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Biochemical Pharmacology (v.72, #7)

Editorial Advisory Board (pp. iii).

Activation of coupled Ah receptor and Nrf2 gene batteries by dietary phytochemicals in relation to chemoprevention by Christoph Köhle; Karl Walter Bock (pp. 795-805).

The Ah receptor (AhR) is a ligand-activated transcription factor and member of the bHLH/PAS (basic helix-loop-helix/Per-Arnt-Sim) family of chemosensors and developmental regulators. It represents a multifunctional molecular switch involved in regulation of endo- and xenobiotic metabolism, in vascular development and in dioxin-mediated toxicities. Recently, the oxidative stress-protecting Nrf2 has been shown to be a downstream target of the AhR [Miao W, Hu L, Scrivens PJ, Batist G. Transcriptional regulation of NF-E2 p45-regulated factor (NRF2) expression by the aryl hydrocarbon receptor-xenobiotic response element signaling pathway. J Biol Chem 2005;280:20340–8]. This finding offers the possibility that distinct but partially overlapping AhR and Nrf2 gene batteries of Phase II xenobiotic-metabolizing enzymes can be synergistically activated by a number of phytochemicals, acting as selective or mixed activators of target genes. In addition, it is conceivable that AhR-mediated oxidative/electrophile stress may be attenuated by coupled Nrf2 activation. The commentary discusses potentials and limitations of (i) selective Nrf2 and of (ii) synergistic AhR plus Nrf2 activation by phytochemicals in efforts towards chemoprevention of cancer and degenerative diseases, and describes clinical trials providing the expectation that chemopreventive measures may favorably modulate unavoidable endo- and exogenous toxin exposures in high risk populations.

Keywords: Abbreviations; ALDH; aldehyde dehydrogenase; ARE; antioxidant response element; GCS; _H; γ-glutamylcysteine synthetase heavy subunit; GCS; _L; γ-glutamylcysteine synthetase light subunit; GSH; glutathione; NQO; NAD(P)H:quinone oxidoreductase; ROS; reactive oxygen species; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; tBHQ; tert butylhydroquinone; TRE; tetradecanoyl phorbol acetate response element; UGT; UDP-glucuronosyltransferase; XRE; xenobiotic response elementAh receptor; Nrf2; Phytochemicals; Mixed Ah receptor/Nrf2 activators; Chemoprevention

The methionine salvage pathway compound 4-methylthio-2-oxobutanate causes apoptosis independent of down-regulation of ornithine decarboxylase by Baiqing Tang; Yuwaraj Kadariya; Maureen E. Murphy; Warren D. Kruger (pp. 806-815).

4-Methylthio-2-oxobutanoic acid (MTOB) is the final compound of the methionine salvage pathway that converts the polyamine byproduct methylthioadenosine to adenine and methionine. Here we find that MTOB inhibits growth of several human cell lines in a dose-dependent manner. Growth inhibition was specific for MTOB as we did not observe any inhibition with other chemically related compounds. MTOB treatment causes apoptosis and reduction of ornithine decarboxylase (ODC) activity but not ODC mRNA. To determine if MTOB exerts its effects primarily via ODC inhibition, we compared the effects of MTOB with the ODC-specific inhibitor difluoromethylornithine (DFMO). We found that MTOB was a more potent inducer of apoptosis than DFMO, lacked activation of caspase 3/7, and was able to induce apoptosis in cells lacking p53. Our results show that MTOB-induced growth inhibition and apoptosis is not simply secondary due to ODC inhibition and implies that MTOB activates apoptosis via other mechanisms.

Keywords: Abbreviations; MTOB; 4-methylthio-2-oxobutanoic acid; ODC; ornithine decarboxylase; DFMO; difluoromethylornithine; MTAP; methylthioadenosine phosphorylase; 7-AAD; 7-amino-actinomycin D; FBS; fetal bovine serumMethionine; Polyamines; Apoptosis; Methylthioadenosine phosphorylase; Keto-acid

Molecular mechanisms underlying the enhanced sensitivity of thiopurine-resistant T-lymphoblastic cell lines to methyl mercaptopurineriboside by Alan Kambiz Fotoohi; Anna Wrabel; Ali Moshfegh; Curt Peterson; Freidoun Albertioni (pp. 816-823).

