Further analysis on lidocaine-induced increase in intracellular Zn2+ concentration : Cytometric model study using FluoZin-3, 5-chloromethylfluorescein, and rat thymocytes

Lidocaine augmented FluoZin-3 fluorescence of rat thymocytes, suggesting the increase in intracellular Zn2+ concentration. However, the mechanism for lidocaine-induced increase in the intensity of FluoZin-3 fluorescence was not elucidated. In this study, in order to reveal the mechanism, the effects of lidocaine on FluoZin-3 and 5-chloromethylfluorescein (5-CMF) fluorescence were examined under various conditions. 5-CMF fluorescence was used as a parameter for cellular content of nonprotein thiols. The lidocaine-induced augmentation of FluoZin-3 fluorescence was observed under external Ca2+- and/or Zn2+-free conditions, suggesting that lidocaine induced intracellular Zn2+ release. Lidocaine attenuated 5-CMF fluorescence, suggesting the decrease in cellular content of nonprotein thiols. Since some peptides consisting of SH-group have been proposed to store Zn2+, the decrease in nonprotein thiol content b

Cytotoxic actions of lidocaine at sublethal concentrations : A model in vitro experiment using rat thymocytes

In order to reveal some cytotoxic features of lidocaine, the effects of lidocaine at sublethal concentrations (10 mM or less) on rat thymocytes were examined by the use of flow-cytometric technique with fluorescent probes. Treatment with lidocaine at 3-10 mM for 1-3 hr significantly increased intracellular Ca2+ and Zn2+ concentrations under normal condition. After the removal of external Ca2+ and Zn2+, lidocaine still increased intracellular Ca2+ and Zn2+ concentrations, suggesting the lidocaine-induced release of intracellular Ca2+ and Zn2+. Treatment with lidocaine at 0.3-1 mM for 24 hr significantly increased the populations of dead cells and shrunken cells. Lidocaine at sublethal concentrations exerts some cellular actions that are supposed to be linked to the cytotoxicity. The mechanism of lidocaine-induced cell death varies in time- and concentration-dependent manners

Modification of cell vulnerability to oxidative stress by N-(3-oxododecanoyl)-L-homoserine-lactone, a quorum sensing molecule, in rat thymocytes

N-(3-oxododecanoyl)-L-homoserine-lactone (ODHL), a quorum sensing molecule, affects intracellular Zn2+ concentration ([Zn2+]i) and cellular levels of nonprotein thiols ([NPT]i) of rat thymic lymphocytes, both of which are assumed to affect cell vulnerability to oxidative stress. Therefore, it is interesting to examine the effects of ODHL on the cells under oxidative stress. ODHL augmented the cytotoxicity of H2O2, but not calcium ionophore A23187. ODHL potentiated the H2O2-induced elevation of [Zn2+]i, wherein, it greatly attenuated the H2O2-induced increase in intracellular Ca2+ concentration. ODHL did not affect [NPT]i in the presence of H2O2. Therefore, we conclude that the elevation of [Zn2+]i is involved in the ODHL-induced potentiation of H2O2 cytotoxicity. Our findings suggest that ODHL modifies cell vulnerability to oxidative stress in host cells

High-Throughput Identification and Screening of Novel Methylobacterium Species Using Whole-Cell MALDI-TOF/MS Analysis

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing

ヒト白血病K562細胞でクロトリマゾールにより誘発される細胞死に対するマイクロモル濃度の亜鉛の影響

Our recent study showed that the simultaneous application of clotrimazole with CdCI2 or PbCI2 exerted potent cytotoxic action in rat thymocytes although respective agents were ineffective. It was also the case of ZnCl2 and clotrimazole in preliminary study using rat thymocytes. Since clotrimazole is supposed to be a candidate for anticancer drug, we examined the effects of clotrimazole, ZnCI2, and their combination on human leukemia K562 cells. The combination of clotrimazole and ZnCl2 exerted potent cytotoxic effects on the growth and lethality of K562 cells by presumably modifying the process of cell death. The result suggests the possibility that endogenous Zn2+ may modify the action of clotrimazole

Decrease in intracellular Zn²⁺ level by propranolol : A model experiment using rat thymocytes

Propranolol is a representative β-blocker used for the treatment of cardiovascular diseases. Because the use of propranolol expands for some clinical purposes that are not related to its β-blocking action, it is necessary to further examine cellular actions of propranolol. We revealed that propranolol decreased the intracellular Zn2+ level in rat thymocytes by the use of cytometric technique with FluoZin-3, a fluorescent indicator of intracellular Zn2+. Propranolol decreased the influx of extracellular Zn2+. However, the agent decreased the intracellular Zn2+ level even in the presence of DTPA, a chelator of extracellular Zn2+. Thus, the decrease in intracellular Zn2+ level by propranolol was not due to the decrease in Zn2+ influx by propranolol. Propranolol increased the cellular content of nonprotein thiol that was estimated by the use of 5-chloromethylfluorescein fluorescence, an indicator for non-proteinous thiol. Since non-proteinous thiols can make a complex with Zn2+, propranolol may increase the cellular content of nonprotein thiol, resulting in the decrease in intracellular Zn2+ level. Since the concentrations of propranolol to affect both intracellular Zn2+ level and cellular content of nonprotein thiol are higher than those reported under clinical conditions, it is difficult to emphasize clinical implications from present results

