Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain-specific lifespan and health effects in Caenorhabditis nematodes.

The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17α-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies-for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions.

Quantitative proteomic analysis of tomato fruit ripening behavior in response to exogenous abscisic acid.

BACKGROUND: To determine how abscisic acid (ABA) affects tomato fruit ripening at the protein level, mature green cherry tomato fruit were treated with ABA, nordihydroguaiaretic acid (NDGA) or sterile water (control, CK). The proteomes of treated fruit were analyzed and quantified using tandem mass tags (TMTs) at 7 days after treatment, and the gene transcription abundances of differently expressed proteins (DEPs) were validated with quantitative real-time polymerase chain reaction. RESULTS: Postharvest tomato fruit underwent faster color transformation and ripening than the CK when treated with ABA. In total, 6310 proteins were identified among the CK and treatment groups, of which 5359 were quantified. Using a change threshold of 1.2 or 0.83 times, 1081 DEPs were identified. Among them, 127 were upregulated and 127 were downregulated in the ABA versus CK comparison group. According to KEGG and protein-protein interaction network analyses, the ABA-regulated DEPs were primarily concentrated in the photosynthesis system and sugar metabolism pathways, and 102 DEPs associated with phytohormones biosynthesis and signal transduction, pigment synthesis and metabolism, cell wall metabolism, photosynthesis, redox reactions, allergens and defense responses were identified in the ABA versus CK and NDGA versus CK comparison groups. CONCLUSION: ABA affects tomato fruit ripening at the protein level to some extent. The results of this study provided comprehensive insights and data for further research on the regulatory mechanism of ABA in tomato fruit ripening. © 2023 Society of Chemical Industry.

Classic Phytochemical Antioxidant and Lipoxygenase Inhibitor, Nordihydroguaiaretic Acid, Activates Phospholipase D through Oxidant Signaling and Tyrosine Phosphorylation Leading to Cytotoxicity in Lung Vascular Endothelial Cells.

Nordihydroguaiaretic acid (NDGA), a dicatechol and phytochemical polyphenolic antioxidant and an established inhibitor of human arachidonic acid (AA) 5-lipoxygenase (LOX) and 15-LOX, is widely used to ascertain the role of LOXs in vascular endothelial cell (EC) function. As the modulatory effect of NDGA on phospholipase D (PLD), an important lipid signaling enzyme in ECs, thus far has not been reported, here we have investigated the modulation of PLD activity and its regulation by NDGA in the bovine pulmonary artery ECs (BPAECs). NDGA induced the activation of PLD (phosphatidic acid formation) in cells in a dose- and time-dependent fashion that was significantly attenuated by iron chelator and antioxidants. NDGA induced the formation of reactive oxygen species (ROS) in cells in a dose- and time-dependent manner as evidenced from fluorescence microscopy and fluorimetry of ROS and electron paramagnetic resonance spectroscopy of oxygen radicals. Also, NDGA caused a dose-dependent loss of intracellular glutathione (GSH) in BPAECs. Protein tyrosine kinase (PTyK)-specific inhibitors significantly attenuated NDGA-induced PLD activation in BPAECs. NDGA also induced a dose- and time-dependent phosphorylation of tyrosine in proteins in cells. NDGA caused in situ translocation and relocalization of both PLD1 and PLD2 isoforms, in a time-dependent fashion. Cyclooxygenase (COX) inhibitors were ineffective in attenuating NDGA-induced PLD activation in BPAECs, thus ruling out the activation of COXs by NDGA. NDGA inhibited the AA-LOX activity and leukotriene C4 (LTC4) formation in cells. On the other hand, the 5-LOX-specific inhibitors, 5, 8, 11, 14-eicosatetraynoic acid and kaempferol, were ineffective in activating PLD in BPAECs. Antioxidants and PTyK-specific inhibitors effectively attenuated NDGA cytotoxicity in BPAECs. The PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), significantly attenuated and protected against the NDGA-induced PLD activation and cytotoxicity in BPAECs. For the first time, these results demonstrated that NDGA, the classic phytochemical polyphenolic antioxidant and LOX inhibitor, activated PLD causing cytotoxicity in ECs through upstream oxidant signaling and protein tyrosine phosphorylation.

