Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

Mercurial-sensitive water transport in barley roots

An isolated barley root was partitioned into the apical and basal part across the partition wall of the double-chamber osmometer. Transroot water movement was induced by subjecting the apical part to a sorbitol solution, while the basal part with the cut end was in artificial pond water. The rate of transroot osmosis was first low but enhanced by two means, infilitration of roots by pressurization and repetition of osmosis. Both effects acted additively. The radial hydraulic conductivity (Lp_r) was calculated by dividing the initial flow rate with the surface area of the apical part of the root, to which sorbitol was applied, and the osmotic gradient between the apical and basal part of the root. Lp_r which was first 0.02–0.04 pm s⁻¹ Pa⁻¹ increased up to 0.25–0.4 pm s⁻¹ Pa⁻¹ after enhancement. Enhancement is assumed to be caused by an increase of the area of the plasma membrane which is avallable to osmotic water movement. The increased Lp_r is in the same order of magnitude as the hydraulic conductivity (Lp) of epidermal and cortical cells of barley roots obtained by Steudie and Jeschke (1983). HgCl₂, a potent inhibitor of water channels, suppressed Lp_r of non-infiltrated and infiltrated roots down to 17% and 8% of control values, respectively. A high sensitivity of Lp_r to HgCl₂ suggests that water channels constitute the most conductive pathway for osmotic radial water movement in barley roots.     

Sucrose consumption in mice: Major influence of two genetic Loci affecting peripheral sensory responses

Individual variability in sucrose consumption is prominent in humans and other species. To investigate the genetic contribution to this complex behavior, we conducted behavioral, electrophysiological, and genetic studies, using male progeny of two inbred mouse strains (C57BL/6ByJ [B6] and 129/J [129]) and their F₂ hybrids. Two loci on Chromosome (Chr) 4 were responsible for over 50% of the genetic variability in sucrose intake. These loci apparently modulated intake by altering peripheral neural responses to sucrose. One locus affected the response threshold, whereas the other affected the response magnitude. These findings suggest that the majority of difference in sucrose intake between male B6 and 129 mice is due to polymorphisms of two genes that influence receptor or peripheral nervous system activity. Received: 27 January 1997 / Accepted: 17 March 1997     

Adenine-nucleotide levels and metabolism-dependent membrane potential in cells of Nitellopsis obtusa Groves

The relationship between adenine-nucleotide levels and metabolism-dependent membrane potential was studied in cells of Nitellopsis obtusa. Effects of ADP and AMP in the presence of ATP on electrogenic pump activity were measured in the dark, using the continuous perfusion method. Both ADP and AMP acte as competitive inhibitors for ATP, the K_i value for either compound being about 0.4 mM. The role of ADP and AMP as regulating factors for the electrogenic pump was investigated under various metabolic conditions. Application of N₂ gas in the dark caused a significant membrane depolarization amounting to 90 mV, but cytoplasmic streaming and membrane excitability were not affected. Under anoxia, the ATP level decreased from 1.6 to 0.5 mM; ADP increased but only slightly, and AMP increased greatly. However, the time course of changes in the adenine nucleotides was not concurrent with that of the membrane-potential changes, thus, the adenine-nucleotide level changes cannot fully account for the N₂-elicited depolarization. Under light, although the membrane hyperpolarized, no significant changes in the adenine-nucleotide levels were observed. Therefore, the light-induced membrane hyperpolarization cannot be explained solely by changes in adenine-nucleotide levels.Abbreviations APW artificial pond water - E_m membrane potential - R_m membrane resistance     

Control of membrane potential and excitability ofChara cells with ATP and Mg2+

Summmary Electric characteristics of internodalChara australis cells, from which the tonoplast had been removed by vacuolar perfusion with media containing EGTA, were studied in relation to intracellular concentrations of ATP and Mg²⁺ using the ordinary microelectrode method and the open-vacuole method developed by Tazawa, Kikuyama and Nakagawa (1975.Plant Cell Physiol. 16:611). The concentration of ATP was decreased by introducing hexokinase and glucose into the cell and that of Mg²⁺ by introducing EDTA or CyDTA. The membrane potential decrease and the membrane resistance increase were both significant when the ATP or Mg²⁺ concentration was decreased. An ATP-dependent membrane potential was also found in other species of Characeae,Nitella axillaris andN. pulchella. Excitability of the membrane was also completely lost by reducing the ATP or Mg²⁺ concentration. Both membrane potential and excitability were recovered by introducing ATP or Mg²⁺ into ATP- or Mg²⁺-depleted cells.The time course of membrane potential recovery was followed by the open-vacuole method. Recovery began as soon as intracellular perfusion with medium containing ATP and Mg²⁺ was started. Reversible transition of the membrane potential between polarized and pepolarized levels by controlling the intracellular concentration of ATP or Mg²⁺ could be repeated many times by the open-vacuole method, when the excitability was suppressed by addition of Pb²⁺ to the external medium.The ineffectiveness of an ATP analog, AMP-PNP, and the synergism of ATP and Mg²⁺ in maintaining the membrane potential and excitability strongly suggest that ATP act via its hydrolysis by Mg²⁺-activated ATPase. The passive nature of the membrane, as judged from responses of the membrane potential to changes of the external K⁺ concentration, was not altered by lowering the ATP concentration in the cell. The mechanism of membrane potential generation dependent on ATP is discussed on the basic of an electrogenic ion pump. Involvement of the membrane potential generated by the ion pump in the action potential is also discussed.     

