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Hepatocyte transmembrane potential (Vm) behaves as an osmometer and varies with changes in extracellular osmotic pressure created by altering the NaCl concentration in the external medium (Howard, L.D. and Wondergem, R. (1987) J. Membr. Biol. 100, 53). We now have demonstrated similar effects on Vm by increasing external osmolality with added sucrose and not altering ionic strength. We also have demonstrated that hyperosmotic stress-induced depolarization of Vm results from changes in membrane K+ conductance, gK, rather than from changes in the K+ equilibrium potential. Vm and aki of hepatocytes in liver slices were measured by conventional and ion-sensitive microelectrodes, respectively. Cell water vols. were estimated by differences in wet and dry weights of liver slices after 10-min incubations. Effect of hyperosmotic medium on membrane transference number for K+, tk, was measured by effects on Vm of step-changes in external [K+]. Hepatocyte Vm decreased 34, 52 and 54% when tissue was superfused with medium made hyperosmotic with added sucrose (50, 100 and 150 mM). Correspondingly, aKi increased 10, 18 and 29% with this hyperosmotic stress of added sucrose. Tissue water of 2.92 ± 0.10 kg H2O/kg dry weight in control solution decreased to 2.60 ± 0.05, 2.25 ± 0.06 and 2.22 ± 0.05 kg H2O/kg dry weight with additions to medium of 50, 100 and 150 mM sucrose, respectively. Adding 50 mM sucrose to medium decreased tK from 0.20 ± 0.01 to 0.05 ± 0.01. Depolarization by 50% with hyperosmotic stress (100 mM sucrose) also occurred in Cl-free medium where Cl- was substituted with gluconate. We conclude that hepatocytes shrink during hyperosmotic stress, and the aKi increases. The accompanying decrease in Vm is opposite to that expected by an increase in aKi, and at least in part results from a concomitant decrease in gK. Changes in membrane Cl- conductance most likely do not contribute to osmotic stress-induced depolarization, since equivalent decreases in Vm occurred with added sucrose in cells depleted of Cl- by superfusing tissue with Cl-free medium.

L'utilisation de champs électriques pulsés (PEF) dans les secteurs de la médecine et de la biotechnologie est devenue de plus en plus courante au cours des dernières décennies. La recherche a montré qu'en ajustant la durée du PEF, nous pouvons prédire quels effets seront observés. Alors que les PEF dans la gamme micro - milliseconde ont été utilisés pour perméabiliser la membrane cellulaire et améliorer l'absorption de médicament ou de protéine, le PEF nanoseconde (nsPEF) a démontré des effets uniques sur les organites intracellulaires. Les deux PEF et nsPEF ont démontré un potentiel thérapeutique pour une variété de pathologies humaines, y compris le traitement du cancer. Utilisant l'imagerie des cellules vivantes, cette thèse a étudié in vitro les effets de champs pulsés d'une durée de 10 ns à 10 ms sur des lignées cancéreuses (U87 glioblastome multiforme) et non cancéreuses (neurones hippocampes de souris (HT22) et cellules ovariennes du hamster chinois (CHO)). Des résultats publiés antérieurement ont démontré que les cellules cancéreuses sont plus sensibles aux champs électriques que les cellules saines. Nos résultats sont en accord avec ces résultats, dans la mesure où les cellules U87 ont subi une dépolarisation significativement plus importante de leur potentiel transmembranaire après une seule impulsion électrique à toutes les durées. Dans un ensemble d'expériences parallèles, malgré des seuils de champ électrique similaires pour la perméabilisation membranaire, les cellules U87 ont démontré une absorption significativement améliorée de YO-PRO par rapport aux autres lignées cellulaires. Bien que les cellules U87 aient subi le plus grand changement dans la dépolarisation membranaire et la perméabilisation membranaire, elles ont également montré la constante de rescellement de la membrane la plus rapide, qui était environ 30 secondes plus rapide que les autres lignées cellulaires. Pour élucider certains des mécanismes sous-jacents par lesquels les cellules U87 répondent aux champs électriques, une série d'expériences a examiné le rôle des canaux ioniques transmembranaires. Plusieurs études récentes ont rapporté que les PEF peuvent agir directement sur les canaux ioniques voltage-dépendants. En utilisant divers modulateurs de canaux ioniques pharmacologiques spécifiques et à action large, nous avons démontré que nous pouvions presque entièrement inhiber la dépolarisation membranaire induite par le champ électrique dans les cellules U87 en bloquant certains canaux cationiques. Ces résultats étaient assez spécifiques, tels que le canal