Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell’s lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.     

Pathology of structured water and associated cations in cells (ther tissue damage syndrome) and its medical treatment

Modern experimental evidence indicates that the cell should be regarded as analogous to an ion exchanger resin granule with structured water in the interstices and with potassium and sodium ions associated with fixed negative charges on the protein matrix. In tissues damaged by disease or trauma, a similar set of changes in properties of cell cations and water is to be expected, for which a similar set of therapies is appropriate. Tissue damage causes a configurational change of the protein matrix from the normal to the damaged state. This leads to loss of association preference for potassium vs. sodium ions and to loss of water structuring, resulting in replacement of cell potassium by sodium and abnormal uptake of water by the cell. Appropriate therapies for reestablishment of the normal configurational state of the proteins of the cell are reestablishment of normal cell ATP production, for which prostaglandin PGBx is the rational approach, plus diets or drugs that decrease sodium and/or increase potassium concentrations in the body. Partial normalization of cell protein configuration by digitalis compounds may also be possible.     

The Development of the Enzyme Complement in Growing Root Cells

Fifteen serial sections, each 1.0 mm. in length, have been takenfrom the tip towards the base of the bean root. On correspondinggroups of sections determinations have been made of water content,protein content, and of dipeptidase, glycine oxidase, phosphatase,and invertase activities. The number of cells in each sectionof the series has also been determined. From these data unitcell values have been calculated which show how each of thequantities involved changes as the cell grows. It has been shown that four phases are involved in the earlydevelopment of the cell. In the first, which is traversed inthe meristematic zone of the root, the volume increases slightly,the protein content decreases, and the activities of three ofthe enzymes studied also decrease. In the second, volume, proteincontent, and all enzyme activities increase considerably. Inthe third, while the volume enlarges by increase in breadthonly, protein content and enzyme activities decrease. In thefourth, growth has ceased and protein content, and enzyme activitiescontinue to decrease. The significance of these changes in discussed.     

Mechanical characteristics of isotonic work and high-energy phosphates in frog ventricle after 1-fluoro-2,4-dinitrobenzene]

The blocking of the creatinphosphokinase by 1-fluoro-2,4-dinitrobenzene (FDNB) allows to investigate the relationship between ATP-supply, contractility and relaxability of the frog's myocardium. In isotonically working isolated ventricles of frogs the time of work, systolic and diastolic volume, velocity of contraction and relaxation as well as the levels of CP, ATP, ADP and AMP were measured at different intervals until termination of each experiment. CP shows a small variation, ATP decreases to 60% and ADP + AMP increase for the same amount under FDNB during the development of a slight inhibition of contractility and a continuously growing inhibition and retardation of relaxation until systolic arrest. ATP content and volume of relaxation correlated strictly. The contracture and the diminished contractility are caused by the decrease of ATP, producing a lack of substrate for Ca transport and actin-myosin-ATPase. This models the course of events during an insufficiency like in angina pectoris and in myocardial infarction.     

Role of AQP2 during apoptosis in cortical collecting duct cells

Background information. A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume decrease, due to the cellular outflow of potassium and chloride ions, followed by osmotically obliged water. In many cells, the ionic pathways triggered during the apoptotic volume decrease may be similar to that observed during a regulatory volume decrease response under hypotonic conditions. However, the pathways involved in water loss during apoptosis have been largely ignored. It was recently reported that in some systems this water movement is mediated via specific water channels (aquaporins). Nevertheless, it is important to identify whether this is a ubiquitous aspect of apoptosis as well as to define the mechanisms involved. The aim of the present work was to investigate the role of aquaporin‐2 during apoptosis in renal‐collecting duct cells. We evaluated the putative relationship between aquaporin‐2 expression and the activation of the ionic pathways involved in the regulatory volume response. Results. Apoptosis was induced by incubating cells with a hypertonic solution or with cycloheximide in two cortical collecting duct cell lines: one not expressing aquaporins and the other stably transfected with aquaporin‐2. Typical features of apoptosis were evaluated with different approaches and the water permeability was measured by fluorescence videomicroscopy. Our results show that the rate of apoptosis is significantly increased in aquaporin‐2 cells and it is linked to the rapid activation of volume‐regulatory potassium and chloride channels. Furthermore, the water permeability of cells expressing aquaporin‐2 was strongly reduced during the apoptotic process and it occurs before DNA degradation. Conclusions. These results let us propose that under apoptotic stimulation aquaporin‐2 would act as a sensor leading to a co‐ordinated activation of specific ionic channels for potassium and chloride efflux, resulting in both more rapid cell shrinkage and more rapid achievement of adequate levels of ions necessary to activate the enzymatic apoptotic cascade.     

