Arrhythmia and sudden death associated with elevated cardiac chloride channel activity

The identification and analysis of several cationic ion channels and their associated genes have greatly improved our understanding of the molecular and cellular mechanisms of cardiac arrhythmia. Our objective in this study was to examine the involvement of anionic ion channels in cardiac arrhythmia. We used a transgenic mouse model to overexpress the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride channel. We used RNase protection and in situ hybridization assays to determine the level of CFTR expression, and radiotelemetry and in vivo electrophysiological study in combination with pharmacological intervention to analyse the cardiac function. Cardiac CFTR overexpression leads to stress-related sudden death in this model. In vivo intracardiac electrophysiological studies performed in anaesthetized mice showed no significant differences in baseline conduction parameters including atrial-His bundle (AH) or His bundle-ventricular (HV) conduction intervals, atrioventricular (AV) Wenckebach or 2:1 AV block cycle length and AV nodal functional refractory period. However, following isoproterenol administration, there was marked slowing of conduction parameters, including high-grade AV block in transgenic mice, with non-sustained ventricular tachycardia easily inducible using programmed stimulation or burst pacing. Our sudden death mouse model can be a valuable tool for investigation of the role of chloride channels in arrhythmogenesis and, potentially, for future evaluation of novel anti-arrhythmic therapeutic strategies and pharmacological agents.     

Sodium chloride resistant cell line from haploidDatura innoxia mill

Summary A cell line resistant to sodium chloride was selected from callus cultures of haploidDatura innoxia by cloning under selective pressure. Cells of the resistant cell line retained their resistance even after subculture in absence of NaCl. Plantlets could be regenerated from resistant cells in the presence as well as absence of NaCl. In contrast, regeneration of plantlets was not possible from normal cells in the presence of NaCl, although regeneration readily occurred in the absence of NaCl.To examine the stability of the resistance in the long-term, callus cultures were initiated in presence of NaCl from stem expiants of the differentiated plantlets. All expiants of plantlets derived from resistant cells showed callus formation. This callus, derived from resistant explants, retained the trait of resistance upon subculture.     

Stretch-induced calcium release in smooth muscle

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) release was not due to calcium-induced calcium release, as release was observed in Ca(2+)-free extracellular solution and when free Ca(2+) ions in the cytosol were strongly buffered to prevent increases in [Ca(2+)](i). Stretch-induced calcium release (SICR) was not affected by inhibition of InsP(3)R-mediated Ca(2+) release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca(2+) release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues.     

Gas chromatographic–mass spectrometric determination of serum mexiletine concentration after derivatization with perfluorooctanoyl chloride, a new derivative

Mexiletine is an antiarrhythmic agent used in the treatment of ventricular arrhythmia. The drug has a narrow therapeutic window which necessitates monitoring its serum concentrations. We describe a gas chromatographic–mass spectrometric analysis of mexiletine using selected ion monitoring. Mexiletine was extracted from alkaline serum with dichloromethane and then derivatized with perfluorooctanoyl chloride. The derivatization reaction was completed in 20 min at 80°C. We used N-propylamphetamine as the internal standard. The ions monitored were m/z 122, 454 and 575 for the derivatized mexiletine and m/z 91, 118, 440 and 452 for the derivatized internal standard. The within-run precision at a serum mexiletine concentration of 1 mg/l was 1.9% (mean=0.98, S.D.=0.019 mg/l, n=7) and the between-run precision was 2.5% (mean=0.99, S.D.=0.025 mg/l, n=7). The assay was linear for serum mexiletine concentrations of 0.2 to 4 mg/l. The detection limit was 0.1 mg/l. The average recoveries of mexiletine and the internal standard were 80% and 84%, respectively at a mexiletine concentration of 1 mg/l. There was no carry over problem in our assay. We observed a good correlation between mexiletine concentrations measured by a reference laboratory (GC) and by our new GC–MS assay.     

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris andbis-Tris at millimolar concentrations. HEPES (pK_a=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES _i ) and this effect increased with [HEPES _i ] so that conductance at the reversal potential was diminished 25% with 10mm HEPES _i )and 70% at very high [HEPES _i ]. HEPES _i block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES _i concentration at constant [HEPES^–] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK_a=7.54), substituted glycine buffers (pK_a=8.1–8.4) andbis-Tris (pK_a=6.46) had no measurable effect on conductance and appear suitable for use with this channel.     

Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline

Hydrogen sulfide (H₂S) has long been considered as a phytotoxin, but nowadays as a cell signal molecule involved in growth, development, and the acquisition of stress tolerance in higher plants. In the present study, hydrogen sulfide donor, sodium hydrosulfide (NaHS), pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize under heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and an accumulation of malondialdehyde (MDA) in coleoptiles of maize seedlings. In addition, pretreatment of NaHS could improve the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and lower proline dehydrogenase (ProDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings. Also, application of proline could enhance endogenous proline content, followed by mitigated accumulation of MDA and increased survival percentage of maize seedlings under heat stress. These results suggest that sodium hydrosulfide pretreatment could improve heat tolerance of maize and the acquisition of this heat tolerance may be involved in proline.     

Cross-resistance to antibiotics of Escherichia coli adapted to benzalkonium chloride or exposed to stress-inducers

AIMS: To study the effects of adaptation and stress on the resistance to benzalkonium chloride (BC) and cross-resistance to antibiotics in Escherichia coli. METHODS AND RESULTS: Precultivation of E. coli ATCC 11775 and E. coli DSM 682 in the presence of subinhibitory concentrations of BC or stress inducers (salicylate, chenodeoxycholate and methyl viologen) resulted in higher minimum inhibitory concentration (MIC) of BC and chloramphenicol (CHL). Adaptation to growth in sixfold of the initial MIC of BC resulted in stable BC resistance and enhanced tolerance to several antibiotics and ethidium bromide (EtBr). The MIC of CHL increased more than 10-fold for both strains. Enhanced efflux of EtBr in adapted E. coli ATCC 11775 indicated that the observed resistance was due to efflux. Changes in outer membrane protein profiles were detected in the BC-adapted cells. There were no indications of lower membrane permeability to BC. CONCLUSIONS: Induction of stress response or gradual adaptation to BC or CHL results in acquired cross-tolerance between BC and antibiotics in E. coli. Enhanced efflux was one of the observed differences in adapted cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Provided not taking due precautions, extensive use of disinfectants could lead to emergence of antibiotic-resistant isolates.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

Cultures of bovine tracheal epithelium with differentiated ultrastructure and ion transport

Summary Tracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of ∼ 10 μm). Short-circuit current (I_sc) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with an air interface, cells showed I_sc equal to or greater than the original tissue, possessed cilia, and had a cell height of ∼ 50 μm. Responses in I_sc to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air interface should prove useful in studies of airway epithelial physiology.