Methylmercaptopurine riboside (meMPR), a cellular metabolite of 6-mercaptopurine (6-MP), is a potent inhibitor of de novo purine synthesis (DNPS). Human MOLT4 T-lymphoblastic leukaemia cells that have acquired resistance to 6-MP or 6-thioguanine (6-TG) as a consequence of defective transport exhibit enhanced sensitivity to meMPR. HPLC-based analysis of the transport of meMPR revealed normal uptake of this compound by our thiopurine-resistant cell sublines, suggesting a route of transport distinct from that for 6-MP and 6-TG. Studies on the wild-type parental leukemic cells showed that adenosine, dipyridamole and nitrobenzylthioinosine inhibit uptake of meMPR to a significant extent, whereas Na⁺ ions have no influence on this process. Transfection of these leukemic cells with small interference RNA molecules targeting the gene encoding the first member of the family of equiliberative nucleoside transporters (ENT1) strongly reduced the initial rate of meMPR transport. Our resistant cell lines exhibited 30–52% reductions ( p<0.005) in their levels of mRNA encoding several proteins involved in de novo purine synthesis, i.e., aminoimidazole carboxamide ribonucleotide formyltransferase, glycinamide ribonucleotide transformylase and guanine monophosphate synthetase. Consequently, the rate of de novo purine synthesis in these resistant sublines was decreased by 50%. Furthermore, the levels of ribonucleoside triphosphates in these cells were significantly lower than in the non-resistant parental cells.In combination, a reduced rate of de novo purine synthesis together with low levels of ribonucleoside triphosphates can explain the enhanced sensitivity of our thiopurine-resistant cell lines to meMPR. In this manner, meMPR bypasses the mechanisms of resistance to thiopurines and is even more cytotoxic towards resistant than towards wild-type cells.

Keywords: Methyl mercaptopurineriboside; De novo; purine synthesis; Equiliberative nucleoside transporter-1; Enhanced sensitivity; 6-Mercaptopurine; 6-ThioguanineAbbreviations; DNPS; de novo; purine synthesis; ENT1; equilibrative nucleoside transporter 1; HGPRT; hypoxanthine-guanine phosphoribosyl transferase; IC; ₅₀; concentration of the drug that inhibits cell growth by 50%; 6-MP; 6-mercaptopurine; meMPR; methylmercaptopurine riboside; meTIMP; methylthioinosine monophosphate; NT(s); human nucleoside transporter(s); NTP; ribonucleoside triphosphate pools; 6-TG; 6-thioguanine; TPMT; thiopurine methyltransferase

Gomisin A alters substrate interaction and reverses P-glycoprotein-mediated multidrug resistance in HepG2-DR cells by Chi-Keung Wan; Guo-Yuan Zhu; Xiao-Ling Shen; Apurba Chattopadhyay; Saibal Dey; Wang-Fun Fong (pp. 824-837).

Through an extensive herbal drug screening program, we found that gomisin A, a dibenzocyclooctadiene compound isolated from Schisandra chinensis, reversed multidrug resistance (MDR) in Pgp-overexpressing HepG2-DR cells. Gomisin A was relatively non-toxic but without altering Pgp expression, it restored the cytotoxic actions of anticancer drugs such as vinblastine and doxorubicin that are Pgp substrates but may act by different mechanisms. Several lines of evidence suggest that gomisin A alters Pgp-substrate interaction but itself is neither a Pgp substrate nor competitive inhibitor. (1) First unlike Pgp substrates gomisin A inhibited the basal Pgp-associated ATPase (Pgp-ATPase) activity. (2) The cytotoxicity of gomisin A was not affected by Pgp competitive inhibitors such as verapamil. (3) Gomisin A acted as an uncompetitive inhibitor for Pgp-ATPase activity stimulated by the transport substrates verapamil and progesterone. (4) On the inhibition of rhodamine-123 efflux the effects of gomisin A and the competitive inhibitor verapamil were additive, so were the effects of gomisin A and the ATPase inhibitor vanadate. (5) Binding of transport substrates with Pgp would result in a Pgp conformational change favoring UIC-2 antibody reactivity but gomisin A impeded UIC-2 binding. (6) Photocrosslinking of Pgp with its transport substrate [¹²⁵I]iodoarylazidoprazosin was inhibited by gomisin A in a concentration-dependent manner. Taken together our results suggest that gomisin A may bind to Pgp simultaneously with substrates and alters Pgp-substrate interaction.