L-929 cells under hyperosmotic conditions. Water, Na+, and K+

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using 14C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of in-folding of the cell surface. Na+ and K+ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na+ and K+ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na+ and K+ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.     

Swelling of Root Cell Walls as an Indicator of Their Functional State

The swelling capacity of cell walls isolated from different parts of lupine root was investigated. The water content in fragments of intact roots (Q) and swelling coefficient of standardized samples of cell walls (K^cw) were determined, and the dependences of Q and Kcw on the distance from the root tip (L) were plotted. It was shown that the change in Q value along the stretch of the lupine root reaches its maximum at distances of 1.5-6 cm or 7-12 cm from the root tip in 7-day-old and 14-day-old seedlings, respectively, whereas the K^cw value distribution over the root length is virtually invariable. In the radial direction, both the Q and K^cw values in cortex tissues are about twice higher than in the central cylinder. In our opinion, the changes of both Q and K^cw in the radial direction are associated with different degrees of cross-linking between polymer chains in cell wall structures of root cortex and central cylinder. The results of measurement of the K^cw value are consistent with the widely accepted mechanisms of water transport in roots in the radial direction. These data show that water transport through apoplast to the border between the cortex and central cylinder is accompanied by an increase in the resistance to water flow. Among other factors, this increase is due to a greater degree of cross-linking between cell wall polymers in the central cylinder. The results of measurement of the swelling coefficient of standardized cell wall samples in water and in 10 mM KCl at different pH values show that the swelling capacity of root cell walls varies according to the physicochemical properties of synthetic ion exchangers. Cell walls shrink (cell wall volume decreases) as ion concentration in solution increases and pH decreases. This causes an increase in the hydraulic resistance (or a decrease in the hydraulic conductivity) of apoplast. It was concluded that swelling is determined by the physicochemical properties of the cell wall, whereas the change in the swelling capacity induced by variation of external or internal conditions is an element of the mechanism of regulation of volume water flow in roots.     

EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts

EPH/EPHRIN signaling is essential to many aspects of tissue self-organization and morphogenesis, but little is known about how EPH/EPHRIN signaling regulates cell mechanics during these processes. Here, we use a series of approaches to examine how EPH/EPHRIN signaling drives cellular self-organization. Contact angle measurements reveal that EPH/EPHRIN signaling decreases the stability of heterotypic cell:cell contacts through increased cortical actomyosin contractility. We find that EPH/EPHRIN-driven cell segregation depends on actomyosin contractility but occurs independently of directed cell migration and without changes in cell adhesion. Atomic force microscopy and live cell imaging of myosin localization support that EPH/EPHRIN signaling results in increased cortical tension. Interestingly, actomyosin contractility also nonautonomously drives increased EPHB2:EPHB2 homotypic contacts. Finally, we demonstrate that changes in tissue organization are driven by minimization of heterotypic contacts through actomyosin contractility in cell aggregates and by mouse genetics experiments. These data elucidate the biomechanical mechanisms driving EPH/EPHRIN-based cell segregation wherein differences in interfacial tension, regulated by actomyosin contractility, govern cellular self-organization.     

A computational model of cell migration coupling the growth of focal adhesions with oscillatory cell protrusions

Cell migration is a highly integrated process where actin turnover, actomyosin contractility, and adhesion dynamics are all closely linked. In this paper, we propose a computational model investigating the coupling of these fundamental processes within the context of spontaneous (i.e. unstimulated) cell migration. In the unstimulated cell, membrane oscillations originating from the interaction between passive hydrostatic pressure and contractility are sufficient to lead to the formation of adhesion spots. Cell contractility then leads to the maturation of these adhesion spots into focal adhesions. Due to active actin polymerization, which reinforces protrusion at the leading edge, the traction force required for cell translocation can be generated. Computational simulations first show that the model hypotheses allow one to reproduce the main features of fibroblast cell migration and established results on the biphasic aspect of the cell speed as a function of adhesion strength. The model also demonstrates that certain temporal parameters, such as the adhesion proteins recycling time and adhesion lifetimes, influence cell motion patterns, particularly cell speed and persistence of the direction of migration. This study provides some elements, which allow a better understanding of spontaneous cell migration and enables a first glance at how an individual cell would potentially react once exposed to a stimulus.     

