Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells

Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba²⁺-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K⁺ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure.     

Visualization of putative ion-transporting epithelia in Amphibalanus amphitrite using correlative microscopy: Potential function in osmoregulation and biomineralization

Thoracican barnacles are a unique suborder of crustaceans typified by their calcified exterior, which provides protection to the sessile juvenile and adult. Biomineralization is mediated by a mantle epithelium that appears to be involved in calcium uptake and the secretion of calcium laden matrix. Larval and adult intertidal Balanomorph barnacles tolerate a wide range of salinities and it is hypothesized that active ion transport is the primary mechanism for osmoregulation. We observed adult Amphibalanus amphitrite producing an electrolyte-rich secretion emanating from the junction of the basis and parietal plates. Further study of this region using silver staining microscopic techniques, verified by scanning electron microscopy-energy dispersive spectroscopy, revealed a chloride ion rich mantle epithelium. A distinctive pattern of silver chloride stained epithelia was revealed in all A. amphitrite life stages. These epithelia were observed to contain mitochondria rich cells in nauplius and cyprid larvae (as shown by DASPMI staining visualized with confocal laser scanning microscopy) and therefore exhibit potential for active ion transport. Rhod-5 N (a low affinity cellular Ca²⁺ indicator) labeling was also observed in all barnacle life stages, in tissues shown to be chloride positive. We suspect that the observed chloride ion rich epithelia facilitate ionic regulation via active transport, and biomineralization via cellular Ca²⁺ uptake, storage and mobilization.     

Selection, Isolation, and Characterization of Cadmium-Resistant Datura innoxia Suspension Cultures

Datura innoxia cells from suspension cultures were selected for their ability to grow and divide rapidly in normally lethal concentrations of cadmium. Cells resistant to 12.5, 25, 50, 100, 160, 200, and 250 micromolar cadmium chloride were isolated and utilized to initiate cell suspension cultures resistant to this toxic metal ion. Variant cell lines retained their ability to grow in cadmium after being grown in its absence for more than 400 generations. Resistance to cadmium was correlated with the synthesis of low molecular weight, cysteine-rich, cadium-binding proteins. Synthesis of these proteins was induced rapidly in cadmium-resistant cells in response to a challenge of cadmium. Induction was detectable within one hour after exposure of the cells to the metal ion. Accumulation of protein bound cadmium reached a maximum eight to twelve hours following exposure. Metal-binding proteins were not detectable in the cadmium sensitive D. innoxia cells from which resistant cells were derived.     

Effects of the venom of the solitary wasp Philanthus triangulum F. on glutamate uptake in the nerve endings of locust muscles—A high resolution autoradiographic study

Using electron microscope autoradiography it was shown that the glutamate uptake in both glia cells and axon in the synaptic region of locust muscles was reduced to ca. 50% under the influence of the venom of the solitary wasp Philanthus triangulum F. However, the ratio of the glutamate accumulation in the glia and the nerves remained identical. Implications are discussed in relation to known postsynaptic effects of the venom of Philanthus triangulum F.     

Loss of Slc4a1b Chloride/Bicarbonate Exchanger Function Protects Mechanosensory Hair Cells from Aminoglycoside Damage in the Zebrafish Mutant persephone

Mechanosensory hair cell death is a leading cause of hearing and balance disorders in the human population. Hair cells are remarkably sensitive to environmental insults such as excessive noise and exposure to some otherwise therapeutic drugs. However, individual responses to damaging agents can vary, in part due to genetic differences. We previously carried out a forward genetic screen using the zebrafish lateral line system to identify mutations that alter the response of larval hair cells to the antibiotic neomycin, one of a class of aminoglycoside compounds that cause hair cell death in humans. The persephone mutation confers resistance to aminoglycosides. 5 dpf homozygous persephone mutants are indistinguishable from wild-type siblings, but differ in their retention of lateral line hair cells upon exposure to neomycin. The mutation in persephone maps to the chloride/bicarbonate exchanger slc4a1b and introduces a single Ser-to-Phe substitution in zSlc4a1b. This mutation prevents delivery of the exchanger to the cell surface and abolishes the ability of the protein to import chloride across the plasma membrane. Loss of function of zSlc4a1b reduces hair cell death caused by exposure to the aminoglycosides neomycin, kanamycin, and gentamicin, and the chemotherapeutic drug cisplatin. Pharmacological block of anion transport with the disulfonic stilbene derivatives DIDS and SITS, or exposure to exogenous bicarbonate, also protects hair cells against damage. Both persephone mutant and DIDS-treated wild-type larvae show reduced uptake of labeled aminoglycosides. persephone mutants also show reduced FM1-43 uptake, indicating a potential impact on mechanotransduction-coupled activity in the mutant. We propose that tight regulation of the ionic environment of sensory hair cells, mediated by zSlc4a1b activity, is critical for their sensitivity to aminoglcyoside antibiotics.     

