Cell shrinkage and monovalent cation fluxes: role in apoptosis

The loss of cell volume or cell shrinkage has been a morphological hallmark of the programmed cell death process known as apoptosis. This isotonic loss of cell volume has recently been term apoptotic volume decrease or AVD to distinguish it from inherent volume regulatory responses that occurs in cells under anisotonic conditions. Recent studies examining the intracellular signaling pathways that result in this unique cellular characteristic have determined that a fundamental movement of ions, particularly monovalent ions, underlie the AVD process and plays an important role on controlling the cell death process. An efflux of intracellular potassium was shown to be a critical aspect of the AVD process, as preventing this ion loss could protect cells from apoptosis. However, potassium plays a complex role as a loss of intracellular potassium has also been shown to be beneficial to the health of the cell. Additionally, the mechanisms that a cell employs to achieve this loss of intracellular potassium vary depending on the cell type and stimulus used to induce apoptosis, suggesting multiple ways exist to accomplish the same goal of AVD. Additionally, sodium and chloride have been shown to play a vital role during cell death in both the signaling and control of AVD in various apoptotic model systems. This review examines the relationship between this morphological change and intracellular monovalent ions during apoptosis.     

Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis

The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.     

Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death.

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.     

Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.

The loss of cell volume is a fundamental feature of apoptosis. We have previously shown that DNA degradation and caspase activity occur only in cells which have shrunken as a result of potassium and sodium efflux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). Furthermore, maintaining a normal intracellular potassium concentration represses the cell death process by inhibiting the activity of apoptotic nucleases and suppressing the activation of effector caspases (Hughes, F. M., Jr., Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 30567-30576). We have now investigated the relationship between cell shrinkage, ion efflux, and changes in the mitochondrial membrane potential, in addition to the role of caspases in these apoptotic events. Treatment of Jurkat cells with a series of inducers which act via distinct signal transduction pathways, resulted in all of the cell death characteristics including loss of cell viability, cell shrinkage, K(+) efflux, altered mitochondrial membrane potential, and DNA fragmentation. Interestingly, only cells which shrunk had a loss of mitochondrial membrane potential and the other apoptotic characteristics. Treatment of Jurkat cells with an anti-Fas antibody in the presence of the general caspase inhibitor z-VAD, abrogated these features. In contrast, when Jurkat cells were treated with either the calcium ionophore A23187 or thapsigargin, z-VAD failed to prevent cell shrinkage, K(+) efflux, or changes in the mitochondrial membrane potential, while effectively inhibiting DNA degradation. Treatment of Jurkat cells with various apoptotic agents in the presence of either the caspase-3 inhibitor DEVD, or the caspase-8 inhibitor IETD also blocked DNA degradation, but failed to prevent other characteristics of apoptosis. Together these data suggest that the cell shrinkage, K(+) efflux, and changes in the mitochondrial membrane potential are tightly coupled, but occur independent of DNA degradation, and can be largely caspase independent depending on the particular signal transduction pathway.     

Cell volume regulation in immune cell apoptosis

The loss of cell volume is an early and fundamental feature of programmed cell death or apoptosis; however, the mechanisms responsible for cell shrinkage during apoptosis are poorly understood. The loss of cell volume is not a passive component of the apoptotic process, and a number of experimental findings from different laboratories highlight the importance of this process as an early and necessary regulatory event in the signaling of the death cascade. Additionally, the loss of intracellular ions, particularly potassium, has been shown to play a primary role in cell shrinkage, caspase activation, and nuclease activity during apoptosis. Thus, an understanding of the role that ion channels and plasma membrane transporters play in cellular signaling during apoptosis may have important physiological implications for immune cells, especially lymphocyte function. Furthermore, this knowledge may also have an impact on the design of therapeutic strategies for a variety of diseases of the immune system in which apoptosis plays a central role, such as oncogenic processes or immune system disorders. The present review summarizes our appreciation of the mechanisms underlying the early loss of cell volume during apoptosis and their association with downstream events in lymphocyte apoptosis.     

