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PubMed2000

Comparison of HOCl traps with myeloperoxidase inhibitors in prevention of low density lipoprotein oxidation.

In this study, the production of the highly toxic oxidant hypochlorous acid (HOCl) by the phagocytic enzyme myeloperoxidase (MPO) was quantitated and the concomitant alterations of low density lipoprotein (LDL) were analyzed in view of the potential role of LDL in atherosclerosis. Using the monochlorodimedone assay, it was found that HOCl is produced in micromolar concentrations. The kinetics of the decrease of tryptophan fluorescence appeared to be a sensitive method to monitor LDL alterations under near in vivo conditions. Therefore, this method was used to subsequently compare the effectiveness of MPO inhibitors that block production of HOCl with compounds that act as HOCl traps. The efficiency of MPO inhibitors to prevent LDL damage increased in the series benzohydroxamic acid < salicylhydroxamic acid < 3-amino-1,2,4-triazole < sodium azide < potassium cyanide < p-hydroxy-benzoic acid hydrazide, while for the HOCl traps the protective efficiency increased in the series glycine < taurine < methionine. We conclude that HOCl traps may have high potential therapeutic impact in vivo due to their low toxicity, although high concentrations of them would have to reach sites of inflammation. In contrast, only low concentrations of a specific MPO inhibitor would be required to irreversibly inhibit the enzyme.

Arteriosclerosis↗

PubMed2000

Identification and characterization of a novel hepatic canalicular ATP diphosphohydrolase.

We have identified and characterized a novel ATP diphosphohydrolase (ATPDase) with features of E-type ATPases from porcine liver. Immunoblotting with a specific monoclonal antibody to this ectoenzyme revealed high expression in liver with lesser amounts in kidney and duodenum. This ATPDase was localized by immunohistochemistry to the bile canalicular domain of hepatocytes and to the luminal side of the renal ductular epithelium. In contrast, ATPDase/cd39 was detected in vascular endothelium and smooth muscle in these organs. We purified the putative ATPDase from liver by immunoaffinity techniques and obtained a heavily glycosylated protein with a molecular mass estimated at 75 kDa. This enzyme hydrolyzed all tri- and diphosphonucleosides but not AMP or diadenosine polyphosphates. There was an absolute requirement for divalent cations (Ca(2+) > Mg(2+)). Biochemical activity was unaffected by sodium azide or other inhibitors of ATPases. Kinetic parameters derived from purified preparations of hepatic ATPDase indicated V(max) of 8.5 units/mg of protein with apparent K(m) of 100 microM for both ATP or ADP as substrates. NH(2)-terminal amino acid sequencing revealed near 50% identity with rat liver lysosomal (Ca(2+)-Mg(2+))-ATPase. The different biochemical properties and localization of the hepatic ATPDase suggest pathophysiological functions that are distinct from the vascular ATPDase/cd39.

Adenosine↗

PubMed1998

780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species.

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine irradiation parameters of a 780 nm low power CW diode laser (6.5 mW) leading to enhanced proliferation of cultured normal human keratinocytes (NHK). The possible role of reactive oxygen species (ROS) in this response was evaluated. STUDY DESIGN/MATERIALS AND METHODS: NHK were exposed to a single dose of 0 to 3.6 J/cm2 (0-180 sec) of irradiation. Proliferation parameters studied were: incorporation of 3H-thymidine during 6-24 hr following irradiation; percentage of dividing cells and number of cells, 24 hr and 48 hr following irradiation, respectively. RESULTS: Proliferation of NHK exposed to 0.45-0.95 J/cm2 was significantly enhanced by 1.3-1.9-folds relative to sham-irradiated controls, as inferred from parameters studied. Exposure to other energy densities was considerably less effective in enhancing proliferation parameters. Added enzymatic antioxidants, superoxide dismutase or catalase, scavenging superoxide anions and H2O2, suppressed this enhanced proliferation. Added scavengers (alpha-tocopherol acetate, scavenging lipid peroxidation, or sodium azide, histidine, mannitol, scavenging singlet oxygen, superoxide anions, and hydroxyl radicals, respectively), or N-acetyl cysteine, the thiol-reducing agent, suppressed the response, but to different extents. CONCLUSIONS: The results indicate that 780 nm low power diode laser irradiation enhanced keratinocytes proliferation in vitro, with an apparent involvement of ROS in this response, and comparably, might be used to promote their proliferation in vivo to enhance wound healing.

