Proton pump inhibitors interfere with the anti-tumor potency of RC48ADC.

Antibody-drug conjugates (ADCs) are a promising modality for cancers, but the interaction between them and proton pump inhibitors (PPIs), the common adjuvant drugs for cancer treatment, has not been understood. Here, the interactions between PPIs and RC48ADC, a novel HER2-targeting ADC, were quantified in vitro. CCK-8 assay showed that RC48ADC displayed a significant inhibitory effect on the proliferation of SK-BR-3, NCI-N87 and SK-OV-3 cells with the IC50 values of 4.91 ± 1.15 ng/mL, 14.54 ± 0.85 ng/mL and 11.28 ± 0.68 ng/mL respectively. PPIs alone had no significant anti-tumor effect in the dose range of 1.37-1000 ng/mL. When used together, PPIs inhibited the anti-tumor activity of RC48ADC in a dose-dependent manner. And 1000 ng/mL (~Cmax) PPIs significantly recovered RC48ADC-inhibited cell proliferation by (32.85 ± 2.81) % (p < 0.05). However, cimetidine, a non-PPIs gastric acid secretion inhibitor, had no significant inhibitory effect on RC48ADC. Furthermore, omeprazole, rather than cimetidine, significantly reduced the activity of vacuolar H+-ATPase and Cathepsin B compared with the control cells. These results, if confirmed in vivo, indicate that PPIs are antagonists of RC48ADC, even all ADCs, appearing to be due to inhibition of vacuolar H+-ATPase activity. Moreover, cimetidine combined with ADCs instead of PPIs can prevent an adverse drug interaction.

Biological activity of quaternary ammonium salts and resistance of microorganisms to these compounds.

Quaternary ammonium salts (QASs) are ubiquitous in nature, being found in organisms ranging from microorganisms to vertebrates (e.g., glycine betaine, carnitine) where they have important cellular functions. QASs are also obtained by chemical synthesis. These compounds, due to their diverse chemical structure (e.g. monomeric QAS or gemini) and their biological properties, are widely used in medicine (as disinfectants, drugs, and DNA carriers), industry, environmental protection and agriculture (as preservatives, biocides, herbicides and fungicides). Discussed chemical compounds reduce the adhesion of microorganisms to various biotic and abiotic surfaces and cause the eradication of biofilms produced by pathogenic microorganisms. The properties of these chemicals depend on their chemical structure (length of the alkyl chain, linker and counterion), which has a direct impact on the physicochemical and biological activity of these compounds. QASs by incorporation into the membranes, inhibit the activity of proteins (H+-ATPase) and disrupt the transport of substances to the cell. Moreover, in the presence of QASs, changes in lipid composition (qualitative and quantitative) of plasma membrane are observed. The widespread use of disinfectants in commercial products can induce resistance in microorganisms to these surfactants and even to antibiotics. In this article we discuss the biological activity of QASs as cationic surfactants against microorganisms and their resistance to these compounds.

Mitochondrial F1FO-ATPase and permeability transition pore response to sulfide in the midgut gland of Mytilus galloprovincialis.

The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F1FO-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg2+ by Ca2+ in the F1FO-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg2+- or Ca2+-dependent F1FO-ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg2+-dependent F1FO-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F1FO-ATPase activities, but desensitizes the mPTP to Ca2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F1FO-ATPase activities and a failed relationship between the Ca2+-dependent F1FO-ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F1FO-ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide.

Coumarin enhances nitrate uptake in maize roots through modulation of plasma membrane H+ -ATPase activity.

Coumarin is one of the simplest plant secondary metabolites, widely distributed in the plant kingdom, affecting root form and function, including anatomy, morphology and nutrient uptake. Although, some plant responses to coumarin have been described, comprehensive knowledge of the physiological and molecular mechanisms is lacking. Maize seedlings exposed to different coumarin concentrations, alone or in combination with 200 µm nitrate (NO3- ), were analysed, through a physiological and molecular approach, to elucidate action of coumarin on net NO3- uptake rate (NNUR). In detail, the time course of NNUR, plasma membrane (PM) H+ -ATPase activity, proton pumping and related gene expression (ZmNPF6.3, ZmNRT2.1, ZmNAR2.1, ZmHA3 and ZmHA4) were evaluated. Coumarin alone did not affect nitrate uptake, PM H+ -ATPase activity or transcript levels of ZmNRT2.1 and ZmHA3. In contrast, coumarin alone increased ZmNPF6.3, ZmNAR2.1 and ZmHA4 expression in response to abiotic stress. When coumarin and NO3- were concurrently added to the nutrient solution, a significant increase in the NNUR, PM H+ -ATPase activity, together with ZmNAR2.1:ZmNRT2.1 and ZmHA4 expression was observed, suggesting that coumarin affected the inducible component of the high affinity transport system (iHATS), and this effect appeared to be mediated by nitrate. Moreover, results with vanadate, an inhibitor of the PM H+ -ATPase, suggested that this enzyme could be the main target of coumarin. Surprisingly, coumarin did not affect PM H+ -ATPase activity by direct contact with plasma membrane vesicles isolated from maize roots, indicating its possible elicitor role in gene transcription.

