Presence Of Metabolic Inhibitors Research Articles

Campylobacter jejuni Response When Inoculated in Bovine In Vitro Fecal Microbial Consortia Incubations in the Presence of Metabolic Inhibitors.

Infection with the foodborne pathogen Campylobacter is the leading bacterial cause of human foodborne illness in the United States. The objectives of this experiment were to test the hypothesis that mixed microbial populations from the bovine rumen may be better at excluding Campylobacter than populations from freshly voided feces and to explore potential reasons as to why the rumen may be a less favorable environment for Campylobacter than feces. In an initial experiment, C. jejuni cultures inoculated without or with freshly collected bovine rumen fluid, bovine feces or their combination were cultured micro-aerobically for 48 h. Results revealed that C. jejuni grew at similar growth rates during the first 6 h of incubation regardless of whether inoculated with the rumen or fecal contents, with rates ranging from 0.178 to 0.222 h-1. However, C. jejuni counts (log10 colony-forming units/mL) at the end of the 48 h incubation were lowest in cultures inoculated with rumen fluid (5.73 log10 CFUs/mL), intermediate in cultures inoculated with feces or both feces and rumen fluid (7.16 and 6.36 log10 CFUs/mL) and highest in pure culture controls that had not been inoculated with the rumen or fecal contents (8.32 log10 CFUs/mL). In follow-up experiments intended to examine the potential effects of hydrogen and hydrogen-consuming methanogens on C. jejuni, freshly collected bovine feces, suspended in anaerobic buffer, were incubated anaerobically under either a 100% carbon dioxide or 50:50 carbon dioxide/hydrogen gas mix. While C. jejuni viability decreased <1 log10 CFUs/mL during incubation of the fecal suspensions, this did not differ whether under low or high hydrogen accumulations or whether the suspensions were treated without or with the mechanistically distinct methanogen inhibitors, 5 mM nitrate, 0.05 mM 2-bromosulfonate or 0.001 mM monensin. These results suggest that little if any competition between C. jejuni and hydrogen-consuming methanogens exists in the bovine intestine based on fecal incubations.

Succinate based polymers drive immunometabolism in dendritic cells to generate cancer immunotherapy.

Boosting the metabolism of immune cells while restricting cancer cell metabolism is challenging. Herein, we report that using biomaterials for the controlled delivery of succinate metabolite to phagocytic immune cells activates them and modulates their metabolism in the presence of metabolic inhibitors. In young immunocompetent mice, polymeric microparticles, with succinate incorporated in the backbone, induced strong pro-inflammatory anti-melanoma responses. Administration of poly(ethylene succinate) (PES MP)-based vaccines and glutaminase inhibitor to young immunocompetent mice with aggressive and large, established B16F10 melanoma tumors increased their survival three-fold, a result of increased cytotoxic T cells expressing RORγT (Tc17). Mechanistically, PES MPs directly modulate glutamine and glutamate metabolism, upregulate succinate receptor SUCNR1, activate antigen presenting cells through and HIF-1alpha, TNFa and TSLP-signaling pathways, and are dependent on alpha-ketoglutarate dehydrogenase for their activity, which demonstrates correlation of succinate delivery and these pathways. Overall, our findings suggest that immunometabolism-modifying PES MP strategies provide an approach for developing robust cancer immunotherapies.

Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

A fundamental phenotype of cancer cells is their metabolic profile, which is routinely described in terms of glycolytic and respiratory rates. Various devices and protocols have been designed to quantify glycolysis and respiration from the rates of acid production and oxygen utilization, respectively, but many of these approaches have limitations, including concerns about their cost-ineffectiveness, inadequate normalization procedures, or short probing time-frames. As a result, many methods for measuring metabolism are incompatible with cell culture conditions, particularly in the context of high-throughput applications. Here, we present a simple plate-based approach for real-time measurements of acid production and oxygen depletion under typical culture conditions that enable metabolic monitoring for extended periods of time. Using this approach, it is possible to calculate metabolic fluxes and, uniquely, describe the system at steady-state. By controlling the conditions with respect to pH buffering, O2 diffusion, medium volume, and cell numbers, our workflow can accurately describe the metabolic phenotype of cells in terms of molar fluxes. This direct measure of glycolysis and respiration is conducive for between-runs and even between-laboratory comparisons. To illustrate the utility of this approach, we characterize the phenotype of pancreatic ductal adenocarcinoma cell lines and measure their response to a switch of metabolic substrate and the presence of metabolic inhibitors. In summary, the method can deliver a robust appraisal of metabolism in cell lines, with applications in drug screening and in quantitative studies of metabolic regulation.