Acetates↗

PubMed2002

Triggering of insulin release by a combination of cAMP signal and nutrients: an ATP-sensitive K+ channel-independent phenomenon.

Nutrient augmentation of Ca(2+)-triggered insulin release occurs in an ATP-sensitive K(+) (K(ATP)) channel--independent manner. Here, using rat islets, we explored the possibility of the K(ATP) channel-independent nutrient triggering of insulin release. In the presence of 250 micromol/l diazoxide, simultaneous application of forskolin and 16.7 mmol/l glucose strongly stimulated insulin release: fourfold and eightfold increases with 1 and 30 micromol/l forskolin, respectively. alpha-Ketoisocaproate (KIC) and 3-isobutylmethylxanthine (IBMX) could be used in place of glucose and forskolin, respectively, to trigger insulin release in the presence of diazoxide. Triggering of insulin release by a combination of nutrients and forskolin was not attenuated by 10 micromol/l nifedipine (a blocker of voltage-dependent Ca(2+) channels) and 2 micromol/l thapsigargin (an inhibitor of intracellular Ca(2+)-ATPase), ascertaining independence of this phenomenon from Ca(2+) entry and from intracellular Ca(2+) liberation. As anticipated, the action of glucose and KIC was greatly (>80%) suppressed by inhibition of mitochondrial metabolism by 2 mmol/l sodium azide (NaN(3)). A combination of palmitate and dimethyl glutamate (a cell-permeable glutamate donor), but not either one alone, weakly but unequivocally triggered insulin release when applied simultaneously with forskolin. In this case, however, mitochondrial poisoning by azide was without effect. The finding suggests that a combination of induced palmitoylation and cytosolic glutamate accumulation partially reconstituted signaling beyond mitochondrial metabolism in the beta-cell upon glucose stimulation. In conclusion, a combination of cAMP signal and nutrients potently triggers insulin release under full activation of the K(ATP) channel, indicating the multiplicity of driving force for insulin exocytosis.

1-Methyl-3-isobutylxanthine↗

PubMed1970

Recovery of viability and radiation resistance by heat-injured conidia of Penicillium expansum Lk. ex Thom.

Spores heated in water at 54 C for up to 1 hr were plated on nutrient agar immediately or held for 3 days in aerated water at 23 C and then plated. Under these conditions, holding was optimal for recovery, increasing survival percentage up to 20-fold over values for immediate plating. Recovery was prevented partially or completely, however, when spores were held in any of the following solutions: glucose, potassium phosphate, ammonium or sodium acetate, sodium azide, or 2,4-dinitrophenol, or in the sodium or potassium salts of pyruvate, and tricarboxylic acid cycle acids. Both anaerobiosis and incubation at 0 C prevented recovery. Survivors of a heat treatment were more sensitive to gamma radiation than were unheated spores. Conditions which affected the recovery of viability had the same effect on restoration of radiation resistance. Thus, many of the processes for restoration of radiation resistance seem involved also in recovery of viability after heating. After a 99% inactivating treatment (about 30 min at 54 C), heated spores respired as fast as unheated spores, or faster. Malate, citrate, succinate, and acetate stimulated respiration in unheated spores and inhibited it in heated spores.

Acetates↗

PubMed2002

Transcriptional profiling identifies two members of the ATP-binding cassette transporter superfamily required for sterol uptake in yeast.