Cationic gradient reversal and cytoskeleton-independent volume regulatory pathways define an early stage of apoptosis

Cell shrinkage, or apoptotic volume decrease (AVD), is a ubiquitous characteristic of programmed cell death that is independent of the death stimulus and occurs in all examples of apoptosis. Here we distinguished two specific stages of AVD based on cell size and a unique early reversal of intracellular ions that occurs in response to activation of both intrinsic and extrinsic cell death signal pathways. The primary stage of AVD is characterized by an early exchange of the normal intracellular ion distribution for sodium from 12 to 113.6 mm and potassium from 139.5 to 30 mm. This early ionic reversal is associated with a 20-40% decrease in cell volume, externalization of phosphatidylserine, loss of mitochondrial membrane potential, and caspase activation and activity along with nuclear condensation that occurs independent of actin cytoskeleton disruption. Disruption of the actin cytoskeleton, however, prevents a secondary stage of AVD in apoptotic cells, characterized by a loss of both potassium and sodium that results in an 80-85% loss in cell volume, DNA degradation, and apoptotic body formation. Together these studies demonstrate that AVD occurs in two distinct stages with the earliest stage reflecting a cellular cationic gradient reversal.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

Non-selective cation channels and oxidative stress-induced cell swelling

Necrosis is considered as a non-specific form of cell death that induces tissue inflammation and is preceded by cell swelling. This increase in cell volume has been ascribed mainly to defective outward pumping of Na+ caused by metabolic depletion and/or to increased Na+ influx via membrane transporters. A specific mechanism of swelling and necrosis driven by the influx of Na+ through nonselective cation channels has been recently proposed (Barros et al., 2001a). We have characterized further the properties of the nonselective cation channel (NSCC) in HTC cells. The NSCC shows a conductance of approximately 18 pS, is equally permeable to Na+ and K+, impermeant to Ca2+, requires high intracellular Ca2+ as well as low intracellular ATP for activation and is inhibited by flufenamic acid. Hydrogen peroxide induced a significant increase in cell volume that was dependent on external Na+. We propose that the NSCC, which is ubiquitous though largely inactive in healthy cells, becomes activated under severe oxidative stress. The ensuing Na+ influx initiates via positive feedback a series of metabolic and electrolytic disturbances, resulting in cell death by necrosis.     

The Na+/K+/Cl- cotransport in C6 glioma cells. Properties and role in volume regulation

The role of the Na+/K+/Cl- cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide greater than bumetanide greater than piretanide greater than furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide-sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl- cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl- cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso-osmotic medium deprived of either Na+, K+ or Cl- also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl- cotransport together with a cell swelling back to the normal volume were observed upon returning ion-deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl- cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.     

Protein kinase C (PKC) inhibits fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. A role for PKC upstream of caspases

Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with G?6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and G?6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with G?6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.     

3-Dimensional microscopy as a method for volume measurement in cells undergoing apoptosis

Different patterns of cell volume perturbations are commonly used for modes of cell death: necrosis (cell swelling) and apoptosis (cell shrinkage). In this study we employed recently developed three dimensional microscopy for the measurement of the volume of attached vascular smooth muscle cells transfected with E1A-adenoviral protein. These cells undergo rapid apoptosis in the absence of growth factors or in the presence of staurosporine. In 30–60 min of serum deprivation the volume of these cells is increased by ～40% that corresponds to the time point of maximal activation of caspase 3 and chromatin cleavage. In 10–15 min swollen cells exhibit morphological collapse indicated by formation of apoptotic bodies. In contrast to serum-deprived cells, staurosporine leads to attenuation of cell volume by 30%. In this case, apoptotic bodies are detected in ～2.5 h after maximal shrinkage. Thus, our results show that cell shrinkage can not be considered as universal hallmark of apoptosis. The role of stimulus-specific cell volume perturbation in the triggering of the cell death machinery should be examined further.     

