Endothelial cells covering the luminal surface of vessels are exposed to at least two different mechanical forces: 1) fluid shear stress produced by the circulation of blood, and 2) periodic stretching and relaxing as a result of the diameter oscillations caused by blood pulsation. In this study we present an apparatus which was constructed to imitate the volume pulse with its typical incisura of the abdominal aorta. Using this apparatus, we exposed cultured endothelial cells to continuously produced cyclic and directional stretching and relaxation for three days. In all experiments cells remained attached and viable when subjected to mechanical stimulation. The vast majority of endothelial cells which underwent mechanical stimulation became elongated and oriented with their longer axis perpendicular to the direction of stretching (angle of cell orientation: alpha = 88.7 degrees +/- 12 degrees; means +/- SD), whereas cells on unstretched membranes had a cobblestone-like appearance and remained in random orientation. In the stretched cells, the factor of elongation was f = 6.8 +/- 1.3; means +/- SD; unstretched cells which exhibited a polygonal shape had a factor of elongation of f = 1.8 +/- 0.8; means +/- SD. In addition, the behavior of cytoskeletal components such as microfilaments and microtubules was examined in the process of cell orientation as both are actively involved in alterations of cell shape and cell migration. Actin filaments were oriented as both are actively involved in alterations of cell shape and cell migration. Actin filaments were oriented in parallel alignment perpendicular to the stretch direction (angle of actin filament orientation: beta = 90.4 degrees +/- 9 degrees; means +/- SD). A distinct orientation of microtubules was not observed, although a noticeable number of microtubules was observed to be in parallel alignment. Furthermore, microtubules of cells which underwent mechanical stimulation exhibited a pronounced asymmetric intracellular distribution with strongly fluorescent cytoplasmic areas in which microtubules seemed to be accumulated. The results indicate that endothelial cell elongation and orientation in vitro can be induced by periodic stretching and relaxation comparable to the periodic oscillations of the vessel wall due to blood pulsation in vivo.

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel (IK channel; Pflügers Arch 432:R87-R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca2+ concentration ([Ca2+]i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca2+]i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca2+]i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca2+]i. We therefore suggest that cell volume - possibly via [Ca2+]i - is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells.

Arterial smooth muscle cells from rabbit aortic media were grown in first subcultures on hydrophilized and collagen-coated silicone membranes which were then subjected to directional cyclic stretches and relaxations at a frequency of 50 times/min. The membranes were stretched 2, 5 and 10% beyond their resting length. Cells on unstretched and stationary membranes in the same chamber served as controls. The cells which were stretched with an amplitude of 2% remained in random orientation after 14 days of continuously performed cyclic stretching. The cells which were stretched 5% for 12 days orientated at an angle of 61 ± 9° to the direction of stretching, while the cells which were stretched with an amplitude of 10% for 6 days orientated at an angle of 76 ± 5°. The cells on the stationary and unstretched membranes remained in random orientation. We were able to confirm that the angle of orientation is reversible, i.e. preorientated cells changed their orientation during application of another stretching amplitude. The results suggest that stretching of the artery wall by blood pulsation may be a factor influencing the orientation of smooth muscle cells within the media of the artery wall and of those smooth muscle cells which proliferate into the subendothelial space after mechanical injury of the endothelium or electrical stimulation of the artery wall. An apparatus is presented which produces cyclic and directional mechanical stimuli similar to those which may occur in the artery wall.

Cell swelling is shown to induce an increase in acridine orange fluorescence intensity, an effect pointing to the alkalinization of acidic vesicles. Since autophagic hepatic proteolysis is accomplished by pH-sensitive proteinases within acidic lysosomes, this effect may contribute to the well-known inhibitory effect of cell swelling on proteolysis. In the present study, the role ofmicrotubules in volume-dependent alterations of pH in acidic vesicles of rat and human hepatocytes was studied. Colcemid and colchicine were used to depolymerize microtubules and vesicular pH was monitored using two dif- Amino acids elicit cell swelling by their concentrative uptake and cellular accumulation, while insulin exerts its effect by activation of ion uptake via Na+, K+, 2C-cotransport and Na+/H+ exchange (1, 2). The antiproteolytic action of insulin and glutamine is fully mimicked by the respective osmotic alterations of cell volume and is abolished if alterations of cell volume are reversed by appropriate alterations of extracellular osmolarity. Further, inhibition of Na+, K+, 2C-cotransport by furosemide or bumetanide not only blunts the swelling effect of insulin but also leads to a proportional inhibition of proteolysis. In the presence of both furosemide and amiloride, insulin-induced cell swelling and its antiproteolytic action are completely abolished. The mechanism linking cell volume alterations to proteolysis remained elusive, however, until recently when it was observed that cell swelling leads to an increase in acridine orange fluorescence intensity. This effect indicated an alkalinization of acidic intracellular compartments (3). Since proteolysis resides largely within acidic lysosomes and is accomplished by pH-sensitive lysosomal proteinases (4), the alkalinization of acidic intracellular compartments could indeed couple cell swelling to the inhibition of proteolysis. The mechanism by which cell volume changes are communicated to the acidic cellular compartments remained, however, unknown. In view of the described interaction between microtubules and intracellular vesicles (5, 6), the present study has been performed to examine a possible involvement of the microtubule network. To this end, the effect of cell swelling on fluorescein isothiocyanate (FITC)-dextran or acridine orange fluorescence has been studied both in intact cells and in cells treated with either colchicine or colcemid, drugs known to depolymerize microtubules (7,8). In addition, a possible effect of y--lumicolchicine, a stereoisomer of colchicine without an inhibitory effect on microtubules (9), was investigated. METHODSCell Culture. Human hepatocytes were prepared from pieces ofliver not appropriate for transplantation. The pieces were perfused through polyethylene catheters inserted into the main portal veins of the cut surface. The subsequent isolation procedure was according to the method ofBerry and Friend (10) with some modifications (11). Rat hepatocytes were prepared by collagenase treatment according to a method previously de...

Recent studies suggest that Spirulina, a unicellular blue-green alga, may have a variety of health benefits and therapeutic properties and is also capable of acting as an antioxidant and antiinflammatory agent. In this study, a cell-free and a cell-based test assay were used to examine the antioxidant and antiinflammatory properties of four selected Spirulina platensis preparations: (1) Biospirulina, (2) SpiruComplex, a preparation with naturally bound selenium, chromium and zinc, (3) SpiruZink, a preparation with naturally bound zinc, (4) Zinkspirulina + Acerola, a preparation with naturally bound zinc and acerola powder. The cell-free test assay used potassium superoxide as a donor for superoxide radicals, whereas the cell-based test assay used the formation of intracellular superoxide radicals of functional neutrophils upon stimulation by phorbol-12-myristate-13-acetate as a model to investigate the potential of Spirulina preparations to inactivate superoxide radicals. In accordance with the recommended daily dosage, test concentrations ranging from 50 to 1000 microg/mL were chosen. The results showed a dose-dependent inactivation of free superoxide radicals (antioxidant effect) as well as an antiinflammatory effect characterized by a dose-dependent reduction of the metabolic activity of functional neutrophils and a dose-dependent inactivation of superoxide radicals generated during an oxidative burst. The results demonstrate that the tested Spirulina preparations have a high antioxidant and antiinflammatory potential. Especially SpiruZink and Zinkspirulina + Acerola might be useful as a supportive therapeutic approach for reducing oxidative stress and/or the generation of oxygen radicals in the course of inflammatory processes.