Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (Vm) and intracellular K+ activity (aKi) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration. Vm increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimental Vm/control Vm)] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8-1.5. The aKi did not change when medium osmolality was decreased 40-70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60 mm K+ or 1 mm quinine HCl or at 27°C resulted in no change in Vm compared with a 20-mV increase in Vm without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume and aKi in response to anisosmotic medium; however, Vm behaves as an osmometer under these conditions. Also, increases in Vm by hypoosmotic stress were abolished by conditions or agents that inhibit K+ conductance.

No description available.

No description available.

No description available.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study evaluated the use of bipolar electrosurgery and laparoscopic clip applier with respect to surgical time, blood loss and inflammatory response during video-assisted ovariohysterectomy with two portals. Two groups (n=10) assessed each of the hemostatic techniques during castration and a third group (GIII, n=6) evaluated changes in serum promoted only by the clinical and anesthetic protocols used in order to exclude the changes made by them. The surgical times, such as the volume of blood loss were significantly lower in Bipolar Group. The inflammatory response was significantly higher throughout the evaluation period after surgery, but no clinical manifestations different than those presented by the Clipador Group. There were no significant changes in packed cell volume between the groups, but among the times evaluated it reduced about 10% from initial value until four hours after the procedure, in the surgical groups and Group III. Both techniques have good execution by the video-assisted procedure, however, the use of bipolar forceps allows minor surgical times, minimal blood loss and shorter learning curve for the surgeon. The bleeding does not result in physiological changes and that one s on packed cell volume are presented because of the clinical and anesthetic protocols. / Este estudo avaliou a utilização da eletrocirurgia bipolar e do clipador laparoscópico em relação ao tempo cirúrgico, perda sanguínea e resposta inflamatória durante a ovariohisterectomia videoassistida com dois portais. Dois grupos (n=10) avaliaram cada uma das técnicas hemostáticas durante as castrações e um grupo (GIII, n=6) avaliou as alterações séricas promovidas somente pelo protocolo clínico e anestésico utilizado, a fim de excluir as alterações promovidas por estes. O tempo cirúrgico, assim como o volume de sangue perdido foram significativamente menores no Grupo Bipolar. A resposta inflamatória apresentou valores significativamente maiores durante todo o período de avaliação pósoperatório, sem manifestações clínicas diferentes das apresentadas pelo Grupo Clipador. Em relação ao hematócrito não houve alterações significativas entre os grupos, mas entre os tempos de avaliação reduziu cerca de 10% do valor inicial, até quatro horas do final do procedimento, tanto nos grupos cirúrgicos como no Grupo III. Ambas as técnicas são de boa execução através do procedimento videoassistido, porém, o uso da pinça bipolar permite menores tempos cirúrgicos, sangramento mínimo e menor curva de aprendizado do cirurgião. O sangramento não acarreta em alterações fisiológicas e as mudanças apresentadas no hematócrito são provenientes dos protocolos clínico e anestésico instituídos.

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ∼60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 μM calyculin A, or 1 μM N6-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl- channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

