Design and synthesis of histamine H3/H4 receptor ligands with a cyclopropane scaffold

We previously designed and synthesized a series of histamine analogues with an imidazolylcyclopropane scaffold and identified potent non-selective antagonists for histamine H₃ and H₄ receptor subtypes. In this study, to develop H₄ selective ligands, we newly designed and synthesized cyclopropane-based derivatives having an indole, benzimidazole, or piperazine structure, which are components of representative H₄ selective antagonists such as JNJ7777120 and JNJ10191584. Among the synthesized derivatives, imidazolylcyclopropanes 12 and 13 conjugated with a benzimidazole showed binding affinity to the H₃ and H₄ receptors comparable to that of a well-known non-selective H₃/H₄ antagonist, thioperamide. These results suggest that the binding modes of the cyclopropane-based H₃/H₄ ligands in the H₄ receptor can be different from those of the indole/benzimidazole-piperazine derivatives.     

Pharmacological characterization of the histamine uptake system in HL-60 human promyelocytic leukemia cells

Serotonin and histamine H₁, H₂ receptor agonists or antagonists inhibited [³H]histamine uptake by HL-60 cells, according to the following order of potency: impromidine >4-MH>histamine>AET>PEA and: cimetidine, histamine>diphenhydramine, serotonin. It is concluded that histamine uptake by HL-60 cells was specifically controlled by the H₂ type histamine receptor and that this active process might be involved in pathophysiological regulations in leukemic and normal granulocytic precursors and in the control of histamine levels in peripheral blood and tissues in man.     

Opposite effects of mepyramine on JNJ 7777120-induced amelioration of experimentally induced asthma in mice in sensitization and provocation

Background

Histamine is detected in high concentrations in the airways during an allergic asthma response. In a murine model of allergic asthma, JNJ 7777120, an antagonist at the histamine H₄ receptor, reduces asthmatic symptoms, while the histamine H₁ receptor-selective antagonist mepyramine is virtually without effect. In the present study, we analyzed the effect of combined antagonism at the histamine H₁ and H₄ receptors in a murine asthma model in relation to the timing of their application, i.e. sensitization or provocation.

Methodology/Principal Findings

Asthma was induced in mice by sensitization and provocation with ovalbumin. JNJ 7777120 and/or mepyramine were injected subcutaneously either during sensitization or during provocation, and typical asthma parameters were analyzed. JNJ 7777120, but not mepyramine, reduced serum concentrations of anti-OVA IgE, inflammatory infiltrations in lung tissue, and eosinophilia in bronchoalveolar-lavage (BAL)-fluids independently of the timing of application. Upon application of JNJ 7777120 plus mepyramine in combination during provocation, mepyramine inhibited the effects of JNJ 7777120. In contrast, when applied during sensitization, mepyramine enhanced the disease-ameliorating effects of JNJ 7777120.

Conclusions/Significance

Our study indicates that both histamine H₁ and H₄ receptors play important roles in the course of murine experimental asthma. Unexpectedly, the contribution of these receptors to the pathogenesis differs between the two phases, sensitization or provocation. Since in human asthma, repeated contact to the allergen is not only provocation but also a boost of sensitization, a combined pharmacological targeting of histamine H₁ and H₄ receptors could be taken into consideration as an option for the prevention of asthma and maybe other allergic diseases.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III

Background

Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H₂DCF) but had no direct impact on the H₂O₂ production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).

Methods

H₂O₂ generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.

Results

We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Q_o site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na⁺ or K⁺ excluding a role for putative mitochondrial K_ATP-channels.

General significance

A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).     

LRRK2, but not pathogenic mutants,protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.     

Addressing the use of PDIF-CN2 molecules in the development of n-type organic field-effect transistors for biosensing applications

Background

There is no doubt that future discoveries in the field of biochemistry will depend on the implementation of novel biosensing techniques, able to record biophysiological events with minimal biological interference. In this respect, organic electronics may represent an important new tool for the analysis of structures ranging from single molecules up to cellular events. Specifically, organic field-effect transistors (OFET) are potentially powerful devices for the real-time detection/transduction of bio-signals. Despite this interest, up to date, the experimental data useful to support the development of OFET-based biosensors are still few and, in particular, n-type (electron-transporting) devices, being fundamental to develop highly-performing circuits, have been scarcely investigated.