In contrast to lipoprotein-mediated sterol uptake, free sterol influx by eukaryotic cells is poorly understood. To identify components of non-lipoprotein-mediated sterol uptake, we utilized strains of Saccharomyces cerevisiae that accumulate exogenous sterol due to a neomorphic mutation in the transcription factor, UPC2. Two congenic upc2-1 strains, differing quantitatively in aerobic sterol uptake due to a modifying mutation in the HAP1 transcription factor, were compared using DNA microarrays. We identified 9 genes as responsive to UPC2 that were also induced under anaerobiosis, when sterol uptake is essential. Deletion mutants in these genes were assessed for sterol influx in the upc2-1 background. UPC2 itself was up-regulated under these conditions and was required for aerobic sterol influx. Deletion of the ATP-binding cassette transporters YOR011w (AUS1) or PDR11, or a putative cell wall protein encoded by DAN1, significantly reduced sterol influx. Sodium azide and vanadate inhibited sterol uptake, consistent with the participation of ATP-binding cassette transporters. We hypothesized that the physiological role of Aus1p and Pdr11p is to mediate sterol uptake when sterol biosynthesis is compromised. Accordingly, expression of AUS1 or PDR11 was required for anaerobic growth and sterol uptake. We proposed similar molecules may be important components of sterol uptake in all eukaryotes.

ATP-Binding Cassette Transporters↗

PubMed2004

Effects of physiologic challenge on the ADC of intracellular water in the Xenopus oocyte.

The biophysical determinants of the intracellular water apparent diffusion coefficient (ADC) in mammalian tissues are poorly understood. Model systems that are more amenable to physical measurements may provide insights into the behavior of more complex systems. Toward that end, we used MRI to evaluate the effects of altered microtubule concentration, nuclear breakdown, and ATP depletion on intracellular water ADC in the Xenopus oocyte. Water ADC did not change in response to polymerization of microtubules with taxol or depolymerization with nocodazole. Water ADC did not change following the breakdown of the nucleus in healthy cells. Short-term depletion of ATP (approximately 20% of normal levels following 4 hr of exposure to sodium azide and 2-deoxy-D-glucose) was not associated with a change in intracellular ADC. Long-term depletion of ATP (approximately 20% of normal levels following 2 days of exposure to antimycin A) was associated with a significant decrease in intracellular water ADC. These findings suggest that intracellular water diffusion in oocytes is not dependent on the state of microtubule polymerization or short-term ATP depletion, although long-term ATP depletion is associated with changes that lead to a decrease in intracellular water ADC.

Adenosine Triphosphate↗

PubMed2003

A single-step procedure for the synthesis of photoreactive and radioactive glycerolipids.

A single-step procedure was developed for the incorporation of iodoazide into oleic acid, triolein, and phosphatidylcholine. Iodoazide was generated using [125I]iodomonochloride and sodium azide that was found to add stereospecifically in a variety of olefins. Photoreactive and radiolabeled triacylglycerol and phosphatidylcholine were synthesized with a moderate yield and high specific activity. The stability of both the radiolabel and the photoreactive group was studied under ultraviolet light under aqueous as well as anhydrous conditions. These synthesized analogs act as substrates in the dark, and as irreversible inhibitors under ultraviolet irradiation for the target hydrolytic enzymes. The synthesized radiolabeled photoprobes were subsequently used to label lipase and phospholipase A(2). The results highlighted the efficiency and rapidity of the method and its potential application in the study of lipid-metabolizing enzymes.

Chlorides↗

PubMed1998

Human in vivo and in vitro hydroquinone topical bioavailability, metabolism, and disposition.