The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H+-ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H+-ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H+-ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H+-ATPase and NADPH oxidase are key factors.

Trichoderma asperellum Induces Maize Seedling Growth by Activating the Plasma Membrane H+-ATPase.

Although Trichoderma spp. have beneficial effects on numerous plants, there is not enough knowledge about the mechanism by which they improves plant growth. In this study, we evaluated the participation of plasma membrane (PM) H+-ATPase, a key enzyme involved in promoting cell growth, in the elongation induced by T. asperellum and compared it with the effect of 10 µM indol acetic acid (IAA) because IAA promotes elongation and PM H+-ATPase activation. Two seed treatments were tested: biopriming and noncontact. In neither were the tissues colonized by T. asperellum; however, the seedlings were longer than the control seedlings, which also accumulated IAA and increased root acidification. An auxin transport inhibitor (2,3,5 triiodobenzoic acid) reduced the plant elongation induced by Trichoderma spp. T. asperellum seed treatment increased the PM H+-ATPase activity in plant roots and shoots. Additionally, the T. asperellum extracellular extract (TE) activated the PM H+-ATPase activity of microsomal fractions of control plants, although it contained 0.3 µM IAA. Furthermore, the mechanism of activation of PM H+-ATPase was different for IAA and TE; in the latter, the activation depends on the phosphorylation state of the enzyme, suggesting that, in addition to IAA, T. asperellum excretes other molecules that stimulate PM H+-ATPase to induce plant growth.

TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls.

Despite being composed of immobile cells, plants reorient along directional stimuli. The hormone auxin is redistributed in stimulated organs leading to differential growth and bending. Auxin application triggers rapid cell wall acidification and elongation of aerial organs of plants, but the molecular players mediating these effects are still controversial. Here we use genetically-encoded pH and auxin signaling sensors, pharmacological and genetic manipulations available for Arabidopsis etiolated hypocotyls to clarify how auxin is perceived and the downstream growth executed. We show that auxin-induced acidification occurs by local activation of H+-ATPases, which in the context of gravity response is restricted to the lower organ side. This auxin-stimulated acidification and growth require TIR1/AFB-Aux/IAA nuclear auxin perception. In addition, auxin-induced gene transcription and specifically SAUR proteins are crucial downstream mediators of this growth. Our study provides strong experimental support for the acid growth theory and clarified the contribution of the upstream auxin perception mechanisms.

F1 -ATP synthase α-subunit: a potential target for RNAi-mediated pest management of Locusta migratoria manilensis.

BACKGROUND: The migratory locust is one of the most destructive agricultural pests worldwide. ATP synthase (F0 F1 -ATPase) uses proton or sodium motive force to produce 90% of the cellular ATP, and the α-subunit of F1 -ATP synthase (ATP5A) is vital for F1 -ATP synthase. Here, we tested whether ATP5A could be a potential target for RNAi-mediated pest management of L. migratoria. RESULTS: Lm-ATP5A was cloned and characterised. Lm-ATP5A is expressed in all tissues. Injection of 100 ng of the double-stranded RNA of ATP5A (dsATP5A) knocked down the transcription of the target gene and caused mortality in 1.5-5 days. The Lm-ATP5A protein level, the oligomycin-sensitive ATP synthetic and hydrolytic activities and the ATP content were correspondingly reduced following dsATP5A injection. CONCLUSION: These findings demonstrated the essential roles of Lm-ATP5A in L. migratoria and identified it as a potential target for insect pest control. © 2015 Society of Chemical Industry.

Impairing effects of angiotensin-converting enzyme inhibitor Captopril on bone of normal mice.