Demonstration of light-induced potential change in Chara cells lacking tonoplast

Chara cells without tonoplasts, prepared by replacing the cellsap with EGTA-containing media, showed essentially the samepattern of light-induced changes in membrane potential and membraneresistance as normal cells although the concentrations of ionsand ATP in the cytoplasm decreased considerably (1/3–1/10)after loss of the tonoplast. Removal of the tonoplast reducedthe rate of photosynthetic O₂ evolution to about 50% of thatof normal cells but did not affect the magnitude of light-inducedpotential change. Not a full but a certain level of electronflow seems necessary to activate the putative electrogenic H⁺-pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Japan. ² Present address: Niigata College of Pharmacy, Niigata 950-21,Japan. (Received September 4, 1978; )     

Determination of serum unconjugated estrone, estradiol-17β and estriol during pregnancy by selected ion monitoring

A simple, rapid and highly specific method by selected ion monitoring (SIM), using 9α,11α-[²H₂]estrone, [2,4-²H₂]estradiol-17β and 2,4-[²H₂]estriol as internal standards, was developed for the determination of serum estrogens during pregnancy. Serum samples were submitted to a simple extraction procedure and were analysed after formation of the trifluoroacetic anhydride derivative. The inter-assay coefficients of variation for estrone, estradiol-17β and estriol were 3.73%, 3.42% and 3.49%, respectively. The results obtained by SIM were compared with analysis performed using radioimmunoassay.     

Existence of common homologous elements in the transcriptional regulatory regions of human nuclear genes and mitochondrial gene for the oxidative phosphorylation system.

Our previous study indicated that nuclear protein factors of HeLa cells specifically bind to three nuclear Mt elements, Mt1, Mt3, and Mt4, located in the 5'-flanking regions of the human nuclear genes for cytochrome c1 and for ubiquinone-binding protein, both of which are subunits of mitochondrial cytochrome bc1 complex (Suzuki, H., Hosokawa, Y., Toda, H., Nishikimi, M., and Ozawa, T. (1990) J. Biol. Chem. 265, 8159-8163). In this study, we examined whether the same nuclear factors could recognize a set of the Mt3 and Mt4 elements that were found in the displacement loop and the promoter region of mammalian mitochondrial genomes. Gel retardation experiments disclosed that the same nuclear protein factors specifically bind to those Mt elements in the human mitochondrial genome as well as to the nuclear Mt3 and Mt4 elements of the two genes, and that the coexistence of both the elements is required for the efficient binding. The nuclear protein factors which recognize the Mt elements located in the regulatory regions of the nuclear and mitochondrial genes may play an important role in coordinate expression of the two physically separated genes during mitochondrial biogenesis.     

Anticalmodulin drugs block the sodium gating current of squid giant axons

Summary The effects of calmodulin (CaM) antagonists (W-7, W-5, trifluoperazine, chlorpromazine, quinacrine, diazepam, propericyazine and carmidazolium) on the sodium and potassium channels were studied on the intracellularly perfused and voltage-clamped giant axon of the squid. It was found that the drugs are more potent blockers of the sodium current than of the potassium current. The drugs also reduce the sodium gating current. The blockage of the sodium and gating current can be explained by assuming that the drugs interact with the sodium gating subunit in one of its closed states. The site of action is probably the intracellular surface of the axolemma where presumably a Ca²⁺-calmodulin complex can be formed.     

Construction of shuttle vectors derived fromAcetobacter xylinum for cellulose-producing bacteriumAcetobacter xylinum

Summary Shuttle vector pUF106 was constructed by ligation ofAcetobacter xylinum plasmid pFF6 toEscherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. pUF106 transformed cellulose-producingA. xylinum ATCC10245 as well asE. coli JM109.