The yeast cAMP protein kinase Tpk3p is involved in the regulation of mitochondrial enzymatic content during growth

During aerobic cell growth, mitochondria must meet energy demand either by adjusting cellular mitochondrial content or by adjusting ATP production flux, allowing a constant growth yield. On respiratory substrate, the Ras/cAMP pathway has been shown to be involved in this process in the yeast Saccharomyces cerevisiae. We show that of the three cAMP protein kinase catalytic subunits, Tpk3p is the one specifically involved in the regulation of cellular mitochondrial content when energy demand decreases. In decreased energy demand, the Deltatpk3 mitochondrial enzymatic content decreases leading to a subsequent decrease in the cellular growth rate. Moreover, enzymatic content decreases in the Deltatpk3 isolated mitochondria, suggesting that the amount of cellular mitochondria is not affected, but rather that the mitochondria are modified. Our study points to an important decrease in the cytochrome c content in the Deltatpk3 mitochondria, which leads to a decrease in the slipping process at the level of cytochrome-c-oxidase.     

The Ionic Dependence of Cardiac Excitability and Contractility

In contrast to the large volume of data supporting the dependence of cardiac excitability and phasic contractility on external Na, Van der Kloot and Rubin (1962) and Singh (1962) have reported the persistence of both electrical and phasic mechanical activity in frog atrial and ventricular preparations soaked in isotonic sucrose solutions. The acute ionic dependence of excitability and contractility in small frog atrial trabeculae has been investigated with the conclusion that excitability and phasic contractions may continue for extended periods of time in sucrose media if the extracellular ionic concentrations remain above 2% of normal. This behavior is attributed to the slow exchange properties of the cell surfaces of the frog cardiac trabeculae and the antagonistic effects of Na, K, and Ca ions on both membrane excitability and fiber contractility.     

NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel

Nitric oxide (NO) donors decrease intraocular pressure (IOP) by increasing aqueous outflow facility in the trabecular meshwork (TM) and/or Schlemm's canal. However, the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study, we investigated the effects of NO donors on outflow facility and NO-induced effects on TM cell volume. We tested the involvement of soluble guanylate cyclase (sGC), cGMP, PKG, and the large-conductance Ca2+-activated K+ (BKCa) channel using inhibitors and activators. Cell volume was measured using calcein AM fluorescent dye, detected by confocal microscopy, and quantified using NIH ImageJ software. An anterior segment organ perfusion system measured outflow facility. NO increased outflow facility in porcine eye anterior segments (0.4884-1.3956 microl.min(-1).mmHg(-1)) over baseline (0.2373-0.5220 microl.min(-1).mmHg(-1)) within 10 min of drug application. These NO-induced increases in outflow facility were inhibited by the the BKCa channel inhibitor IBTX. Exposure of TM cells to NO resulted in a 10% decrease in cell volume, and these decreases were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and IBTX, suggesting the involvement of sGC and K+ eflux, respectively. NO-induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the PKG inhibitor (RP)-8-Br-PET-cGMP-S, suggesting the involvement cGMP and PKG. Additionally, the time course for NO-induced decreases in TM cell volume correlated with NO-induced increases in outflow facility, suggesting that the NO-induced alterations in cell volume may influence outflow facility.     