Dans les tissus, les cellules génèrent et sont soumises en permanence à des forces mécaniques. Les perturbations biochimiques à l'intérieur des cellules, ainsi que les altérations de leur environnement mécanique peuvent modifier l'équilibre physiologique et mener à des pathologies, comme le cancer. Bien que les propriétés mécaniques puissent être modifiées à l'échelle du tissus, la compréhension de la mécanique au niveau de la cellule unique demeure importante. En particulier, la différenciation, la migration des cellules immunitaires et le caractère invasif d'un cancer dépendent fortement des propriétés mécaniques des cellules uniques. Les déformations mécaniques peuvent induire un changement de la surface et du volume cellulaires. Nous nous intéressons particulièrement à la régulation du volume cellulaire chez les cellules mammifères dans le contexte de déformations à différentes échelles de temps. Jusqu'à présent, la régulation du volume dans ce contexte n'a été que très peu étudiée, en raison de la difficulté d'obtention de mesures précises, et du fait que le volume de la cellule est généralement considéré comme constant. Nous avons développé une méthode de mesure du volume cellulaire reposant sur l'exclusion de fluorescence, qui nous permet d'effectuer des mesures de volume précise au niveau de la cellule unique. Dans cette étude, nous nous sommes concentrés sur la régulation du volume cellulaire au cours de l'étalement dynamique sur un substrat (échelle de temps : minutes). Nous avons démontré qu'il existe différents régimes de régulation du volume lors de l'étalement : les cellules réduisent, augmentent ou ne modifient pas leur volume, en fonction de l'état du cortex d'actomyosine et de la vitesse d'étalement. Nous avons constaté que les cellules s'étalant plus vite ont tendance à perdre davantage de volume. Notre hypothèse est que lors d'une extension rapide de lamellipode dépendante d'Arp2/3, l'actine tire sur la membrane et génère une tension et l'activation de transport ionique, s'accompagnant d'une perte de volume compensatoire. L'inhibition de la polymérisation de l'actine ou de sa ramification dépendante d'Arp2/3 réduit la vitesse d'étalement et ainsi la perte de volume. Nous avons ensuite montré que l'inhibition de la contractilité augmente la vitesse d'étalement et la perte de volume. Cependant, l'inhibition d'Arp2/3 dans des cellules à faible contractilité conduit à un étalement rapide sans perte de volume. En effet, l'inhibition d'Arp2/3 induit des bulles de membranes, une déformation rapide n'induirait donc pas de perte de volume car la cellule peut relâcher la tension en dépliant la membrane. Nous avons également montré que la régulation du volume en réponse à une compression mécanique rapide (échelle de temps : millisecondes) indépendante de l'adhérence dépend également de l'état du cortex d'actomyosine. Les cellules perdent jusqu'à 30% de leur volume lorsqu'elles sont confinées, car la membrane plasmique est attachée au cortex et ne peux pas être dépliée en réponse à l'augmentation de la tension. La perturbation du cortex d'actine induit le détachement de la membrane et limite la perte de volume. Enfin, nous avons montré que la réponse du volume à un choc osmotique (échelle de temps : secondes) est plus que complexe que décrite dans la littérature. Nos données indiquent qu'au niveau de la cellule unique, la réponse initiale du volume au changement de l'osmolarité extérieure n'est pas un processus passif uniforme. En utilisant la technique du choc osmotique, nous avons également confirmé que les cellules ont un large excès de membrane repliée dans des réservoirs. Nos résultats montrent que le volume et l'aire cellulaires sont couplés par l'homéostasie de la tension de surface, et, étant donné que les déformations induisent une augmentation de la tension de surface, elles conduisent à des modifications du volume et de l'aire de la cellule. / The field of biomechanics significantly progressed in the last two decades. The importance of the feedback between biochemical signaling and physical properties was revealed in many studies. Cells within tissues constantly generate and experience mechanical forces. Biochemical perturbations inside the cells as well as alterations in the mechanical environment can shift the tiny balance of normal physiological state and lead to pathologies, e.g. cancer. Although the mechanical properties of individual cells can alter when they are within the tissues, the understanding of single cell mechanics is still important. Differentiation, immune cell migration, and cancer invasion strongly depend on the mechanical properties of individual cells. Mechanical deformations can lead to a change in cell surface area and volume. We are particularly interested in single mammalian cell volume regulation in the context of deformations of different timescales. For the moment, volume regulation in this context was out from the research interest, probably due to the difficulties of accurate measurements, and cell volume often considered as a constant parameter. We developed a method for cell volume measurements based on a fluorescent exclusion that allowed us to perform precise volume measurements of individual live cells. In the present study, we mainly focused on cell volume regulation while dynamic spreading on a substrate (timescale – minutes). We demonstrated that there are different regimes for volume regulation while spreading: cells decrease, increase or do not change volume, and a type of the regime depends on the state of the actomyosin cortex and spreading speed. We obtained that faster-spreading cells tend to lose more volume. Our hypothesis is that during fast Arp2/3-driven lamellipodia extension actin pull on the membrane that generates tension and activation of ion transport and regulatory volume loss. Inhibition of actin polymerization or Arp2/3-dependent actin branching decreases spreading speed and volume loss. Next, we showed that inhibition of contractility increases spreading speed and volume loss. However, inhibition of Arp2/3 complex in cells with low contractility leads to fast spreading without volume loss. Our explanation is that inhibition of Arp2/3 induces cell blebbing and even fast deformation does not lead to volume loss as a cell can relax tension by membrane unfolding. We also showed that volume regulation in response to fast mechanical compression (timescale – milliseconds) independent of adhesion also depends on the actomyosin cortex state. Control cells lose up to 30% of volume under confinement, as the cell membrane is attached to the cortex and cannot be unfolded in response to the tension increase. Disruption of actin cortex leads to membrane detachment and prevents volume loss under confinement. Additionally, we showed that cell volume response to the osmotic shock (timescale – seconds) is more complex than it used to be known in the literature. For instance, our data indicate that at the level of individual cells initial volume response to the change of external osmolarity is not a uniform passive process. Using osmotic shock technique, we also confirmed that cells have a large excess of membrane folded in reservoirs. Taken together, our data show that cell volume and surface area are coupled through surface tension homeostasis and as deformations induce surface tension increase, they lead to change volume and surface area.

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.

No description available.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.