Physiological changes and stress responses of heat shock treated Salmonella enterica serovar Typhimurium

Mild heat treatment enhances the ability of microorganisms to endure subsequent heat treatment and other environmental stresses by initiating the heat shock response (HSR). This study was conducted to determine the thermal resistance of foodborne pathogens (Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Cronobacter sakazakii, and Staphylococcus aureus) after various heat shock treatments (HSTs) (50 °C for 30, 60, and 120 min) to subsequent thermal treatment at 60 °C and to investigate the mechanism for the HSR of S. Typhimurium by assessing cellular damage and morphological changes. Heat-shocked (HS) S. Typhimurium treated at 50 °C for 120 min showed the greatest resistance against subsequent heat treatment at 60 °C. Results revealed that HST caused less membrane damage and less heat-induced injury in the presence of metabolic inhibitors, compared with non-heat-shocked (non-HS) cells during thermal treatment. Furthermore, fluorescence microscopic images showed increased death, rather than sublethal injury, in non-HS cells, whereas HST induced mainly sublethal injury during thermal treatment. Analysis of electron micrographs showed that the cytoplasm was the main cellular component damaged by HST. Additionally, HST cross-protected S. Typhimurium against 15% ethanol and 25% NaCl, but not against pH 2.5, 0.01% H2O2, or -20 °C and it also enhanced thermal resistance in foods tested. Therefore, the ability of HS S. Typhimurium to tolerate subsequent thermal treatment and environmental stresses and its altered behavior should be considered when developing microbial control measures for food processing.

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance.

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBFnn ) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N8 -acetylated spermidine, inhibit the hydrolytic activity of TanBFnn and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBFnn and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.

Aqueous Humor Outflow Requires Active Cellular Metabolism in Mice.

PurposeConventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice.MethodsWe measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2.ResultsReducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<<0.001), but reducing temperature did not affect ATP.ConclusionsInhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice.

Enhanced Biosorption of Sb(III) onto Living Rhodotorula mucilaginosa Strain DJHN070401: Optimization and Mechanism.

How to effectively remove excess Sb(III) in the water environment by biosorption is receiving close attention in the international scientific community. To obtain the maximum biosorption efficiency, response surface methodology (RSM) was employed to optimize a total of 13 factors for biosorption of Sb(III) onto living Rhodotorula mucilaginosa DJHN070401. The mechanism of biosorption and bioaccumulation was also studied. The results showed that biosorption reached 56.83% under the optimum conditions. Besides, pH, Fe2+, and temperature are significant influencing factors, and control of Ca2+ and Fe2+ has a beneficial impact on Sb(III) biosorption. The characterization explained that physical adsorption occurred readily on the loose and porous surface of DJHN070401 where carboxyl, amidogen, phosphate group, and polysaccharide C-O functional groups facilitated absorption by complexation with Sb(III), accompanied by ion exchange of Na+, Ca2+ ions with Sb(III). It was also noted that the living cell not only improved the removal efficiency in the presence of metabolic inhibitors but also prevented intracellular Sb(III) being re-released into the environment. The results of this study underpin improved and efficient methodology for biosorption of Sb(III) from wastewater.

Metabolism, ATP production and biofilm generation by Staphylococcus epidermidis in either respiratory or fermentative conditions

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O2]). As [O2] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O2] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O2] S. epidermidis limits its growth and develops the ability to form biofilms.