Computational modeling of cell adhesion and movement using a continuum-kinetics approach

Adhesion of leukocytes to substrate involves the coupling of disparate length and timescales between molecular mechanics and macroscopic transport, and existing models of cell adhesion do not use full cellular information. To address these challenges, a multiscale computational approach for studying the adhesion of a cell on a substrate is developed and assessed. The cellular level model consists of a continuum representation of the field equations and a moving boundary tracking capability to allow the cell to change its shape continuously. At the receptor-ligand level, a bond molecule is mechanically represented by a spring. Communication between the macro/micro- and nanoscale models is facilitated interactively during the computation. The computational model is assessed using an adherent cell, rolling and deforming along the vessel wall under imposed shear flows. Using this approach, we first confirm existing numerical and experimental results. In this study, the intracellular viscosity and interfacial tension are found to directly affect the rolling of a cell. Our results also show that the presence of a nucleus increases the bond lifetime, and decreases the cell rolling velocity. Furthermore, it is found that a cell with a larger diameter rolls faster, and decreases the bond lifetime. This study shows that cell rheological properties have significant effects on the adhesion process contrary to what has been hypothesized in most literature.     

Abnormalities of elastic and transporting properties of red blood cells under development of apoptosis

The functional properties of erythrocytes under development of apoptotic process in these cells were investigated by the low angle light scattering technique. Apoptosis induced by ionomycin was associated with an initial decrease of cell volume and caused formation of echinocytes. After that the cells restored their volume forming rounded erythrocytes with rugged membrane capable to agglomerate with each other. At the late stages of apoptosis, small fragmented cells can be revealed. Preapoptotic red blood cells (at all stages of apoptosis) manifested an enormous tolerance to hypotonic loading, whereas control cells hemolyzed just after reaching a critical volume (∼150 fl). Acidic hemolysis cannot differentiate between control and preapoptotic erythrocytes, the cells being hemolyzed not reaching the critical volume. Placing the control erythrocytes to a medium with ammonia ions instead of sodium ions caused an initial increase of cell volume above the critical point, and then it was also followed by hemolysis. Under ammonia loading, an initial rate of the cell volume growth and a ratio of the hemolyzed cells were significantly reduced in preapoptotic cells.     

Osmotic properties of ehrlich ascites tumor cells during the cell cycle

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.     

Heterogeneity Profoundly Alters Emergent Stress Fields in Constrained Multicellular Systems

Stress fields emerging from the transfer of forces between cells within multicellular systems are increasingly being recognized as major determinants of cell fate. Current analytical and numerical models used for the calculation of stresses within cell monolayers assume homogeneous contractile and mechanical cellular properties; however, cell behavior varies by region within constrained tissues. Here, we show the impact of heterogeneous cell properties on resulting stress fields that guide cell phenotype and apoptosis. Using circular micropatterns, we measured biophysical metrics associated with cell mechanical stresses. We then computed cell-layer stress distributions using finite element contraction models and monolayer stress microscopy. In agreement with previous studies, cell spread area, alignment, and traction forces increase, whereas apoptotic activity decreases, from the center of cell layers to the edge. The distribution of these metrics clearly indicates low cell stress in central regions and high cell stress at the periphery of the patterns. However, the opposite trend is predicted by computational models when homogeneous contractile and mechanical properties are assumed. In our model, utilizing heterogeneous cell-layer contractility and elastic moduli values based on experimentally measured biophysical parameters, we calculate low cell stress in central areas and high anisotropic stresses in peripheral regions, consistent with the biometrics. These results clearly demonstrate that common assumptions of uniformity in cell contractility and stiffness break down in postconfluence confined multicellular systems. This work highlights the importance of incorporating regional variations in cell mechanical properties when estimating emergent stress fields from collective cell behavior.     

Cellular contractility changes are sufficient to drive epithelial scattering

Epithelial scattering occurs when cells disassemble cell–cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial–mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell–cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell–cell interactions occurs by remodeling of cell–cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell–cell adhesion between cells in suspension, suggesting that changes in cell–cell adhesion strength might not accompany epithelial scattering. Instead, cell–substrate adhesion seems to play a bigger role, as cell–substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFβ is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells? ability to scatter. Further consistent with changes in substrate adhesion being required for cell–cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell–cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that induction of myosin-based cellular contractility may be sufficient for cell–cell detachment during epithelial scattering.     

