Apoptosis is a special form of cell death that if non-functional may lead to diseases such as cancer. A reduction of the intracellular potassium ion (K+) content is necessary for activating enzymes important for the execution of apoptosis. Pharmacological modulation of K+ fluxes to reduce intracellular K+ in cancer cells might therefore force the cells into apoptosis and decrease tumour cell mass. Human malignant pleural mesothelioma (MPM) is a form of cancer often caused by asbestos exposure. Although asbestos has been banned in the Western World, the incidence of MPM is expected to increase. Cisplatin is the first-line chemotherapy for MPM, but acquired resistance to the drug is a clinical problem. This thesis is mainly based on work with the human malignant pleural mesothelioma cell line (P31 wt) and a cisplatin-resistant sub-line (P31 res). The aim was to first characterize K+ fluxes in P31 wt and P31 res cells, and then manipulate them in order to reduce intracellular K+ and induce apoptosis with K+ manipulation alone or in combination with cisplatin. Characterization of K+ fluxes in P31 wt cells showed that: 1) ouabain, a digitalis-like drug, and specific blocker of the Na+, K+, ATPase pump, effectively inhibited K+ uptake, 2) bumetanide, a diuretic, and an inhibitor of the Na+, K+, 2Cl-¬-cotransporter, had a transient effect on K+ uptake, and 3) the antifungal drug amphotericin B stimulated K+ efflux. In order to determine intracellular K+ content, the potassium-binding fluorescent probe PBFI-AM was used in a 96-well plate assay. After a 3-h incubation with ouabain, with or without bumetanide, combined with amphotericin B, the intracellular K+ content was reduced in P31 wt cells but not in P31 res cells. Ouabain induced apoptosis in both P31 wt and P31 res cells. P31 res cells were sensitized to cisplatin by ouabain, since 10 mg/L cisplatin in combination with ouabain induced about the same percentage of apoptotic cells as 40 mg/L cisplatin. Apoptosis was executed via caspase-3 activation in both P31 wt and P31 res cells. Amphotericin B enhanced ouabain-induced apoptosis in P31 wt cells via caspase-9 activation, with increased caspase-3 activation and DNA fragmentation as consequences. Ouabain-induced apoptosis in P31 res cells was executed via increased expression of pro-apoptotic Bak. The combination of cisplatin with ouabain and amphotericin B was stressful to both P31 wt and P31 res cells, since SAPK/JNK a known factor in stress-induced apoptosis was activated. In conclusion, K+ flux manipulation with clinical used drugs can induce apoptosis per se and also enhance cisplatin-induced apoptosis in P31 wt and P31 res cells.

Hyperkalemic periodic paralysis (HyperKPP) is characterized by myotonic discharges and weakness/paralysis. It is a channelopathy that is caused by mutation in the SCN4A gene that encodes for the skeletal muscle Na+ channel isoform (Nav1.4) α-subunit. Limb muscles are severely affected while breathing musculature is rarely affected even though diaphragm expresses the Nav1.4 channel. The objective of this study was to investigate the mechanism(s) that render the HyperKPP diaphragm asymptomatic in order to find a novel long lasting therapeutic approach, to treat HyperKPP symptoms. A HyperKPP mouse model carrying the M1592V mutation was used because it has a similar phenotype to that of patients carrying the same mutation. HyperKPP diaphragm, the limb muscles soleus and EDL all had a higher tetrodotoxin (TTX) sensitive Na+ influx than wild type (WT), but only the soleus and EDL had a depolarized resting potential, lower force and greater K+-induced force loss when compared to WT. The lack of a membrane depolarization in HyperKPP diaphragm was because of greater electrogenic contribution of the Na+ K+ ATPase pump compared to WT while such increase was not observed in EDL and soleus. HyperKPP diaphragm also had greater action potential amplitude than EDL and soleus possibly because of higher Na+ K+ ATPase pump maintaining a low [Na+]i. An inhibition of PKA, but not of PKC, increased the sensitivity of the HyperKPP diaphragm to the K+-induced force depression. So, HyperKPP soleus was exposed to forskolin to increase cAMP levels in order to activate PKA to document whether greater activity of PKA will alleviate HyperKPP symptoms. At 4.7 mM K+, forskolin increased force production, but worsened the decrease in force at 8 and 11 mM K+. Forskolin also did not improve membrane excitability. Pinacidil a KATP channel opener, improved force production at all [K+]e by causing a hyperpolarization of resting EM which then allowed for greater action potential amplitude and more excitable fibers. It is concluded that the development of a better therapeutic approach to treat HyperKPP can include a mechanism which activates Na+ K+ ATPase pumps and KATP channels.

Neuronen sind bei der Informationsübertragung des zentralen Nervensystems von entscheidender Bedeutung. Ihre Aktivität liegt der Signalverarbeitung und höheren kognitiven Prozessen zugrunde. Neuronen sind in den extrazellulären Raum eingebettet, der mehrere Teilchen, darunter auch Ionen, enthält. Ionenkonzentrationen sind nicht statisch. Intensive neuronale Aktivität kann intrazelluläre und extrazelluläre Ionenkonzentrationen verändern. In dieser Arbeit untersuche ich das Wechselspiel zwischen neuronaler Aktivität und der Dynamik der Ionenkonzentrationen. Dabei konzentriere ich mich hauptsächlich auf extrazelluläre Kalium- und intrazelluläre Natriumkonzentrationen. Mit Hilfe der Theorie dynamischer Systeme zeige ich, wie moderate Änderungen dieser Ionenkonzentrationen die neuronale Aktivität qualitativ verändern können, wodurch sich möglicherweise die Signalverarbeitung verändert. Dann modelliere ich ein leitfähigkeitsbasiertes neuronales Netzwerk mit Spikes. Das Modell sagt voraus, dass eine moderate Änderung der Konzentrationen, die einen Mikroschaltkreis von Neuronen umgeben, die Leistungsspektraldichte der Populationsaktivität verändern könnte. Insgesamt unterstreicht diese Arbeit die Bedeutung der Dynamik der Ionenkonzentrationen für das Verständnis neuronaler Aktivität auf langen Zeitskalen und liefert technische Erkenntnisse darüber, wie das Zusammenspiel zwischen ihnen modelliert und analysiert werden kann. / Neurons are essential in the information transfer mechanisms of the central nervous system. Their activity underlies both basic signal processing, and higher cognitive processes. Neurons are embedded in the extracellular space, which contains multiple particles, including ions which are vital to their functioning. Ionic concentrations are not static, intense neuronal activity alters the intracellular and extracellular ionic concentrations which in turn affect neuronal functioning. In this thesis, I study the interplay between neuronal activity and ionic concentration dynamics. I focus specifically on the extracellular potassium and intracellular sodium concentrations. Using dynamical systems theory, I illustrate how moderate changes in these ionic concentrations can qualitatively change neuronal activity, potentially altering signal processing. I then model a conductance-based spiking neural network. The model predicts that a moderate change in the concentrations surrounding a microcircuit of neurons could modify the power spectral density of the population activity. Altogether, this work highlights the need to consider ionic concentration dynamics to understand neuronal activity on long time scales and provides technical insights on how to model and analyze the interplay between them.