Inhibitors Iodoacetic Acid Research Articles

Mechanism of uptake of technetium-tetrofosmin. I: Uptake into isolated adult rat ventricular myocytes and subcellular localization

99mTc-labeled tetrofosmin is a new myocardial imaging agent that gives stable heart uptake. However, little is known about the mechanism of uptake in heart tissue. Uptake of 99mTc-labeled tetrofosmin has been examined in isolated adult rat ventricular myocytes. The time course of uptake, efflux rate, and the effect of metabolic and cation channel inhibitors have been assessed. The subcellular localization of radioactivity in ex vivo rat heart tissue was examined by differential centrifugation of ventricular homogenate. Uptake into rat myocytes was found to be rapid and plateaued at approximately 1.5 pmol/10(6) cells/nmol extracellular Tc-labeled tetrofosmin after 60 minutes of incubation. Uptake was temperature dependent but independent of extracellular Tc-labeled tetrofosmin concentration. Uptake at 30 minutes was inhibited by the metabolic inhibitors iodoacetic acid acid and 2,4-dinitrophenol protein but was not affected by cation channel inhibitors. Cells previously incubated with 99mTc-labeled tetrofosmin and then placed into fresh medium were found to have a slow efflux of activity; after 1 hour, 65% of activity was still cell associated. The localization of radioactivity in subcellular fractions indicated that the majority of activity was recovered with the cytosol. However, examination of the distribution of two mitochondrial enzymes indicated that this may have been artifactual. Use of carbonyl cyanide m-chlorophenyl-hydrazone or oligomycin to perturb mitochondrial membrane potential decreased or increased recovery in the mitochondrial fraction, respectively. 99mTc-labeled tetrofosmin uptake by myocytes is by a metabolism-dependent process that does not involve cation channel transport. The most likely mechanism for this is by potential driven diffusion of the lipophilic cation across the sarcolemmal and mitochondrial membranes.

High molecular weight glycosylated protease in calf brain. Purification and partial characterization.

A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.

Adrenergic control of luminescence and cellular metabolism in Porichthys isolated luminous organs

1. Isolated photophores from the luminous fish Porichthys produce light in response to adrenaline and the metabolic inhibitors iodoacetic acid (IAA) or potassium cyanide (KCN). 2. We attempted to analyse the interactions of cellular metabolism and adrenergic stimulation of the photogenic cells. 3. Photophores were treated with IAA in the presence of pyruvate. In these conditions, IAA does inhibit glycolysis without inducing any luminescent activity of the cells. 4. Similarly, other photophores were incubated with KCN in the presence of glucose, in order to inhibit cellular respiration while keeping the luminous system inactive. 5. We observed that adrenergic stimulation of these photophores remained effective and induced a light emission, demonstrating that glycolytic and oxidative metabolism are not absolutely essential to the mechanism underlying adrenergic activation of the luminous system. 6. The comparison of these luminescences with adrenergic responses of control photophores showed that the light emission to adrenaline was markedly inhibited by glycolysis blockade but potentiated by an inhibition of cellular respiration. 7. As the inhibitory effect of IAA does not result from a direct action of IAA on the luminous system, these results suggest that adrenaline activation of adrenergic receptors might interact with glycolysis in photogenic cells. 8. Glyceraldehyde 3-phosphate, or some derivatives, could be implicated in the glycolytic control of luminescence in the photophores.

Buoyant density changes in the cyanobacterium Microcystis aeruginosa due to changes in the cellular carbohydrate content

The cyanobacterium Microcystis aeruginosa was grown in light-limited chemostat cultures with various light—dark rhythms providing a total periodicity length of 24 h. The buoyant density of the cells changed in parallel with the carbohydrate content. Short incubation experiments with different light intensities, and experiments with the inhibitors iodoacetic acid and arsenate, showed that the buoyant density changes were due to variations in the cellular carbohydrate content. It seems likely that the low dark-growth yield on carbohydrate, which had been stored during the light period, served to facilitate buoyancy changes.

Buoyant density changes in the cyanobacteriumMicrocystis aeruginosadue to changes in the cellular carbohydrate content

The cyanobacterium Microcystis aeruginosa was grown in light-limited chemostat cultures with various light—dark rhythms providing a total periodicity length of 24 h. The buoyant density of the cells changed in parallel with the carbohydrate content. Short incubation experiments with different light intensities, and experiments with the inhibitors iodoacetic acid and arsenate, showed that the buoyant density changes were due to variations in the cellular carbohydrate content. It seems likely that the low dark-growth yield on carbohydrate, which had been stored during the light period, served to facilitate buoyancy changes.

