Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27.
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We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO2 and CdCl2), hypertonic stress (sorbitol and NaCI), or anisomycin, an activator of p38 mitogen-activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)-induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl2 but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase-activated protein (MAPKAP) kinase-2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power. (+info)
Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings.
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1. Simultaneous dendritic and somatic patch-clamp recordings were made from Purkinje cells in cerebellar slices from 12- to 21-day-old rats. Voltage responses to current impulses injected via either the dendritic or the somatic pipette were obtained in the presence of the selective I(h) blocker ZD 7288 and blockers of spontaneous synaptic input. Neurons were filled with biocytin for subsequent morphological reconstruction. 2. Four neurons were reconstructed and converted into detailed compartmental models. The specific membrane capacitance (C(m)), specific membrane resistance (R(m)) and intracellular resistivity (R(i)) were optimized by direct fitting of the model responses to the electrophysiological data from the same cell. Mean values were: C(m), 0.77 +/- 0.17 microF cm(-2) (mean +/- S.D.; range, 0.64-1.00 microF cm(-2)), R(m), 122 +/- 18 kOmega cm(2) (98-141 kOmega cm(2)) and R(i), 115 +/- 20 Omega cm (93-142 Omega cm). 3. The steady-state electrotonic architecture of these cells was compact under the experimental conditions used. However, somatic voltage-clamp recordings of parallel fibre and climbing fibre synaptic currents were substantially filtered and attenuated. 4. The detailed models were compared with a two-compartment model of Purkinje cells. The range of synaptic current kinetics that can be faithfully recorded using somatic voltage clamp is predicted fairly well by the two-compartment model, even though some of its underlying assumptions are violated. 5. A model of I(h) was constructed based on voltage-clamp data, and inserted into the passive compartmental models. Somatic EPSP amplitude was substantially attenuated compared to the amplitude of dendritic EPSPs at their site of generation. However, synaptic efficacy of the same quantal synaptic conductance, as measured by the somatic EPSP amplitude, was only weakly dependent on synaptic location on spiny branchlets. 6. The passive electrotonic structure of Purkinje cells is unusual in that the steady-state architecture is very compact, while voltage transients such as synaptic potentials and action potentials are heavily filtered. (+info)
Cadmium-induced accumulation of putrescine in oat and bean leaves.
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The effects of Cd2+ on putrescine (Put), spermidine (Spd), and spermine (Spm) titers were studied in oat and bean leaves. Treatment with Cd2+ for up to 16 hours in the light or dark resulted in a large increase in Put titer, but had little or no effect on Spd or Spm. The activity of arginine decarboxylase (ADC) followed the pattern of Put accumulation, and experiments with alpha-difluoromethylarginine established that ADC was the enzyme responsible for Put increase. Concentrations of Cd2+ as low as 10 micromolar increased Put titer in oat segments. In bean leaves, there was a Cd(2+)-induced accumulation of Put in the free and soluble conjugated fractions, but not in the insoluble fraction. This suggests a rapid exchange between Put that exists in the free form and Put found in acid soluble conjugate forms. It is concluded that Cd2+ can act like certain other stresses (K+ and Mg2+ deficiency, excess NH4+, low pH, salinity, osmotic stress, wilting) to induce substantial increases in Put in plant cells. (+info)
Cholinergic modulation of stellate cells in the mammalian ventral cochlear nucleus.
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The main source of excitation to the ventral cochlear nucleus (VCN) is from glutamatergic auditory nerve afferents, but the VCN is also innervated by two groups of cholinergic efferents from the ventral nucleus of the trapezoid body. One arises from collaterals of medial olivocochlear efferents, and the other arises from neurons that project solely to the VCN. This study examines the action of cholinergic inputs on stellate cells in the VCN. T stellate cells, which form one of the ascending auditory pathways to the inferior colliculus, and D stellate cells, which inhibit T stellate cells, are distinguished electrophysiologically. Whole-cell recordings from stellate cells in slices of the VCN of mice demonstrate that most T stellate cells are excited by cholinergic agonists through three types of receptors, whereas all D stellate cells tested were insensitive to cholinergic agonists. Nicotinic excitation in T stellate cells has two components. The faster component was blocked by alpha-bungarotoxin and methyllycaconitine, suggesting that receptors contained alpha7 subunits; the slower component was insensitive to both. Muscarinic receptors excite T stellate cells by blocking a voltage-insensitive, "leak" potassium conductance. Our results suggest that cholinergic efferent innervation enhances excitation by sounds of T stellate cells, opposing the inhibitory action of cholinergic innervation in the cochlea that is conveyed indirectly through the glutamatergic afferents. The inhibitory action of D stellate cells on their targets is probably not affected by cholinergic inputs. Excitation of T stellate cells by cholinergic efferents would be expected to enhance the encoding of spectral peaks in noise. (+info)
Aristolochic acid impedes endocytosis and induces DNA adducts in proximal tubule cells.