Enforced polarisation and locomotion of fibroblasts lacking microtubules

The polarisation and locomotion of fibroblasts requires an intact microtubule cytoskeleton [1]. This has been attributed to an influence of microtubule-mediated signals on actin cytoskeleton dynamics, either through the generation of active Rac to promote protrusion of lamellipodia [2], or through the modulation of substrate adhesion via microtubule targeting events [3] [4]. We show here that the polarizing role of microtubules can be mimicked by externally imposing an asymmetric gradient of contractility by local application of the contractility inhibitor ML-7. Apolar fibroblasts lacking microtubules could be induced to polarize and to move by application of ML-7 by micropipette to one side of the cell and then to the trailing vertices that developed. The release and retraction of trailing adhesions could be correlated with a relaxation of traction on the substrate and a differential shortening of stress-fibre bundles, with their distal tips relaxed. Although retraction and protrusion in these conditions resembled control cell locomotion, the normal turnover of adhesion sites that form behind the protruding cell front was blocked. These findings show that microtubules are dispensable for fibroblast protrusion, but are required for the turnover of substrate adhesions that normally occurs during cell locomotion. We conclude that regional contractility is modulated by the interfacing of microtubule-linked events with focal adhesions and that microtubules determine cell polarity via this route.     

Cell Shape Dynamics Reveal Balance of Elasticity and Contractility in Peripheral Arcs

The mechanical interaction between adherent cells and their substrate relies on the formation of adhesion sites and on the stabilization of contractile acto-myosin bundles, or stress fibers. The shape of the cell and the orientation of these fibers can be controlled by adhesive patterning. On nonadhesive gaps, fibroblasts develop thick peripheral stress fibers, with a concave curvature. The radius of curvature of these arcs results from the balance of the line tension in the arc and of the surface tension in the cell bulk. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, is not clear. To get insight into the force balance, we inhibit myosin activity and simultaneously monitor the dynamics of peripheral arc radii and traction forces. We use these measurements to estimate line and surface tension. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that both line tension and surface tension dropped, but the line tension decreased to a lesser extent than surface tension. These results suggest that myosin-independent force contributes to tension in the peripheral arcs. We propose a simple physical model in which the peripheral arc line tension is due to the combination of myosin II contractility and a passive elastic component, while surface tension is largely due to active contractility. Numerical solutions of this model reproduce well the experimental data and allow estimation of the contributions of elasticity and contractility to the arc line tension.     

Effect of amiloride on cell volume regulation in renal straight proximal tubules

Amiloride has been shown to impair cell volume regulatory decrease in amphiuma red cells. The present study has been performed to test for the influence of amiloride on volume regulatory decrease and electrical properties in isolated perfused mouse straight proximal tubules. Replacement of 40 mmol/l NaCl with 80 mmol/l mannitol in bath perfusate does not appreciably affect the cell volume or the potential difference across the basolateral cell membrane. Reduction of osmolarity by omission of mannitol leads to cell swelling by 16.7 +/- 0.7% (n = 7), followed by volume regulatory decrease to 107.2 +/- 1.2% (n = 7) of original cell volume within 2 min. 1 mmol/l amiloride (but not 0.1 mmol/l amiloride) in the bath depolarizes the basolateral cell membrane from -63 +/- 1 mV (n = 24) by +16 +/- 1 mV (n = 16), decreases the apparent potassium transference number from 0.69 +/- 0.02 (n = 5) to 0.36 +/- 0.05 (n = 5), and significantly impairs volume regulatory decrease without appreciably modifying cell volume in isotonic solutions. 1 mmol/l amiloride in the luminal perfusate leads to a slight hyperpolarization of the basolateral cell membrane but does not interfere with volume regulatory decrease. Reduction of bath osmolarity depolarizes the basolateral cell membrane within 30 s by +7.8 +/- 0.8 mV (n = 18) in the absence and by +18 +/- 2 mV (n = 8) in the presence of amiloride. In the presence of reduced bath osmolarity and amiloride the potassium transference number amounts to 0.36 +/- 0.04 (n = 8). The hyperpolarization following luminal application of amiloride is most likely due to inhibition of luminal sodium channels, whereas bath amiloride depolarizes the basolateral cell membrane by reduction of basolateral potassium selectivity. As in amphiuma red cells amiloride impairs volume regulatory decrease in proximal straight renal tubules.