The effect of diphenylethane side-chain substituents on dibenzocyclohexadiene formation and their inhibition of α-synuclein aggregation in vitro.

The naturally-occurring di-catechol lignan nordihydroguaiaretic acid (NDGA) and an analog without methyl groups on the butyl linker both undergo intramolecular cyclization at pH 7.4 to form dibenzocyclooctadienes. Both NDGA and these dibenzocyclooctadienes have been shown to prevent in vitro aggregation of α-synuclein, an intrinsically disordered protein associated with Parkinson's disease. NDGA possesses two vicinal methyl groups on the butyl linker and the presence of these methyl groups attenuates the rate of intramolecular cyclization versus the unsubstituted analog, in opposition to the anticipated Thorpe-Ingold effect, likely due to steric repulsions during cyclization. Numerous 1,2-bis-ethane di-catechols are known to inhibit α-synuclein aggregation in vitro and we hypothesize that these compounds undergo a similar intramolecular cyclization and the cyclized products may be responsible for the activity. To test this hypothesis we prepared a series of 1,2-bis-ethane di-catechols with 0, 2 and 4 methyl substituents on the linker. We have confirmed that these compounds undergo intramolecular cyclization to form dibenzocyclohexadienes and that steric interactions between the methyl substituents leads to an increase in the rate of intramolecular cyclization, which is in contrast to what was observed for lignan di-catechols. The rate of cyclization to form six-membered rings is 10-30 times more rapid than formation of eight membered rings and the dibenzocyclohexadienes also prevent in vitro aggregation of α-synuclein.

Macrophages undergo a behavioural switch during wound healing in zebrafish.

In response to wound signals, macrophages are immediately recruited to the injury where they acquire distinct phenotypes and functions, playing crucial roles both in host defense and healing process. Although macrophage phenotypes have been intensively studied during wound healing, mostly using markers and expression profiles, the impact of the wound environment on macrophage shape and behaviour, and the underlying mechanisms deserve more in-depth investigation. Here, we sought to characterize the dynamics of macrophage recruitment and behaviour during aseptic wounding of the caudal fin fold of the zebrafish larva. Using a photo-conversion approach, we demonstrated that macrophages are recruited to the wounded fin fold as a single wave where they switch their phenotype. Intravital imaging of macrophage shape and trajectories revealed that wound-macrophages display a highly stereotypical set of behaviours and change their shape from amoeboid to elongated shape as wound healing proceeds. Using a pharmacological inhibitor of 15-lipoxygenase and protectin D1, a specialized pro-resolving lipid, we investigated the role of polyunsaturated fatty acid metabolism in macrophage behaviour. While inhibition of 15-lipoxygenase using PD146176 or Nordihydroguaiaretic acid (NDGA) decreases the switch from amoeboid to elongated shape, protectin D1 accelerates macrophage reverse migration and favours elongated morphologies. Altogether, our findings suggest that individual macrophages at the wound switch their phenotype leading to important changes in behaviour and shape to adapt to changing environment, and highlight the crucial role of lipid metabolism in the control of macrophage behaviour plasticity during inflammation in vivo.

Nordihydroguaiaretic Acid as a Novel Substrate and Inhibitor of Catechol O-Methyltransferase Modulates 4-Hydroxyestradiol-Induced Cyto- and Genotoxicity in MCF-7 Cells.