Keywords: Abbreviations; [; ¹²⁵; I]IAAP; [; ¹²⁵; I]iodoarylazidoprazosin; MDR; multidrug resistance; Pgp; P-glycoprotein; PI; propidium iodide; Rh-123; rhodamine-123; SDS-PAGE; sodium doedecyl sulphate-polyacrylamide gel electrophoresis; SRB; sulforhodamine BGomisin A; Multidrug resistance; P-glycoprotein; Substrate interaction

Kinetics of nonpeptide antagonist binding to the human gonadotropin-releasing hormone receptor: Implications for structure–activity relationships and insurmountable antagonism by Susan K. Sullivan; Sam R.J. Hoare; Beth A. Fleck; Yun-Fei Zhu; Christopher E. Heise; R. Scott Struthers; Paul D. Crowe (pp. 838-849).

Numerous nonpeptide ligands have been developed for the human gonadotropin-releasing hormone (GnRH) receptor as potential agents for treatment of disorders of the reproductive-endocrine axis. While the equilibrium binding of these ligands has been studied in detail, little is known of the kinetics of their receptor interaction. In this study we evaluated the kinetic structure–activity relationships (SAR) of uracil-series antagonists by measuring their association and dissociation rate constants. These constants were measured directly using a novel radioligand, [³H] NBI 42902, and indirectly for unlabeled ligands. Receptor association and dissociation of [³H] NBI 42902 was monophasic, with an association rate constant of 93±10μM⁻¹min⁻¹ and a dissociation rate constant of 0.16±0.02h⁻¹ ( t_1/2 of 4.3h). Four unlabeled compounds were tested with varying substituents at the 2-position of the benzyl group at position 1 of the uracil (–F, –SO(CH₃), –SO₂(CH₃) and –CF₃). The nature of the substituent did not appreciably affect the association rate constant but varied the dissociation rate constant >50-fold ( t_1/2 ranging from 52min for –SO(CH₃) to >43h for –CF₃). This SAR was poorly resolved in standard competition assays due to lack of equilibration. The functional consequences of the varying dissociation rate were investigated by measuring antagonism of GnRH-stimulated [³H] inositol phosphates accumulation. Slowly dissociating ligands displayed insurmountable antagonism (decrease of the GnRH E_max) while antagonism by more rapidly dissociating ligands was surmountable (without effect on the GnRH E_max). Therefore, evaluating the receptor binding kinetics of nonpeptide antagonists revealed SAR, not evident in standard competition assays, that defined at least in part the mode of functional antagonism by the ligands. These findings are of importance for the future definition of nonpeptide ligand SAR and for the identification of potentially useful slowly dissociating antagonists for the GnRH receptor.

Keywords: Gonadotropin-releasing hormone; G-protein-coupled receptor; Dissociation rate constant; Receptor kinetics; Nonpeptide antagonist; Structure–activity relationship

Suppression of MyD88- and TRIF-dependent signaling pathways of toll-like receptor by (−)-epigallocatechin-3-gallate, a polyphenol component of green tea by Hyung S. Youn; Joo Y. Lee; Shin I. Saitoh; Kensuke Miyake; Keon W. Kang; Yong J. Choi; Daniel H. Hwang (pp. 850-859).

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-κB. (−)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-κB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKβ which is the key kinase in the canonical pathway for NF-κB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.

Keywords: Abbreviations; TRIF; TIR domain-containing adapter inducing IFN-β; IKK; IκB kinase; TBK1; TANK-binding kinase 1; IRF3; IFN-regulatory factor 3EGCG; Flavonoid; Toll-like receptor; MyD88; TRIF; TBK1

Differential production of leukotriene B4 or prostaglandin E₂ by WKYMVm or serum amyloid A via formyl peptide receptor-like 1 by Ha Young Lee; Seong Ho Jo; Chuhee Lee; Suk-Hwan Baek; Yoe-Sik Bae (pp. 860-868).