Methods

Here, films of N,N′-1H,1H-perfluorobutyldicyanoperylene-carboxydi-imide (PDIF-CN₂) molecules, a recently-introduced and very promising n-type semiconductor, have been evaporated on glass and silicon dioxide substrates to test the biocompatibility of this compound and its capability to stay electrically-active even in liquid environments.

Results

We found that PDIF-CN₂ transistors can work steadily in water for several hours. Biocompatibility tests, based on in-vitro cell cultivation, remark the need to functionalize the PDIF-CN₂ hydrophobic surface by extra-coating layers (i.e. poly-l-lysine) to favor the growth of confluent cellular populations.

Conclusions

Our experimental data demonstrate that PDIF-CN₂ compound is an interesting organic semiconductor to develop electronic devices to be used in the biological field.

General significance

This work contributes to define a possible strategy for the fabrication of low-cost and flexible biosensors, based on complex organic complementary metal-oxide-semiconductor (CMOS) circuitry including both p- (hole-transporting) and n-type transistors. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Inhibition of prostaglandin E2 formation and histamine action in cancer immunotherapy

The activity of cell-mediated defense systems is stimulated by consecutive formation of interleukin-1 (IL-1), interleukin-2 (IL-2) and interferon (IFN). The system is inhibited by interleukin-4 (IL-4) and also by prostaglandin E₂ (PGE₂) and histamine, which are released when the immune system is activated. The inhibition is strong in cancer patients, because PGE₂ is formed in many cancer cells and its formation is stimulated by IL-1. The release of histamine is also stimulated by IL-1. Tus PGE₂ and histamine are feedback inhibitors of cell-mediated immunity. This inhibition can be abolished by inhibitors of the cyclo-oxygenase (e. g. indomethacin) and H-2 receptor antagonists (e. g. cimetidine). This may offer a new option to stimulate the immune system to kill cancer cells.     

AD-1, a novel ginsenoside derivative,shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species

Background

Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug.

Methods

The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice.

Results

AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G₀/G₁ cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger — N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10–40 mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index.

Conclusions and general significance

These data support development of AD-1 as a potential agent for lung cancer therapy.     

Evidence for ΔpH surface component (ΔpH) of proton motive force in ATP synthesis of mitochondria

Background

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.

Methods

We measure ?pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.

Results

In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ?pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ?pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ?pH^S, but not with ?Ψ_E.

General Significance

Our data substantiate ?pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ?pH^S and ?Ψ_E in mitochondrial bioenergetics are also presented.     

Signaling Pathway of Histamine H1 Receptor-Mediated Histamine H1 Receptor Gene Upregulation Induced by Histamine in U-373 MG Cells

Histamine H₁ receptor (H1R) is one of the targets of histamine in the nervous system and the peripheral tissues. Protein kinase Cδ (PKCδ) signaling is involved in histamine-induced upregulation of H1R gene expression in HeLa cells. Histamine also upregulates H1R gene expression in U-373 MG cells. However, the molecular signaling of this upregulation is still unclear. Here, we investigated the molecular mechanism of histamine-induced H1R gene upregulation in U-373 MG cells. Histamine-induced H1R gene upregulation was inhibited by H1R antagonist d-chlorpheniramine, but not by ranitidine, ciproxifan, or JNJ77777120, and H2R, H3R, or H4R antagonists, respectively. Ro-31-8220 and Go6976 also suppressed this upregulation, however, the PKCδ selective inhibitor rottlerin and the PKCβ selective inhibitor {"type":"entrez-nucleotide","attrs":{"text":"Ly333531","term_id":"1257370768"}}Ly333531 did not. Time-course studies showed distinct kinetics of H1R gene upregulation in U-373 MG cells from that in HeLa cells. A promoter assay revealed that the promoter region responsible for H1R gene upregulation in U-373 MG cells was different from that of HeLa cells. These data suggest that the H1R-activated H1R gene expression signaling pathway in U-373 MG cells is different from that in HeLa cells, possibly by using different promoters. The involvement of PKCα also suggests that compounds that target PKCδ could work as peripheral type H1R-selective inhibitors without a sedative effect.     