Nordihydroguaiaretic acid (NDGA) is a major lignan metabolite found in Larrea spp., which are widely used in South America to treat various diseases. In breast tissue, estradiol is metabolized to the catechol estrogens such as 4-hydroxyestradiol (4-OHE2), which have been proposed to be cancer initiators potentially involved in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of catechol estrogens to their less toxic methoxy derivatives, such as 4-O-methylestradiol (4-MeOE2). The present study investigated the novel biological activities of NDGA in relation to COMT and the effects of COMT inhibition by NDGA on 4-OHE2-induced cyto- and genotoxicity in MCF-7 human breast cancer cells. Two methoxylated metabolites of NDGA, 3-O-methylNDGA (3-MNDGA) and 4-O-methyl NDGA (4-MNDGA), were identified in the reaction mixture containing human recombinant COMT, NDGA, and cofactors. Km values for the COMT-catalyzed metabolism of NDGA were 2.6 µM and 2.2 µM for 3-MNDGA and 4-MNDGA, respectively. The COMT-catalyzed methylation of 4-OHE2 was inhibited by NDGA at an IC50 of 22.4 µM in a mixed-type mode of inhibition by double reciprocal plot analysis. Molecular docking studies predicted that NDGA would adopt a stable conformation at the COMT active site, mainly owing to the hydrogen bond network. NDGA is likely both a substrate for and an inhibitor of COMT. Comet and apurinic/apyrimidinic site quantitation assays, cell death, and apoptosis in MCF-7 cells showed that NDGA decreased COMT-mediated formation of 4-MeOE2 and increased 4-OHE2-induced DNA damage and cytotoxicity. Thus, NDGA has the potential to reduce COMT activity in mammary tissues and prevent the inactivation of mutagenic estradiol metabolites, thereby increasing catechol estrogen-induced genotoxicities.

Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products.

Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX. Our results suggest a new approach to isoform-specific 5-LOX inhibitor development through exploitation of an allosteric site in 5-LOX.

Cyclized NDGA modifies dynamic α-synuclein monomers preventing aggregation and toxicity.

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson's disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils. This conformational remodeling preserved the dynamic adoption of α-helical conformations, which are essential for physiological membrane interactions. Oxidation-dependent NDGA cyclization was required for the interaction with monomeric α-synuclein. NDGA analog-pretreated α-synuclein did not aggregate even without NDGA-analogs in the aggregation mixture. Strikingly, NDGA-pretreated α-synuclein suppressed aggregation of naïve untreated aggregation-competent monomeric α-synuclein. Further, cyclized NDGA reduced α-synuclein-driven neurodegeneration in Caenorhabditis elegans. The cyclized NDGA analogs may serve as a platform for the development of small molecules that stabilize aggregation-resistant α-synuclein monomers without interfering with functional conformations yielding potential therapies for PD and related disorders.

Nordihydroguaiaretic acid prevents glycation induced structural alterations and aggregation of albumin.

This study demonstrates the antiglycation activity of Nordihydroguaiaretic acid, a lignin from the creosote bush (Larrea tridentate), which has also been proven to assist in the treatment of cancer, neurological disorders, and cardiovascular complications. We determined the antiglycation activity of NDG based on spectroscopic analysis, molecular interactions and circular dichroism studies with albumin. It was also seen that NDG inhibits the aggregation of albumin, after glycation, using Thioflavin T binding and confocal imaging. Results suggest that NDG is a potent inhibitor of advanced glycation end products formation. NDG was found to impart protective effects on albumin by preventing glycation modification of lysine residues (Lys20, Phe36, Lys41, Lys131, and Lys132) due to glycation.

Site-Specific Fluorescence Polarization for Studying the Disaggregation of α-Synuclein Fibrils by Small Molecules.

Fibrillar aggregates of the protein α-synuclein (αS) are one of the hallmarks of Parkinson's disease. Here, we show that measuring the fluorescence polarization (FP) of labels at several sites on αS allows one to monitor changes in the local dynamics of the protein after binding to micelles or vesicles, and during fibril formation. Most significantly, these site-specific FP measurements provide insight into structural remodeling of αS fibrils by small molecules and have the potential for use in moderate-throughput screens to identify small molecules that could be used to treat Parkinson's disease.

Ring substitution influences oxidative cyclisation and reactive metabolite formation of nordihydroguaiaretic acid analogues.