Serum amyloid A (SAA) and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) have been reported as formyl peptide receptor-like 1 (FPRL1) ligands. WKYMVm but not SAA stimulated superoxide generation by human neutrophils. In terms of the downstream signalings triggered by WKYMVm and SAA, both agonists stimulated cytosolic phospholipase A₂-mediated arachidonic acid release, a precursor of leukotriene B4 (LTB4) and prostaglandin E₂ (PGE₂). WKYMVm also strongly stimulated LTB4 production in human neutrophils without affecting PGE₂ production, whereas SAA strongly stimulates cyclooxygenase-2 (COX-2) expression and PGE₂ production but not LTB4 production. In terms of the receptors responsible for the differential actions of these two agonists, we found that FPRL1 is involved in the production of LTB4 by WKYMVm and PGE₂ production by SAA. This study demonstrates that the chemoattractant receptor, FPRL1, can be differentially regulated by distinct ligands to generate different lipid mediators, and thus, different immune responses.

Keywords: Abbreviations; AA; arachidonic acid; LTB4; leukotriene B4; PGE; ₂; prostaglandin E; ₂; GPCR; G-protein coupled receptor; PLA; ₂; phospholipase A; ₂; cPLA; ₂; cytosolic PLA; ₂; iPLA; ₂; calcium independent PLA; ₂; sPLA; ₂; secretory PLA; ₂; 5-LO; 5-lipoxygenase; COX; cyclooxygenase; FPRL1,; formyl peptide receptor-like 1; SAA; serum amyloid A; WKYMVm; Trp-Lys-Tyr-Met-Val-D-Met; BAPTA/AM,; 1,2-bis(2-aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetoxymethyl ester; MAFP; methyl arachidonylfluorophosphonate; BEL; bromoenol lactone; AA-861; 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone; NDGA; nodihydroguaiaretic acid; [Ca; ²⁺; ]; _i; intracellular calcium concentration; PTX; pertussis toxinSerum amyloid A; WKYMVm; Formyl peptide receptor-like 1; Neutrophil; LTB4; PGE; ₂

The synthetic cannabinoid WIN 55,212-2 increases COX-2 expression and PGE₂ release in murine brain-derived endothelial cells following Theiler's virus infection by Leyre Mestre; Fernando Correa; Fabian Docagne; Diego Clemente; Carmen Guaza (pp. 869-880).

Brain endothelial cells infection represents one of the first events in the pathogenesis of TMEV-induced demyelination disease (TMEV-IDD), a model of multiple sclerosis (MS). The fact that cyclooxygenase-2 (COX-2) expression in brain endothelium mediates a wide variety of actions during CNS inflammatory diseases such as MS, and that cannabinoids ameliorate the progression of TMEV-IDD, lead us to investigate the role of cannabinoids on COX-2 expression on murine brain endothelial cell cultures subjected or not to TMEV infection. Murine brain endothelial cells (b.end5) express both cannabinoid receptors CB1 and CB2. However, treatment of b.end5 with the cannabinoid agonist WIN 55,212-2 resulted in up-regulation COX-2 protein and PGE₂ release by a mechanism independent on activation of these receptors. Other cannabinoids such as 2-arachidonoyl glycerol (2-AG) or the abnormal cannabidiol (Abn-CBD) failed to affect COX-2 in our conditions. TMEV infection of murine brain endothelial cell cultures induced a significant increase of COX-2 expression at 8h, which was maintained even increased, at 20 and 32h post-infection. The combination of TMEV infection and Win 55,212-2 treatment increased COX-2 expression to a greater amount than was seen with either treatment alone. 2-AG and Abn-CBD did not modify COX-2 expression after TMEV. COX-2 synthesis involved different signaling pathways when was induced by WIN 55,212-2 and/or by TMEV infection. WIN 55,212-2-induced COX-2 up-regulation involves the PI₃K pathway, whereas COX-2 induction by TMEV needs p38 MAPK activation too. Overexpression of COX-2 and the subsequent increase of PGE₂ could be affecting flow blood and/or immune reactivity.