de potassium de grande conductance (BK), les canaux calciques de type L et T, et le canal cationique non spécifique, TRPM8, étaient capables d'inhiber la dépolarisation tandis que le blocage d'autres canaux ioniques ne produisait aucun changement significatif. . Les travaux de cette thèse ont montré que la lignée cellulaire maligne U87 présentait une plus grande sensibilité aux champs électriques allant de 10 ns à 10 ms par rapport aux lignées cellulaires non cancéreuses étudiées. Des améliorations potentielles aux protocoles de traitement actuels ont été proposées sur la base des résultats présentés ici. / The use of pulsed electric fields (PEF) in medical and biotechnology sectors has become increasingly prevalent over the last few decades. Research has shown that by adjusting the duration of the PEF we can predict what effects will be observed. Whereas PEF in the micro-to-millisecond range have been used to permeabilize the cell membrane and enhance drug or protein uptake, nanosecond PEF (nsPEF) have demonstrated unique effects on intracellular organelles. Both PEF and nsPEF have demonstrated therapeutic potential for a variety of human pathologies, including the treatment of cancer. Using live-cell imaging, this thesis investigated, in vitro, the effects of pulsed fields ranging in duration from 10 ns to 10 ms on cancerous (U87 glioblastoma multiforme) and non-cancerous cell lines (mouse hippocampal neurons (HT22) and Chinese hamster ovary (CHO) cells). Previously published results have demonstrated that cancerous cells have a greater sensitivity to applied electric fields than healthy cells do. Our results are in agreement with these findings, insofar as the U87 cells underwent a significantly greater depolarization of their transmembrane potential following a single electric pulse at all durations. In a parallel set of experiments, despite having similar electric field thresholds for membrane permeabilization, the U87 cells demonstrated significantly enhanced YO-PRO uptake compared to the other cells lines. Although U87 cells underwent the greatest change in both membrane depolarization and membrane permeabilization, they also showed the fastest membrane resealing constant, which was approximately 30 seconds faster than other cell lines. To elucidate some of the underlying mechanisms by which U87 cells respond to electric fields, a series of experiments looked at the role of transmembrane ion channels. Several recent studies have reported that PEFs can act directly on voltage-gated ion channels. Using a variety of specific and broad acting pharmacological ion channel modulators, we demonstrated that we could almost entirely inhibit the electric field-induced membrane depolarization in U87 cells by blocking certain cationic channels. These results were quite specific, such that the big conductance potassium (BK) channel, L- and T-type calcium channels, and the non-specific cationic channel, TRPM8, were able to inhibit depolarization while blocking other ion channels produced no significant change. The work in this thesis showed that the malignant U87 cell line showed a greater sensitivity to electric fields from ranging from 10 ns – 10 ms when compared to the non-cancerous cell lines that were investigated. Potential improvements to current treatment protocols have been proposed based on the findings presented herein.

Simulation of processes in cellular membranes Abstract Many important processes in cells involve ions, e.g., fusion of synaptic vesi- cles with neuronal cell membranes is controlled by a divalent cation Ca2+ ; and the exchange of Na+ and K+ drives the the fast electrical signal transmis- sion in neurons. We have investigated model phospholipid membranes and their interactions with these biologically relevant ions. Using state-of-the-art molecular dynamics simulations, we accurately quantified their respective affinites towards neutral and negatively charged phospholipid bilayers. In order to achieve that, we developed a new model of phospholipids termed ECC-lipids, which accounts for the electronic polarization via the electronic continuum correction implemented as charge rescaling. Our simulations with this new force field reach for the first time a quantitative agreement with the experimental lipid electrometer concept for POPC as well as for POPS with all the studied cations. We have also examined the effects of transmembrane voltage on phospholipid bilayers. The electric field induced by the voltage exists exclusively in the hydrophobic region of the membrane, where it has an almost constant strength. This field affects the structure of nearby water molecules highlighting its importance in electroporation. 1