Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve.
1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization. (+info)
An animal exposure system using ultrasonic nebulizer that generates well controlled aerosols from liquids.
Various aerosol generators have been developed for animal inhalation experiments and the performance tests of measuring instruments and respirators. It has been, however, difficult to generate aerosols from an aqueous solution or suspension keeping the concentration and particle size distribution constant for a long time. Resolving such difficulties, the present study developed an animal exposure system that generates well-controlled and stable aerosols from liquids. The exposure system consists of an aerosol generator using ultrasonic nebulizer, a mixing chamber and an exposure chamber. The validity of this system was confirmed in the generation of NiCl2 and TiO2 aerosol from solution and suspension, respectively. The concentration levels of NiCl2 aerosol were kept at 3.2 mg/m3 and 0.89 mg/m3 for 5 hours with good coefficients of variation (CVs) of 2.5% and 1.7%, respectively. For TiO2 aerosol, the concentration levels of 1.59 mg/m3 and 0.90 mg/m3 were kept for 5 hours with small CVs of 1.3% and 2.0%, respectively. This exposure system could be sufficiently used for inhalation experiments with even high toxic aerosols such as NiCl2 because a momentary high concentration possibly affects results and an extremely stable concentration is required. (+info)
Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.
1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability. (+info)
Structure of the oligonucleotide d(CGTATATACG) as a site-specific complex with nickel ions.
In this paper we explore the application of Ni2+to the crystallization of oligonucleotides. We have determined in this way the structure of a fully alternating (Y-R) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction. This is the first oligonucleotide crystal structure with an alternating 5'-(TA)3-3' central part. Alternating oligonucleotides have a particular interest since they often have a unique structure. In this case the general conformation is B-like with an alternating twist and an end-to-end interaction which involves terminal guanines. The crystal belongs to space group P41212 with a = b = 52.46, c = 101.49 A. This packing imposes a 90 degrees crossing of the symmetry related helices. This is a new way of packing for decamers. The oligonucleotide structure is characterized by the specific association with seven nickel ions, involving the N7 atom of every guanine. One of the Ni2+ions is shared between two guanines of symmetry related molecules. Until now no oligonucleotide has been crystallized in the presence of this metal ion. A novel C.A.T triplet structure has also been tentatively identified. (+info)
Locomotory behaviour of epitheliocytes and fibroblasts on metallic grids.
Behaviour of epitheliocytes and fibroblasts on special discontinuous substrata (metallic grids with square openings of 45x45 microm2) was examined in order to compare the ability of these cells to spread in two mutually perpendicular directions and to stretch over the void spaces. Two cell types with typical fibroblastic morphology, the AGO 1523 line of human foreskin fibroblasts and secondary cultures of mouse embryo fibroblasts, and three cell types with typical epithelial morphology, primary mouse hepatocytes, the IAR-2 line of rat liver cells and the MDCK line of canine kidney epithelial cells (clone 20) were used. We also examined the epitheliocytes (MDCK cells, clone 20) transformed to fibroblast-like morphology by treatment with hepatocyte growth factor/scatter factor (HGF/SF). Time-lapse video microscopy, scanning electron microscopy and immunofluorescence microscopy were used to examine cell reorganizations at various stages of spreading. It was found that early stages of spreading of fibroblasts and epitheliocytes were similar: the cell spread along two bars, perpendicular to each other (bar and crossbar), with the formation of a small triangular lamellar cytoplasm stretched over the opening. Later central parts of the bodies of the fibroblasts retracted from the bars so that the cells remained attached only by their polar lamellae. Successive expansions and partial retractions of these lamellae led to elongation of the cell body crossing several openings of the grid. Epitheliocytes, in contrast to fibroblasts, at the late stages of spreading did not retract their bodies and did not contract polar lamellae. As a result, their central lamellae stretched progressively over the openings. As a result of the treatment of MDCK epitheliocytes with HGF/SF the behaviour of the cells on the grids became similar to that of fibroblasts. It is suggested that these distinct spreading patterns of epitheliocytes and fibroblasts are due to the type-specific differences in the actin-myosin cortex. Experiments with microtubule-specific drugs, colcemid and taxol, indicate that the organization of this cortex is under microtubular control. (+info)
Leucocyte aggregation in subjects with nickel dermatitis.
The effect of nickel sulphate on leuco-aggragation in whole blood buffy coat layers was studied in nickel-sensitive and control subjects. At concentrations of 150 mug and 200 mug nickel sulphate per ml a significant increase in the numbers of leuco-aggregates was noted in the nickel sensitive as compared with the control subjects. (+info)
Effects of nickel and cobalt on kinetics of methanol conversion by methanogenic sludge as assessed by on-line CH4 monitoring.
When metals were added in a pulse mode to methylotrophic-methanogenic biomass, three methane production rate phases were recognized. Increased concentrations of Ni and Co accelerated the initial exponential and final arithmetic increases in the methane production rate and reduced the temporary decrease in the rate. When Ni and Co were added continuously, the temporary decrease phase was eliminated and the exponential production rate increased. We hypothesize that the temporary decrease in the methane production rate and the final arithmetic increase in the methane production rate were due to micronutrient limitations and that the precipitation-dissolution kinetics of metal sulfides may play a key role in the biovailability of these compounds. (+info)
Extracellular heavy-metal ions stimulate Ca2+ mobilization in hepatocytes.
Populations of hepatocytes in primary culture were loaded with fura 2 and the effects of extracellular heavy-metal ions were examined under conditions that allowed changes in fura 2 fluorescence (R340/360, the ratio of fluorescence recorded at 340 and 360 nm) to be directly attributed to changes in cytosolic free [Ca2+] ([Ca2+]i). In Ca2+-free media, Ni2+ [EC50 (concentration causing 50% stimulation) approximately 24+/-9 microM] caused reversible increases in [Ca2+]i that resulted from mobilization of the same intracellular Ca2+ stores as were released by [Arg8]vasopressin. The effects of Ni2+ were not mimicked by increasing the extracellular [Mg2+], by addition of MnCl2, CoCl2 or CdCl2 or by decreasing the extracellular pH from 7.3 to 6.0; nor were they observed in cultures of smooth muscle, endothelial cells or pituitary cells. CuCl2 (80 microM), ZnCl2 (80 microM) and LaCl3 (5 mM) mimicked the ability of Ni2+ to evoke Ca2+ mobilization. The response to La3+ was sustained even in the absence of extracellular Ca2+, probably because La3+ also inhibited Ca2+ extrusion. Although Ni2+ entered hepatocytes, from the extent to which it quenched fura 2 fluorescence the free cytosolic [Ni2+] ([Ni2+]i) was estimated to be <5 nM at the peak of the maximal Ni2+-evoked Ca2+ signals and there was no correlation between [Ni2+]i and the amplitude of the evoked increases in [Ca2+]i. We conclude that extracellular Ni2+, Zn2+, Cu2+ and La3+, but not all heavy-metal ions, evoke an increase in [Ca2+]i in hepatocytes by stimulating release of the hormone-sensitive intracellular Ca2+ stores and that they may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing hepatocytes to contribute to regulation of plasma heavy-metal ions and may mediate responses to Zn2+ released into the portal circulation with insulin. (+info)