ENDOCYTOSIS OF SALMONELLA TYPHIMURIUM 395 MS AND MR10 BY HELA CELLS

Monolayer of HeLa cells were examined for their ability to endocytose Salmonella typhimurium 395 MS (wild) and MR10 (chemotype RD). Monolayers treated with the glycolytic inhibitors iodoacetic acid (IAA) or N-ethylmaleimide (NEM) or the respiratory inhibitor sodium azide (NaN3) or cytochalasin B (CB) were incubated with S. typhimurium. The numbers of cell-associated (intracellular plus cell-membrane attached extracellular) and intracellular bacteria were determined by viable counts, together with the HeLa cell ATP levels. IAA and NEM at concentrations 10(-4)M and 10(-3)M decreased significantly the number of intracellular MR10 and the cellular ATP levels, but did not influence significantly the total number of cell-associated bacteria except for 10(-3)M IAA which slightly increased the association. On the other hand, NaN3 at concentrations 10(-4)M and 10(-3)M did not affect the number of associated or intracellular bacteria, or the cellular ATP levels. CB at concentrations of 5, 10 and 20 microgram/ml increased the number of associated bacteria, decreased the number of intracellular bacteria and caused a small decrease in cellular ATP levels. Thus, HeLa cells may internalize S. typhimurium by an energy-requiring, glycolysis-dependent process. CB had a dose-dependent inhibitory effect on the internalization without influencing significantly the HeLa cell ATP levels. This indicates that CB might affect the internalization process by some means other than decreasing the ATP content.

Effect of selected Inhibitors and Methylene-Blue on a Possible Phosphogluconate Pathway in Washed Board Sperm

The effects of 2mM iodoacetic acid, ImM sodium fluoride and methylene-blue dye were tested on washed boar sperm. Ten treatments (in duplicate) were used in four trials for each of the metabolic inhibitors iodoacetic acid and sodium fluoride on washed boar spermatozoa. The inhibitor or methylene-blue dye plus .5 μc of either glucose 1-14C or glucose 6-14C was paired with control flasks. Oxygen consumption was lower for both iodoacetate-treated (P<001) and sodium fluoride-treated (P<05) sperm. The pH was not significantly influenced by either inhibitor. Motility was lower (P<.01) for iodoacetic acid treatments but was not significantly different from controls for fluoride-treated samples. Iodoacetic acid-treated sperm produced (P<.01) greater 14CO2 from glucose l-14 C and significantly less 14CO2 from glucose 6-14 C. The phosphogluconate pathway was detected in iodoacetic acid-treated samples. Fluoride-treated sperm did not release more 14C02 from glucose 1-14C, but did (P<.01) decrease 14C02 from glucose 6-14C. No phosphogluconate pathway was distinguishable from data for fluoride treatments. The methylene-blue-treated samples had a higher release of 14 CO2 from glucose 1-14 C compared to glucose 6-14 C (P<.05). Data suggest that a phosphogluconate pathway was activated after treatment of sperm with methylene-blue dye.

THE UPTAKE OF PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OF THE BEECH

Summary The temperature coefficients of phosphate absorption by beech mycorrhizas for temperature intervals below 20°C. were found to be above 2 regardless of the external concentration applied. Similar Q1o values were obtained for their constituent fungal and host tissues when exposed separately to phosphate solutions. The proportion of absorbed phosphate passing to the host through the fungal sheath was unaffected by temperature in very high or very low phosphate concentrations and the Q1o values for absorption by both tissues were of the expected magnitude. By contrast the proportion of phosphate passing to the host from intermediate concentrations (3.2 mM) was increased by low temperatures so that the estimates of Q1o for the host were lower than expected. Similarly, the inhibitors sodium azide and iodoacetic acid diminished uptake by both fungus and host in high and low concentrations of phosphate but did not affect the amount absorbed into the host from 3.2 mM phosphate. In concentrations of 3.2 mM phosphate also, separation of the fungal and host tissue increased their absorption rates over short periods but not over long periods of time. In the discussion, these results are considered together with previous experiments and it is concluded that in ecologically probable conditions phosphate passes to the host through the living hyphae of the sheath.