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Intracellular potassium (K+) concentration decrease is not obligatory for apoptosis
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0001-8493-0114
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, The Institute of Technology.
Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.ORCID iD: 0000-0001-6235-7038
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 46, 39823-39828 p.Article in journal (Refereed) Published
Abstract [en]

K+ efflux is observed as an early event in the apoptotic process in various cell types. Loss of intracellular K+ and subsequent reduction in ionic strength is suggested to release the inhibition of proapoptotic caspases. In this work, a new K+-specific microelectrode was used to study possible alterations in intracellular K+ in Xenopus laevis oocytes during chemically induced apoptosis. The accuracy of the microelectrode to detect changes in intracellular K+ was verified with parallel electrophysiological measurements within the same cells. In concordance with previous studies on other cell types, apoptotic stimuli reduced the intracellular K+ concentration in Xenopus oocytes and increased caspase-3 activity. The reduction in intracellular K+ was prevented by dense expression of voltage-gated K (Kv) channels. Despite this, the caspase-3 activity was increased similarly in Kv channel expressing oocytes as in oocytes not expressing Kv channels. Thus, in Xenopus oocytes caspase-3 activity is not dependent on the intracellular concentration of K+.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology , 2011. Vol. 286, no 46, 39823-39828 p.
Keyword [en]
Caspase-3 activation, Electrophysiology, Intracellular K+ concentrations, K+-selective microelectrode, Xenopus laevis oocytes
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-68853DOI: 10.1074/jbc.M111.262725ISI: 000296925700016OAI: oai:DiVA.org:liu-68853DiVA: diva2:421418
Note
Funding agencies|Swedish Research Council||Swedish Heart-Lung Foundation||Swedish Brain Foundation||County Council of Ostergotland, King Gustaf V and Queen Victorias Freemasons Foundation||Swedish Society for Medical Research||Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2014-10-06Bibliographically approved
In thesis
1. Zinc Oxide Nanostructure Based Electrochemical Sensors and Drug Delivery to Intracellular Environments
Open this publication in new window or tab >>Zinc Oxide Nanostructure Based Electrochemical Sensors and Drug Delivery to Intracellular Environments
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The nanoscale science and nanostructure engineering have well established in the fabrication of novel electrochemical biosensors with faster response and higher sensitivity than of planar sensor configurations. Moreover nanostructures are suggested and used as efficient carrier of photosensitizers for cancerous cell treatment. The semi-conductor zinc oxide (ZnO) nanostructures have attracted much interest because of its unique piezoelectric, polar semiconducting, large surface area, catalytic properties, and being biosafe and biocompatible combined with the easiness of growth. This implies that ZnO nanostructures have a wide range of applications in optoelectronics, sensors, transducers, energy conversion and medical sciences. The aim of this study is to highlight recent developments in materials and techniques for electrochemical biosensing, photodynamic therapy, design, operation, and fabrication. The sensors in this study were used to detect and monitor real changes of metal ions and glucose across human fat cells and frog cells using changes in the electrochemical potential at the interface to the intracellular microenvironments. This thesis relates specifically to “zinc oxide nanostructure based electrochemical sensors and drug delivery to intracellular environments” for biological, biochemical and chemical applications.

The first part of the thesis presents extra and intracellular studies on metal ions such as Ca2+, Mg2+, and Na+…..etc selectively sensed by using ZnO nanorods grown on the tip of a borosilicate glass capillary (0.7 μm in diameter) with the aim to produce proto-type electrochemical extra/intracellular biosensors. The single human adipocyte and frog oocyte cells were used to selectively measure the intracellular free metal ions concentration. To make the sensors selective for metal ions with sufficient selectivity and stability, plastic membrane coatings containing specific ionophores were applied. These functionalized ZnO nanorods sensors showed high sensitivity and good stability with linear electrochemical potential versus a wide metal ion concentration range of interest. The measured intracellular values were consistent with values reported in the literature. Furthermore we have successfully determined that the intracellular potassium (K+) concentration decrease is not obligatory for apoptosis. The aim of this study is to show the possibility of using K+ selective microelectrode to detect and monitor intracellular changes of K+ concentration during injection of various test solution and chemically induced apoptosis in Xenopus laevis oocytes parallel with electrophysiological measurements to verify the accuracy.

The second part, presents the calcium ion (Ca2+) detection using functionalized ZnO nanorods attached as an extended gate metal oxide semiconductor field effect transistor (MOSFET). The electrochemical response was coupled directly to the gate of a commercial MOSFET to study the I-V characterization. Here we verified that ZnO nanorods grown on any thin wire can be combined with conventional electronic component to produce a sensitive and selective biosensor.

In the third part, we have performed the experiment to determine glucose concentration intracellularly and in airway surface liquid (ASL) with functionalized ZnO nanorod-coated microelectrodes. In this study, the GOD enzyme was immobilised electrostatically, drawing on the fact that there is a large difference in the isoelectric points of ZnO and glucose oxidase. Insulin has been found to affect the glucose uptake in human adipocytes and frog Xenopus laevis. The large size of these cells makes it possible to microinject specific reagents that interrupt or activate signal transmission to glucose. The measured glucose concentration in human adipocytes or frog oocytes and ASL using our ZnO nanorod sensor was consistent with values of glucose concentration reported in the literature by using other indirect techniques.

The fourth and final part covers the application of ZnO nanorods to cancer cells for photodynamic therapy. The ZnO nanorods were conjugated with protoporphyrin for local mediated photochemistry and efficient treatment of a single cancer cell. The ZnO nanorods were used as an efficient photosensitizer carrier system and at the same time providing intrinsic white light to achieve necrosis of the cancer cell. Breast cancer cells were used to study the catalytic effect of ZnO for treatment. The grown ZnO nanorods were conjugated with protoporphyrin dimethyl ester (PPDME), which absorbs the light emitted by the ZnO nanorods and cause the cytotoxicity which appears to involve the generation of reactive singlet oxygen inside the cell.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2011. 64 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1376
Keyword
ZnO nanorods, Intracellular electrochemical sensor, Functionalization, Metal ions, Glucose, Human Adipocytes, Frog Oocytes, Airway surface liquid
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-68856 (URN)978-91-7393-142-7 (ISBN)
Public defence
2011-09-02, K3, Kåkenhus, Campus Norrköping, Linköpings universitet, Norrköping, 10:15 (English)
Opponent
Supervisors
Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2014-01-15Bibliographically approved
2. The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes
Open this publication in new window or tab >>The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 47 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1424
National Category
Clinical Medicine Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-111045 (URN)10.3384/diss.diva-111045 (DOI)978‐91‐7519‐220‐8 (print) (ISBN)
Public defence
2014-11-07, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 13:15 (English)
Opponent
Supervisors
Available from: 2014-10-06 Created: 2014-10-06 Last updated: 2014-11-14Bibliographically approved

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