A theoretical study concerning the mode of interaction of the histamine H2-agonist dimaprit

A theoretical study was performed to elucidate the mode of interaction of the histamine H₂-agonist dimaprit with the histamine H₂-receptor. For this purpose receptor mapping techniques, including ab initio energy calculations, geometry optimizations and molecular electrostatic potential calculations (MEPs), have been used. The characteristics of dimaprit were compared to those of histamine for which the points of interaction with the H₂-receptor are known, as well as its bioactive conformation. In this comparative study two possible models for the interaction of dimaprit with the H₂-receptor were considered. In one model the two nitrogen atoms of the isothiourea moiety of dimaprit play an essential role in the recognition of the ligand by the receptor and have the same function as the nitrogen atoms of the imidazole ring of histamine; in the second model this role is fulfilled by a sulphur and a nitrogen atom of the same isothiourea moiety. The comparison to histamine was based on geometrical resemblance as well as on similarity in MEPs. Also the conformational energy of dimaprit in the two interaction models was considered. Results of the investigations reveal that the isothiourea moiety of dimaprit most probably interacts with the histamine H₂-receptor through the sulphur and nitrogen atom, the first atom acting as a proton acceptor and the second one as a proton donor. Subsequently, three analogues of dimaprit, namely SK&F 91487, SK&F 91488 and SK&F 92054, were studied. It was possible to explain their pharmacological behavior within the proposed model. Furthermore, the new model for the interaction of dimaprit with the H₂-receptor enabled the design of a structurally new histamine H₂-agonist, 2-amino-5-(2-aminoethyl)thiazole.     

Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide

Background

Hydrogen peroxide (H₂O₂) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H₂O₂ diffusion.

Scope of review

This review summarizes current knowledge about aquaporin-facilitated H₂O₂ diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H₂O₂ diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes.

Major conclusions

Specific aquaporin isoforms facilitate the passive diffusion of H₂O₂ across biological membranes and control H₂O₂ membrane permeability and signaling in living organisms.

General significance

Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H₂O₂, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H₂O₂ levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

Analysis of IP3 receptors in and out of cells

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP₃R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca^2 + channels.

Scope of the review

In this brief review, we first consider a variety of complementary methods that allow the links between IP₃ binding and channel gating to be defined. How does IP₃ binding to the IP₃-binding core in each IP₃R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP₃, Ca^2 + signals and IP₃R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP₃R signalling.

Major conclusions

All IP₃R are regulated by both IP₃ and Ca^2 +. This allows them to initiate and regeneratively propagate intracellular Ca^2 + signals. The elementary Ca^2 + release events evoked by IP₃ in intact cells are mediated by very small numbers of active IP₃R and the Ca^2 +-mediated interactions between them. The spatial organization of these Ca^2 + signals and their stochastic dependence on so few IP₃Rs highlight the need for methods that allow the spatial organization of IP₃R signalling to be addressed with single-molecule resolution.

General significance

A variety of complementary methods provide insight into the structural basis of IP₃R activation and the contributions of IP₃-evoked Ca^2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch

Background

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.

Methods

For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).

Results

Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δ_gelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.

Conclusions

Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.

General Significance

Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.     

Hydogen peroxide-dependent photocytotoxicity by phloxine B,a xanthene-type food colorant

Background

Phloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent.

Methods

We investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants.

Reuslts and conclusions

PhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H₂O₂), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H₂O₂ in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H₂O₂-dependent mechanism.

General significance

Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

Antioxidative activity of the olive oil constituent hydroxy-1-aryl-isochromans in cells and cell-free systems

Background

Hydroxy-1-aryl-isochromans (HAIC) are newly emerging natural polyphenolic antioxidants, enriched in extravirgin olive oil, whose antioxidative potency was only scarcely characterized using cell-free systems and cells.

Methods

We characterized the activity of HAIC to inactivate reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase system, mitochondria (rat brain) and neural cells. ROS levels were estimated using ROS-sensitive probes, such as Amplex Red, MitoSOX^RED.

Results

HAIC (with 2, 3 or 4 hydroxyl substituents) effectively scavenge ROS released from mitochondria. EC₅₀ values estimated with mitochondria and submitochondrial particles were around 20 μM. Moreover, in PC12 and cultured neural primary cells, HAIC buffered cytosolic ROS. Although HAIC permeate biological membranes, HAIC fail to buffer matrix ROS in isolated mitochondria. We show that hydrogen peroxide was effectively abolished by HAIC, whereas the production of superoxide was not affected.

Conclusion

HAIC exert high antioxidative activity to reduce hydrogen peroxide. The antioxidative activity of HAIC is comparable with that of the stilbene-like, polyphenolic resveratrol, but much higher than that of trolox, N-acetylcysteine or melatonin.

General significance

Unlike resveratrol, HAIC do not impair mitochondrial ATP synthesis or Ca²⁺ retention by mitochondria. Thus, HAIC have the decisive advantage to be potent antioxidants with no detrimental side effects on mitochondrial functions.