There are contradicting results about the effects of angiotensin-converting enzyme inhibitors (ACEIs) on bones. This study was aimed to investigate the effect of ACEI, Captopril, on bone metabolism and histology as well as the action of Captopril on skeletal renin-angiotensin system (RAS) and bradykinin receptor pathway in normal male mice. The urine, serum, tibias and femurs from normal control mice and Captopril-treated (10mg/kg) mice were collected for biochemical, histological and molecular analyses after drug administration for eight weeks. The mice after the treatment with Captopril had a significant decrease of serum testosterone level. The histological measurements showed the loss of trabecular bone mass and trabecular bone number, and the breakage of trabecular bone network as well as the changes of chondrocyte zone at epiphyseal plate in Captopril-treated mice. The defect of Captopril on trabecular bone was reflected by the quantitative bio-parameters from micro-CT. The expression of renin receptor and bradykinin B2 receptor (B2R) was significantly up-regulated in tibia of mice upon to the Captopril treatment, which decreased the ratio of OPG/RANKL and the expression of osteoblastic factor RUNX2. Furthermore, Captopril treatment resulted in the increase of pAkt/Akt and pNFκB expression in tibia. The present study revealed the impairing effects of Captopril on bone via interfering with the circulating sex hormone level and B2R pathway, which suggests that the bone metabolism of patients need to be carefully monitored when being prescribed for ACEIs.

Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain.

Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings.

Ion transport in broad bean leaf mesophyll under saline conditions.

MAIN CONCLUSION: Salt stress reduces the ability of mesophyll tissue to respond to light. Potassium outward rectifying channels are responsible for 84 % of Na (+) induced potassium efflux from mesophyll cells. Modulation in ion transport of broad bean (Vicia faba L.) mesophyll to light under increased apoplastic salinity stress was investigated using vibrating ion-selective microelectrodes (the MIFE technique). Increased apoplastic Na(+) significantly affected mesophyll cells ability to respond to light by modulating ion transport across their membranes. Elevated apoplastic Na(+) also induced a significant K(+) efflux from mesophyll tissue. This efflux was mediated predominately by potassium outward rectifying channels (84 %) and the remainder of the efflux was through non-selective cation channels. NaCl treatment resulted in a reduction in photosystem II efficiency in a dose- and time-dependent manner. In particular, reductions in Fv'/Fm' were linked to K(+) homeostasis in the mesophyll tissue. Increased apoplastic Na(+) concentrations induced vanadate-sensitive net H(+) efflux, presumably mediated by the plasma membrane H(+)-ATPase. It is concluded that the observed pump's activation is essential for the maintenance of membrane potential and ion homeostasis in the cytoplasm of mesophyll under salt stress.

Crosstalk between blue-light- and ABA-signaling pathways in stomatal guard cells.

We recently established an immunohistochemical method for the detection of blue light (BL)-induced and phototropin-mediated phosphorylation of plasma-membrane H+-ATPase in stomatal guard cells of Arabidopsis thaliana. This technique makes it possible to detect the phosphorylation/activation status of guard-cell H+-ATPase in the epidermis of a single rosette leaf, without the need to prepare guard-cell protoplasts (GCPs) from a large number of plants. Moreover, it can detect guard-cell responses under more natural and stress-free conditions compared to using GCPs. Taking advantage of these properties, we examined the effect of abscisic acid (ABA) on BL-induced phosphorylation of guard-cell H+-ATPase by using ABA-insensitive mutants. This revealed inhibition of BL-induced phosphorylation of guard-cell H+-ATPase via the early ABA-signaling components PYR/PYL/RCAR-PP2Cs-SnRK2s, which are known to be early ABA-signaling components for a wide range of ABA responses in plants.

cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in Arabidopsis thaliana roots.

3',5'-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na(+)/K(+) ratio and a decrease in gene expression of the plasma membrane (PM) H(+)-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H(2)O(2) or CaCl(2) alleviated the NaCl-induced injury by maintaining a lower Na(+)/K(+) ratio and increasing the PM H(+)-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H(2)O(2) by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H(2)O(2) on ionic homeostasis was abolished when Ca(2+) was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, a Ca(2+) chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H(2)O(2) accumulation in salt stress, and Ca(2+) was necessary in the cGMP-mediated signaling pathway. H(2)O(2), as the downstream component of cGMP signaling pathway, stimulated PM H(+)-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.

Effect of cadmium and lead on the membrane potential and photoelectric reaction of Nitellopsis obtusa cells.