Keywords: Abbreviations; 2-AG; 2-arachidonoyl glicerol; Abn-CBD; abnormal cannabidiol; AEA; anandamide; CB; cannabinoid; CB1; cannabinoid receptor 1; CB2; cannabinoid receptor 2; COX-2; cyclooxygenase-2; IL-1β; interleukin-1 beta; IL-12; interleukin-12; LDH; lactate dehydrogenase; MOI; multiplicity of infection; MS; multiple sclerosis; PGs; prostaglandins; PGE; ₂; prostaglandin E; ₂; SR1; SR141716A; SR2; SR144528; TMEV; Theiler's virus; TMEV-IDD; Theiler's virus-induced demyelination disease; TRPV1; vanilloid receptorCannabinoids; COX-2; PGE2; TMEV; Signaling pathways; Brain endothelial cells

Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Cα in C2C12 myocytes by Ying Wang; Gopa Biswas; Subbuswamy K. Prabu; Narayan G. Avadhani (pp. 881-892).

Protein kinase C (PKC) agonists including phorbol 12-myristate 13-acetate (PMA) not only induce the redistribution of cytosolic PKC to various subcellular compartments but also activate the kinase domain of the protein. In the present study we have investigated the nature of mitochondrial PKC pool and its effects on mitochondrial function in cells treated with PMA. Treatment of C2C12 myoblasts, C6 glioma and COS7 cells with PMA resulted in a dramatic redistribution of intracellular PKCα pool, with large fraction of the protein pool sequestered in the mitochondrial compartment. We also observed mitochondrial PKCδ accumulation in a cell restricted manner. The intramitochondrial localization was ascertained by using a combination of protection against protease treatment of isolated mitochondria and immunofluorescence microscopy. PMA-induced mitochondrial localization of PKCα was accompanied by increased mitochondrial PKC activity, altered cell morphology, disruption of mitochondrial membrane potential, decreased complex I and pyruvate dehydrogenase activities, and increased mitochondrial ROS production. All of these changes could be retarded by treatment with PKC inhibitors. These results show a direct role for PMA-mediated PKCα translocation to mitochondria in inducing mitochondrial toxicity.

Keywords: Abbreviations; CcO; cytochrome; c; oxidase; PDH; pyruvate dehydrogenase; PKC; protein kinase C; PMA; phorbol 12-myristate 13-acetate; ROS; reactive oxygen species

Cytochrome P450 reductase dependent inhibition of cytochrome P450 2B1 activity: Implications for gene directed enzyme prodrug therapy by Johannes Lengler; Markus Omann; Dana Düvier; Harry Holzmüller; Wolfgang Gregor; Brian Salmons; Walter H. Günzburg; Matthias Renner (pp. 893-901).

Cytochrome P450 (P450) enzymes are often used in suicide gene cancer therapy strategies to convert an inactive prodrug into its therapeutic active metabolites. However, P450 activity is dependent on electrons supplied by cytochrome P450 reductase (CPR). Since endogenous CPR activity may not be sufficient for optimal P450 activity, the overexpression of additional CPR has been considered to be a valuable approach in gene directed enzyme prodrug therapy (GDEPT). We have analysed a set of cell lines for the effects of CPR on cytochrome P450 isoform 2B1 (CYP2B1) activity. CPR transfected human embryonic kidney 293 (HEK293) cells showed both strong CPR expression in Western blot analysis and 30-fold higher activity in cytochrome c assays as compared to parental HEK293 cells. In contrast, resorufin and 4-hydroxy-ifosfamide assays revealed that CYP2B1 activity was up to 10-fold reduced in CPR/CYP2B1 cotransfected HEK293 cells as compared to cells transfected with the CYP2B1 expression plasmid alone. Determination of ifosfamide-mediated effects on cell viability allowed independent confirmation of the reduction in CYP2B1 activity upon CPR coexpression. Inhibition of CYP2B1 activity by CPR was also observed in CYP2B1/CPR transfected or infected pancreatic tumour cell lines Panc-1 and Pan02, the human breast tumour cell line T47D and the murine embryo fibroblast cell line NIH3T3. A CPR mediated increase in CYP2B1 activity was only observed in the human breast tumour cell line Hs578T. Thus, our data reveal an effect of CPR on CYP2B1 activity dependent on the cell type used and therefore demand a careful evaluation of the therapeutic benefit of combining cytochrome P450 and CPR in respective in vivo models in each individual target tissue to be treated.