Hydroquinone is a ubiquitous chemical readily available as monographed in cosmetic and nonprescription forms for skin lightening, and is an important industrial chemical. The in vivo bioavailability for 24-h application in humans was 45.3+/-11.2% of dose from a 2% cream formulation containing [14C]hydroquinone, with the majority of radioactivity excreted in the first 24 h. Timed skin wash and skin tape-stripping sequences showed a rapid and continuous movement of hydroquinone into the stratum corneum of human volunteers. Plasma levels taken both ipsilateral and contralateral to the topical dosing site contained radioactivity at the first 0.5-h sampling time. Peak plasma radioactivity was at 4 h in the 8-h blood sampling period. In vitro percutaneous absorption with fresh viable human skin gave a bioavailability of 43.3% of dose, and flux was calculated at 2.85 microg/cm2/h. In vitro, some of the skin samples were pretreated with the metabolic inhibitor sodium azide, which had no effect on percutaneous absorption. Receptor fluid accumulations and 24-h skin samples were extracted and the extracts subjected to thin-layer chromatography (TLC). Control [14C]hydroquinone extraction and TLC had one radioactivity peak, hydroquinone. Receptor fluid and skin extraction had a second peak with the same Rf as benzoquinone, which was decreased with azide treatment. No other peaks were found. Ethyl acetate extraction of urine from the in vivo study showed all radioactivity to be only water-soluble, free hydroquinone released following glucuronidase treatment. Risk assessment should not only involve the bioavailability of intact topical hydroquinone, but also consider phase I and phase II metabolism in both humans and any animal for which toxicity potential was assessed.

Administration, Topical↗

PubMed2004

Cell membrane fluidity changes and membrane undulations observed using a laser scattering technique.

Local transversal micromotions of cell membranes have been reported. These may provide the physical basis for changes in membrane fluidity. In this study, tissue scattering properties are used to measure the magnitude of transversal micromotions under varying stimuli. Laser light is directed at the cell surface at a low incident angle of 12 degrees so that reflected or refracted light does not enter the microscope objective. Scattered light showed strong low-frequency intensity fluctuations with random-walk behavior. Fluctuations were quantified by the median coefficient of variation over the entire observed cell area. Incubation of the cells with protein crosslinkers (paraformaldehyde) and metabolic suppressors (sodium azide) suppressed these fluctuations by 62 and 44%, respectively. The application of hypoosmotic media caused an increase of fluctuation magnitude by 23% (p < 0.005). Agents that increase membrane fluidity (2% ethanol and xenon) increased fluctuation magnitude by 15 and 31%, respectively (p < 0.05). Cessation of the ethanol and xenon exposure led to partial recovery of the fluctuation magnitude, which was nonsignificant for ethanol. This study shows a strong link between membrane fluidity and transversal membrane undulations and provides an important step in the understanding of the mechanosensing function of the cell membrane.

Cell Line, Tumor↗

PubMed2004

Overexpression of short heterodimer partner recovers impaired glucose-stimulated insulin secretion of pancreatic beta-cells overexpressing UCP2.

The short heterodimer partner (SHP) (NR0B2) is an orphan nuclear receptor whose function in pancreatic beta-cells is unclear. Mitochondrial uncoupling protein (UCP2) in beta-cells is upregulated in obesity-related diabetes, causing impaired glucose-stimulated insulin secretion (GSIS). We investigated whether SHP plays a role in UCP2-induced GSIS impairment. We overexpressed SHP in normal islet cells and in islet cells overexpressing UCP2 by an adenovirus-mediated infection technique. We found that SHP overexpression enhanced GSIS in normal islets, and restored GSIS in UCP2-overexpressing islets. SHP overexpression increased the glucose sensitivity of ATP-sensitive K+ (KATP) channels and enhanced the ATP/ADP ratio. A peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist, GW9662, did not block the SHP effect on GSIS. SHP overexpression also corrected the impaired sensitivity of UCP2-overexpressing beta-cells to methylpyruvate, another energy fuel that bypasses glycolysis and directly enters the Krebs cycle. KATP channel inhibition mediated by dihydroxyacetone, which gives reducing equivalents directly to complex II of the electron transport system, was similar in Ad-Null-, Ad-UCP2- and Ad-UCP2+Ad-SHP-infected cells. The mitochondrial metabolic inhibitor sodium azide totally blocked the effect of SHP overexpression on GSIS. These results suggest that SHP positively regulates GSIS in beta-cells and restores glucose sensitivity in UCP2-overexpressing beta-cells by enhancing mitochondrial glucose metabolism, independent of PPARgamma activation.