ヒト白血病K562細胞におけるアドリアマイシン作用のクレモフォールELによる修飾

Adriamycin and paclitaxel are simultaneously used for cancer treatment in some cases. The formula of paclitaxel contains cremophor EL as a solvent. Since this solvent exerts diverse biological actions, the modification of adriamycin action by cremophor EL has been studied on human leukemia K562 cells. Cremophor EL did not significantly affect the concentration-response relation for antiproliferative action of adriamycin and the cell cycle changed by adriamycin. However, the induction of morphological change by adriamycin was significantly augmented by cremophor EL. The simultaneous application of cremophor EL increased the intensity of fluorescence from adriamycin trapped inside the cells in a concentration-dependent manner, suggesting an increase in intracellular concentration of adriamycin by cremophor EL. Adriamycin alone at concentrations higher than those to completely inhibit the growth induced morphological change in K562 cells. Therefore, cremophor EL may potentiate some of actions induced by adriamycin when adriamycin and paclitaxel are simultaneously applied

Hyperpolarization by N-(3-oxododecanoyl)-L-homoserine-lactone, a quorum sensing molecule, in rat thymic lymphocytes

To study the adverse effects of N-(3-oxododecanoyl)-L-homoserine-lactone (ODHL), a quorum sensing molecule, on mammalian host cells, its effect on membrane potential was examined in rat thymic lymphocytes using flow cytometric techniques with a voltage-sensitive fluorescent probe. As 3–300 μM ODHL elicited hyperpolarization, it is likely that it increases membrane K+ permeability because hyperpolarization is directly linked to changing K+ gradient across membranes, but not Na+ and Cl- gradients. ODHL did not increase intracellular Ca2+ concentration. ODHL also produced a response in the presence of an intracellular Zn2+ chelator. Thus, it is unlikely that intracellular Ca2+ and Zn2+ are attributed to the response. Quinine, a non-specific K+ channel blocker, greatly reduced hyperpolarization. However, because charybdotoxin, tetraethylammonium chloride, 4-aminopyridine, and glibenclamide did not affect it, it is pharmacologically hypothesized that Ca2+-activated K+ channels, voltage-gated K+ channels, and ATP-sensitive K+ channels are not involved in ODHL-induced hyperpolarization. Although the K+ channels responsible for ODHL-induced hyperpolarization have not been identified, it is suggested that ODHL can elicit hyperpolarization in mammalian host cells, disturbing cellular functions

Nitroprusside increases intracellular Zn2+ concentration without affecting cellular thiol content : A model experiment using rat thymocytes and FluoZin-3

Nitric oxide (NO) is cytotoxic under some conditions although it has physiological roles. It is recently proposed that the cytotoxicity of NO is resulted from its interaction with glutathione and zinc. Since we have revealed that a decrease in cellular content of non-protein thiols, presumably glutathione, induces intracellular Zn2+ release, there is a possibility that the cytotoxicity of nitroprusside, a donor of NO, is resulted from the interaction of NO with cellular thiols, leading to an increase in intracellular Zn2+ concentration. To test the possibility, the effects of nitroprusside on cell lethality, intracellular thiol content, and intracellular Zn2+ concentration were examined in rat thymocytes by using a flow cytometer with propidium iodide and FluoZin-3. Nitroprusside at concentrations of 0.3 mM or more (up to 10 mM) significantly augmented FluoZin-3 fluorescence, indicating an increase in intracellular Zn2+ concentration. It was also the case under external Zn2+-free condition, suggesting nitroprusside-induced release of intracellular Zn2+. However, nitroprusside at 10 mM did not affect cell lethality and cellular thiol content. Thus, it can be concluded that nitroprusside-induced increase in intracellular Zn2+ concentration is not related to its cytotoxicity

Myocardial sympathetic denervation prevents chamber-specific alteration of beta-adrenergic transmembrane signaling in rabbits with heart failure

Objectives.The purpose of this study was to assess the effect of myocardial sympathetic denervation on the chamber-specific alteration of beta-adrenergic signaling in left ventricular failure in rabbits.Background.Local abnormalities in sympathetic nerve terminals, including the neuronal reuptake of norepinephrine, are thought to be responsible for the chamber-specific regulation of beta-adrenergic signaling in heart failure.Methods.Sixteen rabbits were given 6-hydroxydopamine, 25 mg/kg body weight intravenously on days 1 and 2 and 50 mg/kg intravenously on days 7 and 8. Another 16 rabbits received vehicle. Aortic regurgitation was induced in eight of the 6-hydroxydopamine—treated and eight of the vehicle-treated rabbits on day 14. Another eight of the 6-hydroxydopamine—treated and eight of the vehicletreated rabbits underwent a sham operation. The hearts were excised for biochemical analysis on day 21.Results.Hemodynamic characteristics on day 21 showed left ventricular failure in both the aortic regurgitation groups. The plasma norepinephrine concentration on day 21 was higher in both the aortic regurgitation groups than in the sham groups. The beta-adrenoceptor densities and isoproterenol plus 5′guanylylimidodiphosphate-, 5′-guanylylimidodiphosphate- and sodium fluoride-stimulated adenylyl cyclase activities were decreased only in the failing left ventricle of the vehicle-pretreated aortic regurgitation group, but in both ventricles of the 6-hydroxydopamine-pretreated aortic regurgitation group. The basal and forskolin-stimulated adenylyl cyclase activities were similar in both the aortic regurgitation groups and in the sham groups.Conclusions.Sympathetic denervation prevented chamberspecific alterations in beta-adrenergic signaling in acute left ventricular failure. Local loss of sympathetic nerve endings, and especially the defective neuronal norepinephrine reuptake, are likely to be responsible for the chamber-specific alteration of the beta-adrenoceptor-G protein-adenylyl cyclase system in heart failure in rabbits