Activation of calcium-sensitive potassium channels in L6 skeletal myocytes by arginine vasopressin requires extracellular calcium

The effects of extracellular application of arginine vasopressin (AVP) upon membrane currents in L6 skeletal myocytes was investigated using the whole-cell configuration of the patch-clamp technique. At O mV AVP produced large amplitude, transient outward currents that reversed when the clamping potential was changed to -100 mV (negative to EK) The effects of alterations in the extracellular K+ concentration upon the current reversal potential suggested that the current elicited by AVP was carried mainly by K+ ions. Intracellular dialysis with 10 μM inositol 1,4,5-trisphosphate (InsP3) elicited similar currents but only in 6/14 cells. Inclusion of 5 mg ml-1 heparin in the intracellular solutions was ineffective at inhibiting the current responses to AVP. The AVP-induced current was totally abolished when the intracellular EGTA concentration was increased from 0.05 mM to 10 mM or Ca2+ was removed from the extracellular perfusing solution. These results suggest that AVP produces activation of a Ca2+-sensitive K+ conductance in L6 skeletal myocytes by a process dependent upon extracellular Ca2+ and not intracellular Ca2+ release. © 1995 Academic Press. All rights reserved.

Mechanisms of ion channel activation in primary cultured and clonal cell lines

The effects of hypotonic shock upon membrane C1 permeability of ROS 17/2.8 osteoblast-like cells was investigated using the patch-clamp technique. Hypotonic shock produced cell swelling that was accompanied by large amplitude, outwardly rectifying, currents that were active across the entire physiological range of membrane potentials (-80 to +100 mV). At strong depolarisations (> +50 mV) the currents exhibited time-dependent inactivation that followed a monoexponential time course. The currents were anion selective and exhibited a selectivity sequence of SCN- > I > Br- > Cl- > F- > gluconate. Current activation was unaffected by inhibitors of protein kinase (A (H-89) and tyrosine kinase (tyrphostin A25), and could not be mimicked by elevation of intracellular Ca2+ or activation of protein kinase C. Similarly, disruption of actin filaments by dihydrocytochalsin B, or generation of membrane tension by dipyridamole failed to elicit significant increases in cell chloride permeability. The mechanism of current activation is as yet undetermined. The currents were effectively inhibited by the chloride channel inhibitors NPPB and DIDS but resistant to DPC. A Cl- conductance with similar characteristics was found to be present in mouse primary cultured calvarial osteoblasts. The volume-sensitive Cl- current in ROS 17/2.8 cells was inhibited by arachidonic acid in two distinct phases. A rapid block that developed within 10 s, preceding a slower developing inhibitory phase that occurred approximately 90 s after onset of arachidonate superfusion. Arachidonic acid also induced kinetic modifications of the current which were evident as an acceleration of the time-dependent· inactivation exhibited at depolarised potentials. Inhibitors of cyclo-oxygenases, lipoxygenases and cytochrome P-4S0 were ineffectual against arachidonic acid's effects sugtgesting that arachidonic acid may elicit it's effects directly. Measurements of cell volume under hypotonic conditions showed that ROS 17/2,8 cells could effectively regulate their volume, However, effective inhibitors of the volume-sensitive CI" current drastically impaired this response suggesting that physiologically this current may have a vital role in cell volume regulation, In L6 skeletal myocytes, vasopressin was found to rapidiy hyperpolarise cells. This appears to occur as the result of activation of Ca2+ -sensitive K+ channels in a process dependent upon the presence of extracellular Ca2+.

Nucleoside diphosphate kinase--a component of the [Na+1]- and [Cl-]-sensitive phosphorylation cascade in human and murine airway epithelium

We have shown that proteins within apically enriched fractions of human nasal respiratory epithelium vary their phosphohistidine content with ambient [Cl-] and other anion concentrations. This membrane-delimited phosphorylation cascade includes a multifunctional protein histidine kinase - nucleoside diphosphate kinase (NDPK). NDPK is itself a cascade component in both human and ovine airway, the self-phosphorylation of which is inhibited selectively by [Na+] in the presence of ATP (but not GTP). These findings led us to propose the existence of a dual anion-/cation-controlled phosphorylation-based "sensor" bound to the apical membrane. The present study showed that this cascade uses ATP to phosphorylate a group of proteins above 45 kDa (p45-group, identities unknown). Additionally, the Cl- dependence of ATP (but not GTP) phosphorylation is conditional on phosphatase activity and that interactions exist between the ATP- and GTP-phosphorylated components of the cascade under Cl--free conditions. As a prelude to studies in cystic fibrosis (CF) mice, we showed in the present study that NDPK is present and functionally active in normal murine airway. Since NDPK is essential for UTP synthesis and regulates fetal gut development, G proteins, K+channels, neutrophil-mediated inflammation and pancreatic secretion, the presence of ion-regulated NDPK protein in mouse airway epithelium might aid understanding of the pathogenesis of CF.

Role of epithelial Na+ channels in endothelial function

An increasing number of mechano-sensitive ion channels in endothelial cells have been identified in response to blood flow and hydrostatic pressure. However, how these channels respond to flow under different physiological and pathological conditions remains unknown. Our results show that epithelial Na+ channels (ENaCs) colocalize with hemeoxygenase-1 (HO-1) and hemeoxygenase-2 (HO-2) within the caveolae on the apical membrane of endothelial cells and are sensitive to stretch pressure and shear stress. ENaCs exhibited low levels of activity until their physiological environment was changed; in this case, the upregulation of HO-1, which in turn facilitated heme degradation and hence increased the carbon monoxide (CO) generation. CO potently increased the bioactivity of ENaCs, releasing the channel from inhibition. Endothelial cells responded to shear stress by increasing the Na+ influx rate. Elevation of intracellular Na+ concentration hampered the transportation of l-arginine, resulting in impaired nitric oxide (NO) generation. Our data suggest that ENaCs that are endogenous to human endothelial cells are mechano-sensitive. Persistent activation of ENaCs could inevitably lead to endothelium dysfunction and even vascular diseases such as atherosclerosis.

Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis

Objective-We previously demonstrated that upregulation of intermediate-conductance Ca2+ -activated K+ channels (KCa 3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of KCa3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results-Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific KCa3.1 blocker TRAM-34. Expression of KCa3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. KCa3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented KCa3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion-Blockade of KCa3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis. © 2008 American Heart Association, Inc.

Direct inhibitory effects of an antisense oligodeoxynucleotide upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells

The effects of a 15-mer antisense c-myc phosphorothioate modified oligodeoxynucleotide (OdN) upon the volume-sensitive Cl- current in ROS 17/2.8 cells were investigated using the whole-cell configuration of the patch clamp technique. At 5 microM, the OdN reversibly inhibited the current in a voltage- and time-dependent fashion. This was evident from the reduction in the peak current as assessed at the termination of each voltage pulse and an acceleration of the time-dependent inactivation present at strongly depolarised potentials. The kinetic modifications induced by the OdN suggest it may act by blocking the pore of open channels when the cell membrane potential is depolarised.

Domestic violence and child abuse: developing sensitive policies

Domestic violence is everywhere and nowhere. No statutory organization or health service has work with either perpetrators or survivors of domestic violence (usually women and children) as the primary focus of their service, yet all agencies will have very significant numbers among their clients/service users. It is therefore crucial that the policy framework is developed both within and between agencies to address the need, and scope, of intervention in this area and particularly the impact on children. Currently, significant steps have been taken by some agencies in the UK to address this previously neglected issue, though the developments are patchy. This paper draws on a UK-wide research study which mapped the extent and range of service provision for families where there is domestic violence and also developed a framework of good practice indicators for ‘Mapped the extent and range of service provision for families where there is domestic violence and also developed a framework of good practice indicators’ provision in this area. This article examines one of the indicators of good practice arising from the research—that of policy development—within social service departments and within the multi-agency arena.

Studies in the marketing strategy of a U.K. producer of conveyor belts

Research was undertaken in the field of marketing strategy, its formulation and implementation in Dunlop Belting Division. Emphasis was placed on marketing channel strategy, but other strategies including product strategy were studied. The research has resulted in changes in management practice in the client organisation. The relevance of theories of company organisation, planning and strategy, and marketing channels was examined in the light of the research evidence. The technique of action-research was used to gain admittance to and effect change within the client organisation. Case study material was collected for subsequent analysis. The factors affecting marketing strategy formulation in the client organisation were studied. Both the external and the internal business environments were considered. The operation of the observed marketing channels was compared with channel theory. Market segmentation and penetration, and the selling and technical resources of the channels were analysed. Recommendations were made to (a) enlarge and resite the client's distribution unit to locate it centrally in England (b) use the resited unit to secure local advantage (c) obtain greater integration of field sales activities with and from the centre. A new ex-stock distribution unit was established. Improvements to the client's ex-stock marketing in Scotland were also recommended, including improvements to the Scottish distributor's stock control procedure, as well as to Dunlop-Distributor relationships at all levels. The influence of company organisation structure and formalised procedures and systems on the formulation of strategy were considered with respect to channel and product strategy, and other aspects of marketing. Conclusions were drawn that the action research resulted in successful implementation of .agreed changes in the client organisation; that theories of strategy formulation and planning, of the operation of decentralised companies, and of industrial market segmentation required modification; that the theory of marketing channels was found relevant and useful.

Highly radiation sensitive type IA FBGs for future dosimetry applications

We have studied Co60 gamma-irradiation effect on the characteristics of Type IA fiber Bragg gratings. A record Bragg peak shift of 190 pm was observed for a grating written in Fibercore PS-1250/1500 photosensitive fiber at a radiation dose of 116 kGy. Type IA and Type I gratings show different kinetics under radiation and during post-radiation annealing, which can be used for the design of a grating based dosimetry system.

A practical phase sensitive amplification scheme for two channel phase regeneration

A "black-box" phase sensitive amplifier is presented achieving simultaneous suppression of deterministic phase distortion on two independent 42.66 Gbit/s DPSK modulated signal wavelengths.

Phase synchronization scheme for a practical phase sensitive amplifier of ASK-NRZ signals

We present a phase locking scheme that enables the demonstration of a practical dual pump degenerate phase sensitive amplifier for 10 Gbit/s non-return to zero amplitude shift keying signals. The scheme makes use of cascaded Mach Zehnder modulators for creating the pump frequencies as well as of injection locking for extracting the signal carrier and synchronizing the local lasers. An in depth optimization study has been performed, based on measured error rate performance, and the main degradation factors have been identified.

Optimal packing for cascaded regenerative transmission based on phase sensitive amplifiers

We investigate the transmission performance of advanced modulation formats in nonlinear regenerative channels based on cascaded phase sensitive amplifiers. We identify the impact of amplitude and phase noise dynamics along the transmission line and show that after a cascade of regenerators, densely packed single ring PSK constellations outperform multi-ring constellations. The results of this study will greatly simplify the design of future nonlinear regenerative channels for ultra-high capacity transmission