Role of chloride ion as an allosteric activator of angiotensin-converting enzyme

The nature of chloride ion as an activator of angiotensin-converting enzyme was studied by a series of kinetic experiments with hog plasma enzyme preparation. The enzyme required the presence of chloride ion for its full catalytic activity, but its requirement of monovalent anion was not absolute. The K_A value for the enzymechloride binding was estimated to be about 150 mm in all cases regardless of the peptide substrates employed. In the presence of chloride ion, the activity of the enzyme was increased, but its optimum pH was shifted gradually to the alkaline region up to pH 8.2 depending on the concentration of chloride ion. In addition, in the presence of chloride ion, the apparent K_m values were reduced markedly while the V_max values were not much altered; for example, for the hydrolysis of angiotensin I decapeptide, the K_m value decreased by a factor of 50 while only an 18% increase in V_max was observed when the enzyme was saturated with chloride ion. The result suggests that chloride ion acts as a conformational modifier inducing the affinity of synergistic binding of substrate.     

Effects of lead chloride on human erythrocyte membranes and on kinetic anion sulphate and glutathione concentrations

Our study concerns the effects of exposure to lead chloride on the morphology, K⁺ efflux, SO₄ ^? influx and GSH levels of the human erythrocyte. Blood was collected in heparinized tubes and washed three times. The cells were suspended at 3% hematocrit and incubated for 1 h at 25°C in a medium containing increasing concentrations of lead chloride (0, 0.3, 0.5 and 1 ??M). After incubation, the suspensions were centrifuged and the erythrocyte pellets were divided into three aliquots for testing. The results show: an increase in the permeability of erythrocytes treated with lead chloride with consequent damage and cellular death, especially in the presence of high concentrations; an increase in potassium ion efflux; alterations in the morphology and membrane structure of the red blood cells; and a decrease in sulphate uptake, due either to the oxidative effect of this compound on the band 3 protein, which loses its biological valence as a carrier of sulphate ions, or to a decrease in the ATP erythrocyte concentration. In conclusion, the exposure of erythrocytes to Pb²⁺ ions leads to a reduction in the average lifetime of the erythrocytes and the subsequent development of anemia. These data are discussed in terms of the possible effect of lead on the reduction-oxidation systems of the cell. Oxidant agents, such as lead, are known to cross-link integral membrane proteins, leading to K/Cl-cotransport. The increased K⁺ efflux affects the altered redox state.     

Antigen recognition by T lymphocytes. 3. Evidence for two populations of thymus-dependent rosette-forming cells in spleen and lymph nodes

Rosette-forming cells, present in normal spleen, are composed of 70% theta-positive cells with “high” in vitro sensitivity to Azathioprine (AZ) (1 μg/ml) and 30% theta-negative cells with “low” sensitivity to AZ (500 μg/ml). Theta-positive RFC are also found in the thymus (with “high” sensitivity to AZ) and in lymph nodes and peripheral blood (with “intermediate” sensitivity to AZ:50 μg/ml). RFC with “high” sensitivity to AZ (T1) are eliminated from the spleen by adult thymectomy in less than 6 days; it takes more than 48 hours exposure to antilymphocyte serum (ALS) in vivo to suppress them, whereas RFC with “intermediate” sensitivity to AZ (T2), present in lymph nodes and peripheral blood, disappear 6 hours after in vivo ALS treatment but not after adult thymectomy.     

Osmotic Fragility and Viability of Lysostaphin-induced Staphylococcal Spheroplasts

When Staphylococcus aureus FDA 209P cells were treated with lysostaphin (1 unit/ml) in hypertonic sodium chloride or sucrose environments, viable, osmotically fragile spheroplasts were produced. Turbidimetric studies indicated that 64% (w/v) sucrose or 20 to 28% (w/v) sodium chloride gives maximal protection against lysis of the lysostaphin-treated cells. The NaCl appeared to give greater protection than the sucrose and proved to be much more suitable for viability and related studies. Viability of both shocked and nonshocked treated cells was determined by S. aureus colony counts on agar plates overlayered with the test dilution of the cells suspended in 4 ml of semisolid agar containing 72% sucrose. The difference in the counts represented the number of revertible spheroplasts. Under these conditions, 30 to 50% of the test cells were recovered as osmotically fragile, but revertible, spheroplasts after 5 to 10 min of exposure to lysostaphin in 24% NaCl. This rewere obtained after 5 to 10 min of exposure to lysostaphin in 24% NaCl. This recovery rate fell off rapidly with prolonged exposure. In view of residual turbidity of 30- and even 60-min exposure preparations, it appeared probable that most of the osmotically fragile cells were eventually converted to protoplasts by the prolonged lysostaphin treatment. Osmotically fragile cells were converted to osmotic stability by fixation with 4% (v/v) Formalin.     

Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system

The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO₃⁻ was faster than that of Cl⁻. Phosphate and sulfate crossed the tonoplast only slowly. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p-chloromercuribenzene sulfonate and HgCl₂ stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K_m for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP.     