The effects of Cd and Pb on membrane potential (E(m)) and photoelectric reaction of Nitellopsis obtusa cells were investigated. It was found that Cd and Pb at 1.0 mM caused a depolarization of the E(m), whereas both metals at lower concentrations changed the E(m) in a different way. Pb at 0.1 mM and 0.01 mM hyperpolarized the E(m), whereas Cd at the same concentrations depolarized and did not change the E(m), respectively. In the presence of 0.01 mM Pb, the light-induced hyperpolarization of the E(m) was by 18% higher as compared to the control, whereas at 1.0 mM Pb it was by 40% lower. Pb at 0.1 mM and Cd at 0.01 mM or 5 × 0.01 mM did not change the light-induced membrane hyperpolarization. However, in the presence of Cd at 0.1 mM and 1.0 mM this hyperpolarization was 2-fold lower or was completely abolished, respectively. These results suggest that at high Cd and Pb concentrations both depolarization of the E(m) and decrease of light-induced membrane hyperpolarization in Nitellopsis obtusa cells are probably due to inhibition of the plasma membrane H(+)-ATPase activity, whereas both metals at lower concentrations differ in mechanism of membrane potential changes.

Aluminum regulates oxalate secretion and plasma membrane H+-ATPase activity independently in tomato roots.

We demonstrated that aluminum (Al)-induced oxalate secretion and plasma membrane (PM) H(+)-ATPase activity in tomato (Lycopersicon esculentum 'Hezuo903') roots were poorly correlated. In addition, vanadate, an inhibitor of PM H(+)-ATPase, had no effect on Al-induced oxalate secretion, but significantly inhibited enzyme activity. An anion channel inhibitor phenylglyoxal inhibited oxalate secretion, but not PM H(+)-ATPase activity. Exposure of tomato roots to 10 µM LaCl(3) also stimulated PM H(+)-ATPase activity; however, La failed to induce oxalate secretion. Furthermore, Al-induced changes of PM H(+)-ATPase activity were not associated with oxalate secretion in two tomato cultivars differing in the ability to secrete oxalate under Al stress. These results indicate that Al independently regulates oxalate secretion and PM H(+)-ATPase activity in tomato roots. Analysis of expression levels of PM H(+)-ATPase genes by real-time reverse transcription-PCR and protein by Western blot and immunodetection revealed that the regulation of PM H(+)-ATPase in response to Al was subjected to transcriptional and posttranscriptional control. However, since neither transcriptional level of genes nor translational level of proteins directly relate to the enzyme activity, posttranslational modification of PM H(+)-ATPase under Al stress likely contributes to changes in activity of this protein.

Isoforms of plasma membrane H(+)-ATPase in rice root and shoot are differentially induced by starvation and resupply of NO3⁻ or NH4+.

The goal of this study was to evaluate the effects of nitrogen starvation and resupply in 10 PM H+-ATPase isoforms and the expression of NO3⁻ and NH4+ transporters in rice. The net uptake of both forms of NO3⁻-N or NH4+-N was increased with its resupply. Resupply of NO3⁻ resulted in induction of the following PM H+-ATPase isoforms, OsA1, OsA2, OsA5 and OsA7 in the shoots and OsA2, OsA5, OsA7 and OsA8 in the roots. Resupply of NH4+ resulted in the induction of the following OsA1, OsA3 and OsA7 isoforms in the roots while OsA1 was induced in the shoots. It was observed that increased PM H+-ATPase activity also resulted in increased net uptake of NO3⁻ and NH4+. In the roots, OsNRT2.1 and OsNRT2.2 were induced by NO3⁻ resupply, while OsAMT1.1 and OsAMT1.2 were induced by NH4+ deficiency. The results showed that the expression of PM H+-ATPase isoforms is related to NO3⁻ and NH4+ transporters as well as in which section of the plant it takes place. PM H+-ATPase isoforms OsA2 and OsA7 displayed the strongest induction in response to N resupply, therefore indicating that these genes could be involved in N uptake in rice.

Role of ATP synthase alpha subunit in low-dose endothelial monocyte-activating polypeptide-II-induced opening of the blood-tumor barrier.

This study was performed to determine whether the α subunit of ATP synthase (α-ATP synthase) on brain microvascular endothelial cells (BMECs) serves as the functional target for endothelial monocyte-activating polypeptide-II (EMAP-II)-induced increase in blood-tumor barrier (BTB) permeability. Using a rat C6 glioma model, we found that low-dose (80 ng/kg) EMAP-II significantly decreased the mRNA and protein expression levels of tight junction (TJ)-related proteins claudin-5, occludin, and ZO-1 on BMECs. Meantime, radioimmunity and Western blot assay showed a significant decrease in the expression levels of cAMP and catalytic subunit of protein kinase A (PKAcs) of tumor tissues. Also, pretreatment with specific α-ATP synthase antibody significantly blocked the effects of EMAP-II on TJ-related proteins, cAMP, and PKAcs. In addition, double immunofluorescence assay identified that EMAP-II was co-localized with α-ATP synthase on BMECs. This in vivo study demonstrated that α subunit of ATP synthase on BMECs serves as the functional target for EMAP-II selective opening of the BTB, and that cAMP/PKA signaling transduction pathway might be involved in the modulating process.