Involvement of a Novel Organic Cation Transporter in Paeonol Transport Across the Blood-Brain Barrier.

Paeonol has neuroprotective function, which could be useful for improving central nervous system disorder. The purpose of this study was to characterize the functional mechanism involved in brain transport of paeonol through blood-brain barrier (BBB). Brain transport of paeonol was characterized by internal carotid artery perfusion (ICAP), carotid artery single injection technique (brain uptake index, BUI) and intravenous (IV) injection technique in vivo. The transport mechanism of paeonol was examined using conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) as an in vitro model of BBB. Brain volume of distribution (VD) of [3H]paeonol in rat brain was about 6-fold higher than that of [14C]sucrose, the vascular space marker of BBB. The uptake of [3H]paeonol was concentration-dependent. Brain volume of distribution of paeonol and BUI as in vivo and inhibition of analog as in vitro studies presented significant reduction effect in the presence of unlabeled lipophilic compounds such as paeonol, imperatorin, diphenhydramine, pyrilamine, tramadol and ALC during the uptake of [3H]paeonol. In addition, the uptake significantly decreased and increased at the acidic and alkaline pH in both extracellular and intracellular study, respectively. In the presence of metabolic inhibitor, the uptake reduced significantly but not affected by sodium free or membrane potential disruption. Similarly, paeonol uptake was not affected on OCTN2 or rPMAT siRNA transfection BBB cells. Interestingly. Paeonol is actively transported from the blood to brain across the BBB by a carrier mediated transporter system.

Silicon Affects Transcellular and Apoplastic Uptake of Some Nutrients in Plants

The positive effects of silicon (Si) on growth of plants have been well documented; however, the impact of Si on plant nutrient uptake remains unclear. The growth, nutrient content and uptake of wheat (Triticum aestivum L.), canola (Brassica napus L.) and cotton (Gossypium hirsutum L.) plants were evaluated with or without application of 1.5 mmol L−1 Si. Application of Si increased dry weights by 8%, 30% and 30% and relative growth rate (RGR) by 10%, 13% and 17% in the cotton, canola and wheat plants, respectively. The plant relative water content (RWC) was also increased, but the plant transpiration was decreased by Si application. The uptake and content of Ca2+ were 19% and 21% lower in the cotton and wheat plants with Si than those without Si, respectively; however, Si application increased both K+ and Fe uptake and contents in all plant species. Silicon application reduced B uptake and content only in cotton and increased P and Zn2+ contents in all three plant species. The decrease in Ca2+ uptake by Si application was sustained even in the presence of metabolic inhibitors 2,4-dinitrophenol and sodium cyanide. Uptake of Ca2+ by Si application was enhanced or did not change when plant shoots were saturated with water vapor or their roots were exposed to low temperature. Thus, Si application increased the uptake of transcellularly transported elements like K+, P, Zn2+ and Fe. In contrast, Ca2+ uptake which occurred via both apoplastic and transcellular pathways was decreased by Si application, possibly through reduction of apoplastic uptake. More efficient nutrient uptake might be another promoting effect of Si on plant growth.

Carrier-mediated uptake of nobiletin, a citrus polymethoxyflavonoid, in human intestinal Caco-2 cells

The mechanism of intestinal absorption of nobiletin (NBL) was investigated using Caco-2 cells. The uptake of NBL from the apical membranes of Caco-2 cells was rapid and temperature-dependent and the presence of metabolic inhibitors, NaN3 and carbonylcyanide p-trifluoromethoxyphenylhydrazone, did not cause a decrease in NBL uptake. The relationship between the initial uptake of NBL and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The Km and uptake clearance (Vmax/Km) values for NBL were 50.6 and 168.1μl/mg protein/min, respectively. This clearance value was about 9-fold greater than that of the non-saturable uptake clearance (Kd: 18.5μl/mg protein/min). The presence of structurally similar compounds, such as quercetin and luteolin, competitively inhibited NBL uptake. These results suggest that uptake of NBL from the apical membranes of Caco-2 cells is mainly mediated by an energy-independent facilitated diffusion process.