Adenosine Diphosphate↗

PubMed1999

BPD-MA-mediated photosensitization in vitro and in vivo: cellular adhesion and beta1 integrin expression in ovarian cancer cells.

Benzoporphyrin derivative monoacid (BPD-MA) photosensitization was examined for its effects on cellular adhesion of a human ovarian cancer cell line, OVCAR 3, to extracellular matrix (ECM) components. Mild BPD-MA photosensitization (approximately 85% cell survival) of OVCAR 3 transiently decreased adhesion to collagen IV, fibronectin, laminin and vitronectin to a greater extent than could be attributed to cell death. The loss in adhesiveness was accompanied by a loss of beta1 integrin-containing focal adhesion plaques (FAPs), although beta1 subunits were still recognized by monoclonal antibody directed against human beta1 subunits. In vivo BPD-MA photosensitization decreased OVCAR 3 adhesiveness as well. Photosensitized adhesion was reduced in the presence of sodium azide and enhanced in deuterium oxide, suggesting mediation by singlet oxygen. Co-localization studies of BPD-MA and Rhodamine 123 showed that the photosensitizer was largely mitochondrial, but also exhibited extramitochondrial, intracellullar, diffuse cytosolic fluorescence. Taken together, these data show that intracellular damage mediated by BPD-PDT remote from the FAP site can affect cellular-ECM interactions and result in loss of FAP formation. This may have an impact on long-term effects of photodynamic therapy. The topic merits further investigation.

Animals↗

PubMed2002

Characterization and cytochemical localization of an ATP diphosphohydrolase from Leishmania amazonensis promastigotes.

An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.

Adenosine Diphosphate↗

PubMed2003

Polarised transport of monocarboxylic acid type drugs across rat jejunum in vitro: the effect of mucolysis and ATP-depletion.

The transport characteristics of monocarboxylic acid type drugs (ketoprofen, ibuprofen and gemfibrozil) across the excised jejunal segments and artificial (octanol impregnated) membrane in side-by-side diffusion cells were studied. All three model drugs permeated faster across the intestinal tissue in the mucosal-to-serosal direction than in the opposite direction. No polarised transport of tested drugs was observed when the mucosal side of the intestine was treated with mucus disrupting agent, L-cysteine 1% (w/v), which significantly increased the microclimate pH at the mucosal surface of the intestine. Similar effects on the transport characteristics of model drugs and microclimate pH were observed when metabolic inhibitor, sodium azide (10mM), was present in the incubation medium. Furthermore, the direction of proton gradient across the artificial membrane was shown to significantly influence the transport of model drugs across this membrane. The results of this study indicate that the inwardly directed proton gradient maintained by the acidic microclimate pH at the intestinal surface could be considered as a driving force for the transport of monocarboxylic acid type drugs across the intestinal epithelia and could explain rapid absorption of these drugs after oral application.

Adenosine Triphosphate↗

PubMed1999

Photo-oxidative killing of human colonic cancer cells using indocyanine green and infrared light.

Despite of the approval of Photofrin in various countries, chemically defined sensitizers for photodynamic therapy (PDT) are still needed for the absorption of light in the infrared spectrum, which provides a maximal penetration of light into tissue. Therefore, both the efficacy and the mechanism of action of the clinically approved dye indocyanine green (ICG) and laser irradiation were investigated in vitro. For the investigation of phototoxic effects, HT-29 cells were incubated 24 h prior to irradiation by using different concentrations of ICG (10-500 microM). In each experiment, cells were irradiated using a continuous wave (cw)-diode laser (lambda(ex) = 805 nm, 30 J cm(-2), 40 mW cm(-2)). After laser irradiation, cell viability of dark control and of cells incubated with 500 microM ICG was 1.27+/-0.11 or 0.28+/-0.05 respectively. Using 100 microM ICG and D2O, cell viability was further decreased from 0.46+/-0.03 (H2O) to 0.11+/-0.01 (D2O). Using D2O and 100 microM ICG, the concentration of malondialdehyde, a marker of lipid peroxidation, increased from 0.89+/-0.10 nmol 10(-6) cells to 11.14+/-0.11 nmol 10(-6) cells. Using 100 microM ICG and laser irradiation sodium azide or histidine (50 mM), quenchers of singlet oxygen reduced the cell killing significantly. In contrast, when using mannitol, a quencher of superoxide anion and hydroxyl radical, cell killing was not inhibited. According to the present results, photoactivated ICG seems to kill colonic cancer cells due to the generation of singlet oxygen and the subsequent formation of lipid peroxides. Therefore, ICG might present a promising photosensitizer for PDT; first clinical results confirm these findings.