Keywords: Cytochrome P450 reductase; Cytochrome P450; CYP2B1; Ifosfamide; P450 GDEPT

Caffeine metabolites are inhibitors of the nuclear enzyme poly(ADP-ribose)polymerase-1 at physiological concentrations by Liesbeth Geraets; Harald J.J. Moonen; Emiel F.M. Wouters; Aalt Bast; Geja J. Hageman (pp. 902-910).

The activity of the nuclear enzyme poly(ADP-ribose)polymerase-1 (E.C.2.4.2.30), which is highly activated by DNA strand breaks, is associated with the pathophysiology of both acute as well as chronic inflammatory diseases. PARP-1 overactivation and the subsequent extensive turnover of its substrate NAD⁺ put a large demand on mitochondrial ATP-production. Furthermore, due to its reported role in NF-κB and AP-1 mediated production of pro-inflammatory cytokines, PARP-1 is considered an interesting target in the treatment of these diseases.In this study the PARP-1 inhibiting capacity of caffeine and several metabolites as well as other (methyl)xanthines was tested using an ELISA-assay with purified human PARP-1. Caffeine itself showed only weak PARP-1 inhibiting activity, whereas the caffeine metabolites 1,7-dimethylxanthine, 3-methylxanthine and 1-methylxanthine, as well as theobromine and theophylline showed significant PARP-1 inhibiting activity. Further evaluation of these compounds in H₂O₂-treated A549 lung epithelial and RF24 vascular endothelial cells revealed that the decrease in NAD⁺-levels as well as the formation of the poly(ADP-ribose)polymer was significantly prevented by the major caffeine metabolite 1,7-dimethylxanthine. Furthermore, H₂O₂-induced necrosis could be prevented by a high dose of 1,7-dimethylxanthine. Finally, antioxidant effects of the methylxanthines could be ruled out with ESR and measurement of the TEAC.Concluding, caffeine metabolites are inhibitors of PARP-1 and the major caffeine metabolite 1,7-dimethylxanthine has significant PARP-1 inhibiting activity in cultured epithelial and endothelial cells at physiological concentrations. This inhibition could have important implications for nutritional treatment of acute and chronic inflammatory pathologies, like prevention of ischemia-reperfusion injury or vascular complications in diabetes.

Keywords: Poly(ADP-ribose)polymerase-1; Oxidative stress; NAD; ⁺; Necrosis; Methylxanthines; Inhibition

Effect of the mitochondrial transition pore inhibitor, S-15176, on rat liver mitochondria: ATP synthase modulation and mitochondrial uncoupling induction by Didier Morin; Roland Zini; Alain Berdeaux; Jean-Paul Tillement (pp. 911-918).

S-15176 is a new inhibitor of the permeability transition pore (PTP) which has been shown to display anti-ischemic properties. We show here that S-15176 prevented PTP, cytochrome c release and maintained mitochondrial membrane potential when low concentrations of S-15176 were used (not exceeding 50nmol/mg protein). For higher concentrations S-15176 is able to collapse mitochondrial potential. This effect was reversed by the recoupling agent 6-ketocholestanol (6-KCh) suggesting that S-15176 has uncoupling properties. In addition, S-15176 is able to inhibit ATP synthase activity and to stimulate the hydrolytic activity of the enzyme but none of these effects appears to be related to its PTP inhibiting property. These data demonstrate that S-15176 interacts with several targets in mitochondria and these pharmacological properties should be considered in the examination of its health benefits as well as its potential cytotoxicity.

Keywords: Abbreviations; CsA; cyclosporin A; CCCP; carbonyl cyanide; m; -chlorophenylhydrazone; PTP; permeability transition pore; 6-KCh; 6-ketocholestanolATP synthase; Uncoupler; Mitochondrial membrane potential; Permeability transition pore; 6-Ketocholestanol; S-15176

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Biochemical Pharmacology (v.72, #7)