A Thorough QTc Study of 3 Doses of Iloperidone Including Metabolic Inhibition Via CYP2D6 and/or CYP3A4 and a Comparison to Quetiapine and Ziprasidone

The potential for iloperidone, a D2/5-HT2A antipsychotic, to affect the heart rate-corrected QT interval (QTc) was assessed in the absence and presence of metabolic inhibitors in a randomized, open-label, multicenter study. QT interval prolongation by medications, including both conventional and atypical antipsychotic drugs, can predispose patients to cardiac arrhythmias and result in sudden death. Adults with schizophrenia or schizoaffective disorder and normal electrocardiograms at baseline (N = 188) were randomized 1:1:1:1:1 to iloperidone, 8 mg twice daily (BID), 12 mg BID, 24 mg once daily (QD); quetiapine, 375 mg BID; or ziprasidone, 80 mg BID during period 1 (no metabolic inhibitors present). Iloperidone BID produced mean changes in QTc Fridericia correction (QTcF) interval (8.5-9.0 milliseconds [ms]) similar to those produced by ziprasidone (9.6 ms) and higher than those produced by quetiapine (1.3 ms). Iloperidone, 24 mg QD, produced a mean QTcF change of 15.4 ms. Coadministration of metabolic inhibitors with iloperidone during periods 2 (paroxetine) and 3 (paroxetine and ketoconazole) resulted in greater increases in the QTc interval. Increased QTc was observed in individuals with specific cytochrome P450 2D6 polymorphisms. Up to 10% of patients on iloperidone experienced QTc intervals of 60 ms or longer in the presence of metabolic inhibition and QD dosing. However, no patients experienced QTc changes of clinical concern (QTc ≥ 500 ms). The most common adverse events with iloperidone were headache, anxiety, and dyspepsia. The only cardiovascular adverse events with iloperidone were non-concentration-dependent tachycardia that was mild in most patients and did not lead to further sequelae. Pharmacogenetics and recommendations are discussed.

Regulatory role of osteopontin in malignant transformation of endometrial cancer

Osteopontin (OPN) involves in the tumor-promoting or metastasis in human endometrial cancer. Depletion of OPN gene expression in endometrial cancer cells was significantly decreased in cell viability and the cells undergo apoptotic cell death. The status of OPN in THESC, RL95, Hec1A and Ishikawa cell lines were analyzed by RT-PCR and western blot. After OPN-siRNA transfection, mRNA and protein expression levels of OPN were determined in Hec1A and Ishikawa cells. Cell proliferation and cell cycle distribution were observed by MTT and flow cytometry analysis. DNA fragmentation assay was used to measure cell apoptosis. Cell migration was assessed by wound healing assay. Depletion of OPN gene expression in endometrial cancer cell lines (Hec1A and Ishikawa cells) reproducibly changed their ability of proliferation. Concomitant changes were seen in the expression of OPN binding cell surface receptors, cell cycle-regulatory genes, cell invasion and colony formation nature of the tumor cells. Decreased colonizing potential in the absence of OPN was reversed in the presence of recombinant OPN. Inhibition of anchorage-independent growth was observed in the presence of metabolic inhibitors of the PI3K, Src and integrin signaling cascades, which was ameliorated in the presence of exogenously added OPN. Our result showed the role of OPN in endometrial cancer, in particular on the malignancy-promoting aspects of OPN that may pave way for new approaches to the clinical management of endometrial cancer.