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BACKGROUND: Aristolochic acid (AA), present in Aristolochia plants, appears to be the toxin responsible for Chinese herbs nephropathy (CHN), a rapidly progressive tubulointerstitial nephritis. One of the earliest sign of CHN is the urinary excretion of low-molecular-weight proteins (LMWP), suggesting that AA is toxic to proximal tubules (PT). METHODS: The effects of AA on PT functions including reabsorption of LMWP were investigated on the well-established opossum kidney (OK) cell line, a model for PT, and compared with those of the classical PT toxin cadmium chloride (CdCl2). RESULTS: OK cell monolayers internalized albumin and beta2-microglobulin by receptor-mediated endocytosis, both proteins apparently competing for the same receptor, a complex of megalin and cubulin. The process was significantly impaired by 24-hour preincubation with AA (10 or 20 micromol/L) or CdCl2 (15 micromol/L). Furthermore, 24-hour exposure to AA followed by its removal during one to six days led to a persistent inhibition of the uptake of albumin, in contrast to the substantial recovery observed after CdCl2 removal. Neither AA nor CdCl2 affected cell viability, Na+-glucose cotransport or total rate of protein synthesis. AA significantly decreased megalin expression and formed specific DNA adducts in OK cells, similar to those found in kidneys from CHN patients. CONCLUSIONS: The present data support the involvement of AA in the early PT dysfunction found in CHN; furthermore, they suggest a causal relationship between DNA adduct formation, decreased megalin expression, and inhibition of receptor-mediated endocytosis of LMWP. (+info)
Cadmium-induced changes in the growth and oxidative metabolism of pea plants.
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The effect of growing pea (Pisum sativum L.) plants with CdCl(2) (0-50 microM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 microM CdCl(2), showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl(2) can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium. (+info)
Zinc-metallothionein levels are correlated with enhanced glucocorticoid responsiveness in mouse cells exposed to ZnCl(2), HgCl(2), and heat shock.
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Metallothioneins (MTs) are the major low molecular weight, zinc-binding proteins in mammalian cells. It has been hypothesized that they play a role in the function of zinc-dependent signal transduction proteins and transcription factors. We investigated the capacity of zinc and other metal ions and conditions to increase both Zn-associated MT levels and the receptiveness of cells to transcriptional activation mediated by the zinc-dependent glucocorticoid receptor (GR). We studied, in a GR-responsive mouse mammary-tumor cell line, the ability of dexamethasone (DEX) to stimulate transcription of a chloramphenicol acetyltransferase (CAT) gene controlled by a mouse mammary-tumor virus promoter. In cells pretreated with 20 to 100 microM ZnCl(2), DEX-induced CAT activity correlated with zinc-induced MT levels. However, 0.05 to 0.5 microM CdCl(2) had no effect on CAT activity, despite an increase in Cd-associated MT. Copper-associated MT was detected in cells treated with 20 microM CuCl(2,) but there was no change in the level of Zn-MT, nor was CAT activity altered in cells exposed to 5 to 20 microM CuCl(2). These results may reflect a functional difference between zinc-associated MT, and MT associated with other metals. Significantly more CAT activity was observed in both heat-shocked cells and in cells exposed to 40 or 50 nM HgCl(2). Although absolute amounts of MT were unchanged by these two treatments, a higher percentage of total cellular zinc was associated with the MT protein fractions after treatment. Changes in GR levels could not account for variations in CAT activity. These data indicate that hormonal signalling can be altered by exposure to metal salts and heat shock, and the effect is correlated with the level of Zn-MT. (+info)
Overexpression of metallothionein-II sensitizes rodent cells to apoptosis induced by DNA cross-linking agent through inhibition of NF-kappa B activation.
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DNA cross-linking agents such as mitomycin C (MMC) and cisplatin are used as chemotherapeutic agents in cancer treatment. However, the molecular mechanism underlying their antitumor activity is not entirely clear. Critical steps in cytotoxicity toward cross-linking agents can involve DNA repair efficiency, inhibition of replication, cell-cycle checkpoints, regulation, and induction of apoptosis. The complexity of the mechanisms of the mammalian cell defense against cross-linking agents is reflected by the existence of many complementation groups identified in rodent cells that are specifically sensitive to MMC. We recently showed that increased induction of apoptosis contributes to the MMC sensitivity of the group represented by the V-H4 hamster mutant cell line. In this study, through the analyses of a substractive library, we discovered that sensitive V-H4 cells display a 40-fold increase of steady-state expression of metallothionein II (MT-II) mRNA compared with resistant parental V79 cells. Down-regulation of MT-II by antisense oligonucleotides partially restores MMC resistance in V-H4 cells, indicating that MT-II overexpression is directly involved in MMC hypersensitivity of these cells. MTs have been reported to regulate the activation of NF-kappaB, one of the key proteins that modulates the apoptotic response. Here we found that NF-kappaB activation by MMC is impaired in V-H4 cells and is partially restored following down-regulation of MT-II by antisense oligonucleotides. All these data suggest that the overexpression of MT-II in V-H4 cells impairs NF-kappaB activation by MMC, resulting in decreased cell survival and enhanced induction of apoptosis. (+info)