Lead transport in IEC-6 intestinal epithelial cells

This study evaluated the use of IEC-6 cells as a model for studying lead (Pb) transport by intestinal epithelial cells (IECs) and examined potential transport mechanisms for Pb uptake and extrusion. Pb accumulation in IEC-6 cells exposed to 5 and 10 μM Pb for up to 60 min was time- and dose-dependent. Reduction of incubation temperature significantly reduced the total cellular Pb content of IEC-6 cells. Simultaneous exposure of cells to zinc (Zn) and Pb resulted in decreased total cellular Pb contents compared to total cellular Pb contents of cells exposed to Pb only. IEC-6 cells treated with ouabain (1 mM) or sodium azide (1 mM) and 5 μM Pb accumulated more Pb than cells exposed to Pb only. Cells treated withp-chloromercuribenzensulfonic acid (50 μM),p-chloromercuribenzoic acid (50 μM), or iodoacetimide (50 μM) accumulated less Pb than cells treated with Pb only. We conclude that Pb uptake by IEC-6 cells depends on the extracellular Pb concentration. Our data suggest that the mechanism of Pb uptake by IECs is complex, and that Pb transport in IEC-6 cells is time- and temperature-dependent, involves sulfhydryl groups, and is decreased by the presence of Zn. Extrusion of Pb is at least partially dependent on metabolic energy.     

Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having “neuron-like” morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = ?80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl^? ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.     

Effect on waterhyacinth ofNeochetina eichhorniae [Col.: Curculionidae] combined withOrthogalumna terebrantis [Acari: Galumnidae]

Seven hundred mottled waterhyacinth weevils,Neochetina eichhorniae Warner, were released in a Fort Lauderdale, Florida, canal on a mat of waterhyacinth,Eichhornia crassipes (Mart.) Solms-Laubach. At the time of weevil release, the mat contained ca. 100 adult waterhyacinth mites,Orthogalumna terebrantis Wall-work/plant, and ca. 15 immature waterhyacinth mites/cm². Waterhyacinth weevil populations increased to ca. 4 adults and 5 larvae/plant after 50 weeks, and were unaffected by weather. Waterhyacinth mite populations reached a peak of ca. 840/plant after 16 weeks, at which time they were reduced in number by low temperatures. Decrease in size and density of waterhyacinth closely followed buildup of weevil populations, with petiole length decreasing by 35% and plant density reduced by 45% over a 50 week period. The phytopathogenic fungusAcremonium zonatum (Saw.) Gams., which causes zonate leaf spot disease of waterhyacinth, developed in waterhyacinth mite tunnels after the adult mite created an emergence hole, but was not observed in weevil feeding spots.     

Outward-rectifying chloride channels in cultured adult and fetal human nasal epithelial cells

Summary The patch-clamp technique was used to characterize ion channels in the apical membranes of cultured human nasal epithelial cells, dissociated from fetal nasal mucosa and from adult nasal polyps. Outward-rectifying chloride channels were found in 4.3% of the cell-attached patches from fetal cells (n=258) and in 3.1% of the patches from adult cells (n=320). After exeision the number of patches containing active chloride channels increased threefold to 13% of the patches from the fetal cells and 10% from adult cells. The single-channel conductance at 0 mV in symmetrical 150mm NaCl solutions was 24.3 ±0.9 pS (n=28) and 26.0 ± 1.2 pS (n=30), respectively, in adult and fetal cells and showed outward rectification in the potential range from –80 to +80 mV. In fetal cells as well as in adult cells the channels were anion selective, and were almost impermeable for larger anions and monovalent cations. In cell-free patches the channels were Ca²⁺ independent. In most of the channels the open probability was voltage independent and high (±0.86); in 20% of the channels, however, the open probability increased with depolarization. In conclusion, fetal nasal epithelial cells contain chloride channels in their apical membranes with singlechannel properties and regulatory mechanisms similar to those found in cells from adults.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Cadmium induced changes in subcellular glutathione contents within glandular trichomes of Cucurbita pepo L.

Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca Greb. Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 µM cadmium chloride (CdCl₂) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100?kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.     

Heat and starvation induced hormesis in longevity of Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) adult females

Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) is an important endoparasitoid of Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) larvae and pupae. Previous studies have showed that environmental stress induced hormesis in a variety of organisms. In the present study, we investigated the effects of heat and starvation stress on the survival and induced hormesis in longevity of O. sokolowskii adult females under laboratory conditions. Results showed that temperature and exposure time significantly affected the survival rate of O. sokolowskii adult females with the extreme temperatures and/or longer durations proving to be more lethal. When O. sokolowskii adult females were heat-treated for 0.5, 1, 2, 4, and 8 h, LTemp₅₀ were >50.00, 43.24, 38.82, 38.32, and 38.17 °C, respectively. LTime₅₀ at the threshold temperature of 38 °C was 3.90 h. The sub-lethal temperature exposure for a certain time period reduced the survival numbers, but increased the longevity of survived adult females at the condition of starvation. The exposure for 2 h at 36 and 38 °C reduced the survival numbers by 26.67% and 46.67%, but extended the longevity of the survived adult females by 65.89% and 55.37% in comparison with those in the control, respectively. These results suggest O. sokolowskii adult female has the potential of thermal resistance, and its longevity will increase via heat and starvation treatment.