Abstract 226: Platelet Mitochondria Regulate Thrombotic and Inflammatory Function

Introduction: Cells require energy provided by glycolytic and mitochondrial sources. Obesity, a health issue worldwide, affects mitochondrial function. Platelets possess mitochondria although their role in platelet function is unclear. We assessed the hypothesis that platelet mitochondria contribute to cellular processes and the mitochondrial disorder in obesity may also affect platelet functions. Methods: Platelet aggregation and adhesion were assessed to estimate the effects of selective metabolic inhibitors on platelet functions. Mitochondrial ultrastructure was analyzed by transmission electron microscopy in the presence of these inhibitors. C57BL/6J mice were used to investigate the mitochondrial contribution to the effects of high fat diet (HFD) on platelet adhesion. mRNA expression in mouse platelets was measured by RT-PCR. Results: The disruption of the mitochondrial function by complementary inhibitors resulted in a reduction of platelet aggregation to 80.9 ± 7.7% (antimycin A), 77.5 ± 9.5% (oligomycin), 59.1 ± 3.2% (carbonyl cyanide 3-chlorophenylhydrazone, CCCP), and 37.2 ± 4.3% (potassium cyanide); n=3, p &lt;0.0001. Significant morphological changes in platelet mitochondria, including mitochondrial cristae swelling and accumulation of dense masses in the presence of metabolic inhibitors oligomycin and CCCP were observed. A HFD enhanced thrombin-induced platelet adhesion (44.2 ± 8.7% vs normal chow 30.6 ± 1.3%), which was abrogated in the presence of metabolic inhibitors (19.4 ± 7.1% vs normal chow 14.6 ± 5.4% in the presence of oligomycin; 4.5 ± 2.1% vs normal chow 6.6 ± 2.0% in the presence of CCCP; n=7, p &lt;0.001). Mitochondrial, inflammatory, and thrombotic transcripts were all affected in platelets by a HFD. Expression of mitochondrial (mitofusin 2)- and thrombotic (CD41)-related genes were downregulated, while inflammatory-related genes TLR2 and IL1R1 were upregulated in mouse platelets after 3 weeks of a HFD. In conclusion, platelet mitochondria maintain homeostasis and regulate thrombotic and inflammatory function. Mitochondrial dysfunction in obesity affects platelet function, potentially contributing to cardiovascular disease risks associated with the metabolic syndrome.

Mechanisms of Efficient Arsenite Uptake by Arsenic Hyperaccumulator Pteris vittata

Arsenate (AsV) and arsenite (AsIII) are two dominant arsenic species in the environment. While arsenate uptake is via phosphate transporter in plants, including arsenic hyperaccumulator Pteris vittata , AsIII uptake mechanisms by P. vittata are unclear. In this study, we investigated AsIII uptake by P. vittata involving root radial transport from external medium to cortical cells and xylem loading. In the root symplastic solution, AsIII was the predominant species (90-94%) and its concentrations were 1.6-21 times those in the medium. AsIII influx into root symplast followed Michaelis-Menten kinetics with K(m) of 77.7 μM at external AsIII concentrations of 2.6-650 μM. In the presence of metabolic inhibitor 2,4-dinitrophenol (DNP), arsenic concentrations in the root symplast were reduced to the levels lower than in the medium, indicating that a transporter-mediated active process was mainly responsible for AsIII influx into P. vittata roots. Unlike radial transport, AsIII loading into xylem involved both high- and low-affinity systems with K(m) of 8.8 μM and 70.4 μM, respectively. As indicated by the effect of 2,4-DNP, passive diffusion became more important in arsenic loading into xylem at higher external AsIII. The unique AsIII uptake system in P. vittata makes it a valuable model to understand the mechanisms of arsenic hyperaccumulation in the plant kingdom.

Physiological responses to temperature and haeme synthesis modifiers in earthworm Lumbricus terrestris (Annelida: Oligochaeta)