Cell Death↗

PubMed2006

Biosynthetic requirements for the repair of sublethal membrane damage in Escherichia coli cells after pulsed electric fields.

AIMS: The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Escherichia coli cells after exposure to pulsed electric fields (PEF). METHODS AND RESULTS: The partial loss of the barrier and homeostatic functions of the cytoplasmic membrane was examined by adding sodium chloride to the recovery media. More than 4 log10 cycles of survivors were sublethally injured after PEF. Repair of such sublethal membrane damages occurred when survivors to PEF were incubated in peptone water for 2 h. Two different types of sublethally injured cells were detected. Whereas a small proportion (<5%) repaired after PEF in less than 2 min, the repair of the remaining 95% injured cells lasted 2 h and was dependent on biosynthetic requirements. The addition of inhibitors such as chloramphenicol, cerulenin, penicillin G, rifampicin and sodium azide to the liquid repair medium showed that the repair required energy and lipid synthesis, and was not dependent on protein, peptidoglican or RNA synthesis. CONCLUSIONS: Cell survival after PEF is dependent on the repair of the cytoplasmic membrane. Requirement of lipid synthesis for the repair of sublethally injured cells confirms that the cytoplasmic membrane is a target directly involved in the mechanism of inactivation by PEF. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the damages inflicted by PEF might help in the design of more efficient treatments.

Anti-Bacterial Agents↗

PubMed2006

Critical role of 5'-AMP-activated protein kinase in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.

5'-AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptation under conditions of ATP depletion, such as hypoxia and inhibition of oxidative phosphorylation. Whether activation of AMPK is critical for stimulation of glucose transport in response to inhibition of oxidative phosphorylation is unknown. Here we found that treatment of Glut1-expressing Clone 9 cells with sodium azide (5 mM for 2 h) or the AMPK activator 5'-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 2 mM for 2 h) stimulated the rate of glucose transport by two- to fourfold. Use of small interference RNA (siRNA) directed against AMPKalpha(1) or AMPKalpha(1) + AMPKalpha(2) (total AMPKalpha) resulted in a significant inhibition of the glucose transport response and the content of phosphorylated AMPKalpha(1) + phosphorylated AMPKalpha(2) (total p-AMPKalpha) and phosphorylated acetyl-CoA carboxylase (p-ACC) in response to azide. Transfection with siRNA directed against AMPKalpha(2) did not affect the glucose transport response. The efficacy of transfection with siRNAs in reducing AMPK content was confirmed by Western blotting. Incubation of cells with compound C, an inhibitor of AMPK, abrogated the glucose transport response and abolished the increase in total p-AMPK in azide-treated or hypoxia-exposed cells. Simultaneous exposure to azide and AICAR did not augment the rate of transport in response to AICAR alone. There was no evidence of coimmunoprecipitation of total p-AMPKalpha with Glut1. However, LKB1 was associated with total p-AMPKalpha. We conclude that activation of AMPK plays both a sufficient and a necessary role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.

AMP-Activated Protein Kinase Kinases↗

PubMed1999

Mitochondria present in excised patches from pancreatic B-cells may form microcompartments with ATP-dependent potassium channels.

Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K(ATP)) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or alpha-ketoisocaproic acid (10 mM) reduced the open probability of K(ATP) channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K(ATP) channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K(ATP) channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K(ATP) channels thus forming functional microcompartments.

Adenosine Triphosphate↗