ATP synthase and the actions of inhibitors utilized to study its roles in human health, disease, and other scientific areas.

ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and P(i), the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology.

The mitochondrion is a site of trypanocidal action of the aromatic diamidine DB75 in bloodstream forms of Trypanosoma brucei.

Human African trypanosomiasis (HAT) is a fatal tropical disease caused by infection with protozoans of the species Trypanosoma brucei gambiense and T. b. rhodesiense. An oral prodrug, DB289, is a promising new therapy undergoing phase III clinical trials for early-stage HAT. DB289 is metabolically converted to the active trypanocidal diamidine DB75 [2,5-bis(4-amidinophenyl)furan]. We previously determined that DB75 inhibits yeast mitochondrial function (C. A. Lanteri, B. L. Trumpower, R. R. Tidwell, and S. R. Meshnick, Antimicrob. Agent Chemother. 48:3968-3974, 2004). The purpose of this study was to investigate if DB75 targets the mitochondrion of T. b. brucei bloodstream forms. DB75 rapidly accumulates within the mitochondria of living trypanosomes, as indicated by the fluorescent colocalization of DB75 with a mitochondrion-specific dye. Fluorescence-activated cell sorting analysis of rhodamine 123-stained living trypanosomes shows that DB75 and other trypanocidal diamidines (pentamidine and diminazene) collapse the mitochondrial membrane potential. DB75 inhibits ATP hydrolysis within T. brucei mitochondria and appears to inhibit the oligomycin-sensitive F 1 F 0-ATPase and perhaps other ATPases. DB75 is most likely not an inhibitor of electron transport within trypanosome mitochondria, since DB75 fails to inhibit mitochondrial respiration when glycerol-3-phosphate is used as the respiratory substrate. However, DB75 inhibits whole-cell respiration (50% inhibitory concentration, 20 microM) at drug concentrations and incubation durations that also result in the dissipation of the mitochondrial membrane potential. Taken together, these findings suggest that the mitochondrion is a target of the trypanocidal action of DB75.

Akt activation improves oxidative phosphorylation in renal proximal tubular cells following nephrotoxicant injury.

Previously, we showed that protein kinase B (Akt) activation increases intracellular ATP levels and decreases necrosis in renal proximal tubular cells (RPTC) injured by the nephrotoxicant S-(1, 2-dichlorovinyl)-l-cysteine (DCVC) (Shaik ZP, Fifer EK, Nowak G. Am J Physiol Renal Physiol 292: F292-F303, 2007). This study examined the role of Akt in improving mitochondrial function in DCVC-injured RPTC. Our data show a novel observation that phosphorylated (active) Akt is localized in mitochondria of noninjured RPTC, both in mitoplasts and the mitochondrial outer membrane. Mitochondrial levels of active Akt decreased in nephrotoxicant-injured RPTC, and this decrease was associated with mitochondrial dysfunction. DCVC decreased basal, uncoupled, and state 3 respirations; ATP production; activities of complexes I, II, and III; the mitochondrial membrane potential (DeltaPsi(m)); and F(0)F(1)-ATPase activity. Expressing constitutively active Akt in DCVC-injured RPTC increased the levels of phosphorylated Akt in mitochondria, reduced the decreases in basal and uncoupled respirations, increased complex I-coupled state 3 respiration and ATP production, enhanced activities of complex I, complex III, and F(0)F(1)-ATPase, and improved DeltaPsi(m). In contrast, inhibiting Akt activation by expressing dominant negative (inactive) Akt or using 20 microM LY294002 exacerbated decreases in electron transport rate, state 3 respiration, ATP production, DeltaPsi(m), and activities of complex I, complex III, and F(0)F(1)-ATPase. In conclusion, our data show that Akt activation promotes mitochondrial respiration and ATP production in toxicant-injured RPTC by 1) improving integrity of the respiratory chain and maintaining activities of complex I and complex III, 2) reducing decreases in DeltaPsi(m), and 3) restoring F(0)F(1)-ATPase activity.