Earthworms (Lumbricus terrestris) acclimated at 2° and 6°C above their average habitat temperature (10°C) had respectively 15 and 40% higher rate of respiration than those at habitat temperature. At 14°C, the rate of respiration and blood hemoglobin (Hb) concentration both increased by ∼60 and 50%, respectively, of the values at habitat temperature. At higher temperatures the rate of respiration and Hb synthesis started decreasing. At 20-23°C, the respiration and Hb concentration decreased respectively by about 85% and 35% of that at 14°C. Decrease in blood Hb concentration at higher temperatures appeared to be due to the lowering of the activity of blood enzyme δ-aminolaevulinic acid dehydratase (ALAD). Exposure of 20-23°C-acclimated pale worms to ALAD inhibitor (lead), lowered the already compromised rate of respiration and blood Hb concentration; while exposure to hexachlorobenzene (HCB, inducer of haeme synthesis) and ferric chloride (enhancer of haeme synthesis) did not overcome the inhibitory effect of high temperature on Hb synthesis. At 20-23°C the affinity of Hb for oxygen also decreased as indicated by the lowering of oxy-Hb (HbO) concentration in blood. The lowering of concentration of blood Hb and its affinity for oxygen may lower the amount of oxygen delivered to cells, which may limit the level of aerobic metabolism (glycolysis, oxidative phosphorylation), as indicated by an increase in blood glucose concentration and a decrease in in vitro activities of mitochondrial electron transport system components (ETS) namely NADH-cytochrome c reductase, succinate dehydrogenase, cytochrome c oxidase, and ATPases. Although the oxygen concentration in air, at sea level, does not decrease significantly from 6° to 20-23°C (lack of hypoxia), lowering of both Hb and HbO concentrations by high temperature may cause significant hypoxemia. The latter may lead to inhibition of the activity of muscle mitochondrial respiratory enzymes (ETS). The resulting inhibition of ATP synthesis and hydrolysis may cause deficit of energy needed for peristalsis/fictive locomotion of body and heart muscles (as indicated by a decrease in heart rate) to facilitate diffusion and transport of gases. The upper critical temperature (20-23°C) also slows down the heart rate and causes hyperosmotic stress (hypovolemia). Thus, a rise in soil temperature above 18°C, which inhibits Hb synthesis, Hb oxygenation, and mitochondrial ETS activity, and slows down the heart rate and causes hyperosmotic stress, can make this and higher temperatures lethal to populations of these earthworms, especially in the presence of metabolic inhibitors and respiratory poisons.

Function and Expression of ATP-Binding Cassette Transporters in Cultured Human Y79 Retinoblastoma Cells

The aim of this study was to reveal the expression and function of P-glycoprotein and multidrug resistance-associated proteins (MRP), members of the ATP-binding cassette (ABC) superfamily of drug transporters, in cultured human Y79 retinoblastoma cells. ABC transporter mRNA expression was evaluated by conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses. Cellular accumulation of rhodamine 123 (P-glycoprotein substrate), calcein (MRP substrate), and doxorubicin (P-glycoprotein/MRP substrate) was analyzed by fluorometry. Conventional RT-PCR analysis showed the expression of multidrug resistance 1 (MDR1), MRP1, MRP2 and lung resistance-related protein (LRP) mRNAs. Real-time RT-PCR analysis revealed that the expression levels of the MDR1 and MRP2 genes in Y79 cells were much lower than those in human intestinal cell line Caco-2, while the expression level of MRP1 was higher than that in Caco-2 cells. The accumulation of rhodamine 123 was not enhanced by verapamil or reversin 205, inhibitors of P-glycoprotein, indicating no function of P-glycoprotein in Y79 cells. The accumulation of calcein was significantly increased by various MRP inhibitors including probenecid, indicating that MRP functions in Y79 cells. The accumulation of doxorubicin was increased in the presence of metabolic inhibitors (10 mM 2-deoxyglucose and 5 mM sodium azide). However, most MRP inhibitors such as probenecid and indomethacin did not affect doxorubicin accumulation, while cyclosporin A and taclorimus significantly increased doxorubicin accumulation. These results suggest that MRP, but not P-glycoprotein, functions in Y79 cells, and that the efflux of doxorubicin from Y79 cells may be due to an ATP-dependent transporter, which has not been identified yet.

Investigations on the motility of human spermatozoa in a defined medium in the presence of metabolic inhibitors and of carnitine.