Nordihydroguaiaretic acid (NDGA) is a natural polyphenol with a broad spectrum of pharmacological properties. However, its usefulness is hindered by the lack of understanding of its pharmacological and toxicological pathways. Previously we showed that oxidative cyclisation of NDGA at physiological pH forms a dibenzocyclooctadiene that may have therapeutic benefits whilst oxidation to an ortho-quinone likely mediates toxicological properties. NDGA analogues with higher propensity to cyclise under physiologically relevant conditions might have pharmacological implications, which motivated this study. We synthesized a series of NDGA analogues which were designed to investigate the structural features which influence the intramolecular cyclisation process and help to understand the mechanism of NDGA's autoxidative conversion to a dibenzocyclooctadiene lignan. We determined the ability of the NDGA analogues investigated to form dibenzocyclooctadienes and evaluated the oxidative stability at pH 7.4 of the analogues and the stability of any dibenzocyclooctadienes formed from the NDGA analogues. We found among our group of analogues the catechols were less stable than phenols, a single catechol-substituted ring is insufficient to form a dibenzocyclooctadiene lignan, and only compounds possessing a di-catechol could form dibenzocyclooctadienes. This suggests that quinone formation may not be necessary for cyclisation to occur and the intramolecular cyclisation likely involves a radical-mediated rather than an electrophilic substitution process. We also determined that the catechol dibenzocyclooctadienes autoxidised at comparable rates to the parent catechol. This suggests that assigning in vitro biological activity to the NDGA dibenzocyclooctadiene is premature and requires additional study.

In vitro inhibitory profile of NDGA against AChE and its in silico structural modifications based on ADME profile.

Acetylcholinesterase (AChE) inhibitors are currently in focus for the pharmacotherapy of Alzheimer's disease (AD). These inhibitors increase the level of acetylcholine in the brain and facilitate cholinergic neurotransmission. AChE inhibitors such as rivastigmine, galantamine, physostigmine and huperzine are obtained from plants, indicating that plants can serve as a potential source for novel AChE inhibitors. We have performed a virtual screening of diverse natural products with distinct chemical structure against AChE. NDGA was one among the top scored compounds and was selected for enzyme kinetic studies. The IC(50) of NDGA on AChE was 46.2 µM. However, NDGA showed very poor central nervous system (CNS) activity and blood-brain barrier (BBB) penetration. In silico structural modification on NDGA was carried out in order to obtain derivatives with better CNS activity as well as BBB penetration. The studies revealed that some of the designed compounds can be used as lead molecules for the development of drugs against AD.

Phenolic compound production in relation to differentiation in cell and tissue cultures of Larrea divaricata (Cav.).

The lignan nordihydroguaiaretic acid (NDGA) and its derivatives existing in Larrea divaricata species show a wide range of pharmacological activities which makes this genus an interesting target to consider the plant in vitro cultivation systems as a feasible alternative source for their production. These compounds are potentially useful in treating diseases related to heart condition, asthma, arteriosclerosis, viral and bacterial infections, inflammation and cancer. In the present study, calli, cell suspension cultures, and in vitro and wild plants of L. divaricata were investigated for their potential to synthesize phenolic compounds. Calli, both with and without organogenesis, produced NDGA and quercetin, as did plantlet and wild plants. NDGA was also produced by the cell suspension cultures, together with p-coumaric acid, ferulic acid and sinapyl alcohol. The capacity of undifferentiated tissues to form phenolic compounds is very limited, but when the calli underwent organogenesis, developing mainly adventitious shoots, the phenolic compound production increased significantly. Plantlets regenerated from adventitious shoots of L. divaricata calli did not show the same phenolic pattern as wild plants, with levels of NDGA and quercetin being 3.6- and 5.9-fold lower, respectively.

Glucose-induced delay of seed germination in rice is mediated by the suppression of ABA catabolism rather than an enhancement of ABA biosynthesis.

