replacement of F483 by alanine resulted in a 30-fold lower V_max for bufuralol 1′-hydroxylation, while the K_m was hardly affected. The V_max for 3,4-methylenedioxy-methylamphetamine O-demethylenation on the other hand decreased only two-fold, whereas the effect on the K_m was much larger. For dextromethorphan, in addition to dextrorphan ( O-demethylation) and 3-methoxymorphinan ( N-demethylation), two other metabolites were formed that could not be detected for the wild-type. The substrate 7-methoxy-4-(aminomethyl)-coumarin was not metabolised at all by CYP2D6[F483A], a phenomenon that was reported also for CYP2D6[F120A]. The presented data show that next to F120, residue F483 plays a very important role in the metabolism of typical CYP2D6 substrates. The influence of F483 on metabolism was found to be strongly substrate-dependent.

Keywords: Abbreviations; CYP; cytochrome P450; CPR; cytochrome P450 reductase; FU; fluorescence units; HAMC; 7-hydroxy-4-(aminomethyl)-coumarin; MAMC; 7-methoxy-4-(aminomethyl)-coumarin; MDMA; 3,4-methylenedioxy-methylamphetamine; MDA; 3,4-methylenedioxy-amphetamine; 3,4-OH-MA; 3,4-dihydroxy-methylamphetamineCytochrome P450 2D6; Active site; Site-directed mutagenesis; Phenylalanine 483; Substrate selectivity

Effects of alkyl gallates on P-glycoprotein function by Shuji Kitagawa; Tomohiro Nabekura; Shizu Kamiyama; Tomoharu Takahashi; Yutaka Nakamura; Yoshiki Kashiwada; Yasumasa Ikeshiro (pp. 1262-1266).

In this study, we examined the effects of the food antioxidants, alkyl gallates, on the function of P-glycoprotein (P-gp) and elucidated the importance of alkyl chains and gallic acid moieties on the activity of P-gp. We examined the effects of three alkyl ( n-butyl, n-octyl and n-dodecyl) gallates and their related compounds on the cellular accumulation and efflux of rhodamine 123 and daunorubicin in P-gp overexpressing KB-C2 cells. Alkyl gallates increased the cellular accumulation of these P-gp substrates dependent on their alkyl chain lengths by inhibiting the efflux of the substrates. n-Dodecylresorcinol also increased the accumulation, but its effect was less than that of n-dodecyl gallate. However, either lauric acid or n-dodecyl-β-d-maltoside, which does not have a phenol group, did not increase the accumulation. The results indicated that both the gallic acid moiety and a long alkyl chain play important roles in the modification of P-gp function. The cytotoxicity of daunorubicin was recovered in the presence of alkyl gallates possibly due to their inhibition of P-gp function.

Keywords: Abbreviations; D-MEM; Dulbecco's modified Eagle medium; EGCG; (−)epigallocatechin gallate; MDR; multidrug resistance; MRP; multidrug resistance protein; PBS; phosphate-buffered saline; P-gp; P-glycoproteinAlkyl gallates; P-glycoprotein; Polyphenols; KB-C2 cells; MDR

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Biochemical Pharmacology (v.70, #8)

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Biochemical Pharmacology (v.70, #8)