When washed human sperm were incubated in a modified Krebs-Ringer buffer solution in the absence of exogenous metabolizable substrates at 30 degrees C they maintained progressive motility for at least six hours. Under these conditions the spermatozoa apparently utilize endogenous substrates but addition of exogenous substrates (glucose, fructose, acetate, short-chain fatty acids, branched chain amino acids) did not affect the % progressive motility or % total motility of the cells. The phospholipase inhibitors, quinacrine and Upjohn No. 1002, inhibited progressive motility when added to sperm utilizing endogenous substrate, and subsequent addition of oxidative or glycolytic substrates did not reverse the inhibition. In contrast, the inhibition by KCN of progressive motility based upon utilization of endogenous substrate was reversed upon addition of glycolyzable compounds (glucose or fructose). The addition of carnitine or its acetyl-, propionyl-, isobutyryl-, valeryl- or isovaleryl esters did not consistently affect progressive or total motility of sperm samples. The inhibitor, octylsulfobetaine, inhibited sperm motility at a concentration higher than that required for inhibition of carnitine acetyltransferase or translocase activity. On the basis of these results it does not appear that exogenous carnitine has an effect on the motility of human sperm incubated under the conditions described here.

The use of microelectrode array (MEA) to study the protective effects of potassium channel openers on metabolically compromised HL-1 cardiomyocytes

The microelectrode array (MEA) was used to evaluate the cardioprotective effects of adenosine triphosphate sensitive potassium (KATP) channel activation using potassium channel openers (KCOs) on HL-1 cardiomyocytes subjected to acute chemically induced metabolic inhibition. Beat frequency and extracellular action potential (exAP) amplitude were measured in the presence of metabolic inhibitors (sodium azide (NaN3) or 2-deoxyglucose (2-DG)) or KCOs (pinacidil (PIN, a cyanoguanidine derivative, activates sarcolemmal KATP channels) or SDZ PCO400 (SDZ, a benzopyran derivative, activates mitochondrial KATP channels)). The protective effects of these KCOs on metabolically inhibited HL-1 cells were subsequently investigated. Signal shapes indicated that NaN3 and 2-DG reduced the rate of the sodium (Na+) influx signal as reflected by a reduction in beat frequency. PIN and SDZ appeared to reduce both rate of depolarization and extent of the Na+ influx signals. Pre-treating cardiomyocytes with PIN (0.1 mM), but not SDZ, prevented the reduction of beat frequency associated with NaN3- or 2-DG-induced metabolic inhibition. The exAP amplitude was not affected by either KCO. The cardioprotective effect of PIN relative to SDZ may be due to the opening of different KATP channels. This metabolic inhibition model on the MEA may provide a stable platform for the study of cardiac pathophysiology in the future.

Nonlinear Intestinal Absorption of (1.RAR.3)-.BETA.-D-Glucan Caused by Absorptive and Secretory Transporting System

The mechanism of the nonlinear concentration dependence of intestinal absorption of (1-->3)-beta-D-glucan was studied using in situ rat intestinal perfusion, as well as the in vitro Ussing-type chamber method mounted with rat intestinal tissue. The intestinal absorption rate constant of a (1-->3)-beta-D-glucan, laminaran, evaluated by the loop method increased significantly with increasing concentration of laminaran up to 0.5 muM in a nonlinear fashion and tended to decrease at higher concentrations. Mucosal-to-serosal directed permeation of the laminaran across rat ileal sheets evaluated by the in vitro Ussing-type chamber method also decreased in a dose-dependent fashion. Serosal-to-mucosal directed permeation decreased in a concentration-dependent manner. In addition, the serosal-to-mucosal flux was reduced in the presence of metabolic inhibitor, 2,4-di-nitrophenol. These results suggest that laminaran is secreted into the intestinal lumen predominantly by the efflux transporting system. We conclude that intestinal transport of (1-->3)-beta-D-glucan involves specialized transporter or something similar in both absorptive and secretory directions, and complex nonlinear intestinal absorption characteristics can be ascribed to the participation of multiple transport mechanism.