Both glucose and ABA play crucial roles in the regulation of seed germination and post-germination development. In Arabidopsis thaliana, up-regulation of ABA biosynthesis is suggested as one of the possible mechanisms mediating the glucose-induced delay in seed germination. Since the endogenous ABA level is controlled by the equilibrium between ABA biosynthesis and catabolism, we investigated how this equilibrium is related to the regulation of seed germination by glucose in rice. When ABA biosynthesis was inhibited by nordihydroguaiaretic acid (NDGA), an inhibitor of the ABA anabolic enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), rice seed germination showed no response. In contrast, inhibition of ABA catabolism by diniconazole significantly arrested seed germination, suggesting that the regulation of ABA catabolism plays a major role. Further experiments indicated that the expression of OsABA8ox3, a key gene in ABA catabolism and encoding ABA 8'-hydroxylase in rice, was significantly increased during the first 6 h of imbibition, which was consistent with the decline of ABA content in the imbibed seeds. Expression of OsABA8ox genes, especially OsABA8ox2 and OsABA8ox3, was sensitively suppressed in the presence of exogenously supplied glucose. In contrast, the expression profiles of OsNCED genes that control the limiting step of ABA biosynthesis showed no significant changes in response to low levels of glucose. Our results demonstrated that the glucose-induced delay of seed germination is a result of the suppression of ABA catabolism rather than any enhancement of ABA biosynthesis during rice seed germination.

Larrea tridentata (Creosote bush), an abundant plant of Mexican and US-American deserts and its metabolite nordihydroguaiaretic acid.

Although controversial, Creosote bush, Larrea tridentata (Sesse and Moc. ex DC) Coville, is used to treat a variety of illnesses including infertility, rheumatism, arthritis, diabetes, gallbladder and kidney stones, pain and inflammation. Recently, it has been used as a nutritional supplement. The primary product extracted from this common plant of the arid regions of northern Mexico and Southwestern United States is the potent antioxidant nordihydroguaiaretic acid (NDGA). It was widely used during the 1950s as a food preservative and to preserve naturals fibers. Later it was banned after reports of toxicity during the early 1960s. Renal and hepatotoxicity are also reported for chronic use of creosote bush and NDGA. This article reviews traditional and contemporary uses and pharmacology, including toxicology of this plant widely used in Mexican traditional medicine.

Adipocyte differentiation of multipotent cells established from human adipose tissue.

In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.e., expression of specific molecular markers, lipolytic response to agonists of beta-adrenoreceptors (beta2-AR agonist > beta1-AR agonist >> beta3-AR agonist) and to the atrial natriuretic peptide, insulin-stimulated glucose transport, and secretion of leptin and adiponectin. hMADS cells were able to respond to drugs as inhibition of adipocyte differentiation was observed in the presence of prostaglandin F2alpha, tumour necrosis factor-alpha, and nordihydroguaiaretic acid, a natural polyhydroxyphenolic antioxidant. Thus, for the first time, human adipose cells with normal karyotype and indefinite life span have been established. They represent a novel and valuable tool for studies of fat tissue development and metabolism.

Estrogenic activity of an antioxidant, nordihydroguaiaretic acid (NDGA).

Nordihydroguaiaretic acid (NDGA), an antioxidant which has been used to preserve oils and foods, has recently become well known as a putative anticancer agent. Although NDGA is a member of the lignan family, its hormonal activities have not been well investigated. Here we show that NDGA has in vitro estrogenic activity in the ERE-luc reporter system using NIH3T3 cells, and in the estrogen-responsive cell growth assay in a pituitary cell line, MtT/E-2. Uterotropic assay in rats indicated that NDGA has estrogenic activity in vivo as well, albeit weak. Interestingly it preferentially induced ERalpha mediated ERE-luc activity, although it showed similar binding affinity to both ERalpha and ERbeta. With ERbeta, on the other hand, NDGA showed only weak agonistic action, and antagonized the estrogenic action of 17beta-estradiol when the two were coadministered. These data suggest that NDGA is an estrogenic agonist for ERalpha : ERE transcription and a mixed agonist/antagonist for ERbeta mediated ERE transactivation.

The role of oxidant-mediated pathways in the cytotoxicity of endothelial cells exposed to mesenteric lymph from rats subjected to trauma-hemorrhagic shock.

Because gut-derived factors carried in mesenteric lymph are implicated in multiple organ dysfunction syndrome and have been shown to injure endothelial cells, we investigated several cellular pathways by which this process could occur. To accomplish this, mesenteric lymph (5%, v/v) collected at 1 to 3 h postshock from male rats undergoing trauma (5-cm laparotomy) and hemorrhagic shock (90 min of mean arterial pressure [MAP] of 30 mmHg; T/HS) was tested for endothelial cell cytotoxicity on human umbilical vein endothelial cells (HUVECs). Over 30 pharmacologic agents that had been reported to inhibit endothelial cell death were tested for their ability to prevent T/HS lymph-induced HUVEC cell death. These included agents documented to protect against oxidant-mediated, calcium-mediated, and arachidonic acid pathway-mediated endothelial cell injury and death. These pharmacologic inhibitors were preincubated with HUVECs for 1 h or were added to the HUVECs simultaneously with lymph, and were then incubated for 18 h. Controls were lymph alone, inhibitor alone, or medium alone. Mitochondrial tetrazolium (MTT) and LDH release assays were used to determine cell viability. The inhibitors that significantly protected HUVECs from the cytotoxicity of T/HS lymph (P < 0.001) included the antioxidant combination of vitamins C and E and the antioxidant-lipooxygenase inhibitor nordihydroguaretic acid (NDGA). These agents were equally effective when added simultaneously with lymph or preincubated with the HUVECs, suggesting an extracellular or membrane-bound process. In summary, the inhibitors that provided protection from toxic lymph appear to work at the membrane and are involved in limiting membrane peroxidation. Based on this study, it appears that an oxidant pathway is involved in T/HS lymph-induced endothelial cell injury and death.

Nitric oxide triggers the toxicity due to glutathione depletion in midbrain cultures through 12-lipoxygenase.

Glutathione (GSH) depletion is the earliest biochemical alteration shown to date in brains of Parkinson's disease patients. However, data from animal models show that GSH depletion by itself is not sufficient to induce nigral degeneration. We have previously shown that non-toxic inhibition of GSH synthesis with l-buthionine-(S,R)-sulfoximine in primary midbrain cultures transforms a nitric oxide (NO) neurotrophic effect, selective for dopamine neurons, into a toxic effect with participation of guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG) (Canals, S., Casarejos, M. J., de Bernardo, S., Rodríguez-Martín, E., and Mena, M. A. (2001) J. Neurochem. 79, 1183-1195). Here we demonstrate that arachidonic acid (AA) metabolism through the 12-lipoxygenase (12-LOX) pathway is also central for this GSH-NO interaction. LOX inhibitors (nordihydroguaiaretic acid and baicalein), but not cyclooxygenase (indomethacin) or epoxygenase (clotrimazole) ones, prevent cell death in the culture, even when added 10 h after NO treatment. Furthermore, the addition of AA to GSH-depleted cultures precipitates a cell death process that is indistinguishable from that initiated by NO in its morphology, time course, and 12-LOX, GC, and PKG dependence. The first AA metabolite through the 12-LOX enzyme, 12-hydroperoxyeicosatetraenoic acid, induces cell death in the culture, and its toxicity is greatly enhanced by GSH depletion. In addition we show that if GSH synthesis inhibition persists for up to 4 days without any additional treatment, it will induce a cell death process that also depends on 12-LOX, GC, and PKG activation. In this study, therefore, we show that the signaling pathway AA/12-LOX/12-HPETE/GC/PKG may be important in several pathologies in which GSH decrease has been documented, such as Parkinson's disease. The potentiating effect of NO over such a signaling pathway may be of relevance as part of the cascade of events leading to and sustaining nerve cell death.

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid.

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP<-->Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.