Stimulation of Kv1.3 potassium channels by death receptors during apoptosis in Jurkat T lymphocytes

The loss of intracellular potassium is a pivotal step in the induction of apoptosis but the mechanisms underlying this response are poorly understood. Here we report caspase-dependent stimulation of potassium channels by the Fas receptor in a human Jurkat T cell line. Receptor activation with Fas ligand for 30 min increased the amplitude of voltage-activated potassium currents 2-fold on average. This produces a sustained outward current, approximately 10 pA, at physiological membrane potentials during Fas ligand-induced apoptosis. Both basal and Fas ligand-induced currents were blocked completely by toxins that selectively inhibit Kv1.3 potassium channels. Kv1.3 stimulation required the expression of Fas-associated death domain protein and activation of caspase 8, but did not require activation of caspase 3 or protein synthesis. Furthermore, Kv1.3 stimulation by Fas ligand was prevented by chronic stimulation of protein kinase C with 20 nm phorbol 12-myristate 13-acetate during Fas ligand treatment, which also blocks apoptosis. Thus, Fas ligand increases Kv1.3 channel activity through the same canonical apoptotic signaling cascade that is required for potassium efflux, cell shrinkage, and apoptosis.     

A selective requirement for elevated calcium in DNA degradation, but not early events in anti-Fas-induced apoptosis

Jurkat cells undergo apoptosis in response to anti-Fas antibody through a caspase-dependent death cascade in which calcium signaling has been implicated. We have now evaluated the role of calcium during this death cascade at the single cell level in real time utilizing flow cytometric analysis and confocal microscopy. Fluo-3 and propidium iodide were employed to evaluate calcium fluxes and to discriminate between viable and non-viable cells, respectively. Anti-Fas treatment of Jurkat cells resulted in a sustained increase in intracellular calcium commencing between 1 and 2 h after treatment and persisting until subsequent loss of cell membrane integrity. The significance of this rise in calcium was evaluated by buffering intracellular calcium with BAPTA and/or removing calcium from the extracellular medium and monitoring the effects of these manipulations on calcium signaling and components of the apoptotic process. Complete inhibition of the anti-Fas induced rise in intracellular calcium required both chelation of [Ca(2+)](i) and removal of extracellular calcium. Interestingly, this condition did not abrogate several events in Fas-induced apoptosis including cell shrinkage, mitochondrial depolarization, annexin binding, caspase activation, and nuclear poly(A)DP-ribose polymerase cleavage. Furthermore, calcium-free conditions in the absence of anti-Fas antibody weakly induced these apoptotic components. In marked contrast, calcium depletion did not induce DNA degradation in control cells, and inhibited apoptotic DNA degradation in response to anti-Fas. These data support the concept that the rise in intracellular calcium is not a necessary component for the early signal transduction pathways in anti-Fas-induced apoptosis in Jurkat cells, but rather is necessary for the final degradation of chromatin via nuclease activation.     

Regulation of sheep erythrocyte volume in anisotonic media.

Sheep erythrocytes of high and low potassium types were incubated in non-haemolytic hypotonic and hypertonic media for 4-5 h at 30 degrees. After initial swelling or shrinking, they readjusted their volume toward their initial isotonic volume. The volume regulation was associated with specific changes in cation fluxes. In the swollen cells, efflux of both sodium and potassium was increased and influx of both cations was slightly decreased; the converse was true for the shrunken cells. All four fluxes were changed in a direction that led to return to normal volume. The difference in the response of the two types of sheep erythrocytes to changes of extracellular fluid osmolality resided in the different activity of their cation transport systems. It is concluded that sheep erythrocytes possess some means of regulating their volume in vitro which is linked to cation permeability. The exact nature of the physical mechanisms by which they accomplish this remains to be elucidated.     

Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium

The relationship between receptor-mediated increases in the intracellular free calcium concentration [( Ca]i) and the stimulation of ion fluxes involved in fluid secretion was examined in the rat parotid acinar cell. Agonist-induced increases in [Ca]i caused the rapid net loss of up to 50-60% of the total content of intracellular chloride (Cli) and potassium (Ki), which is consistent with the activation of calcium-sensitive chloride and potassium channels. These ion movements were accompanied by a 25% reduction in the intracellular volume. The relative magnitudes of the losses of Ki and the net potassium fluxes promoted by carbachol (a muscarinic agonist), phenylephrine (an alpha-adrenergic agonist), and substance P were very similar to their characteristic effects on elevating [Ca]i. Carbachol stimulated the loss of Ki through multiple efflux pathways, including the large-conductance Ca-activated K channel. Carbachol and substance P increased the levels of intracellular sodium (Nai) to more than 2.5 times the normal level by stimulating the net uptake of sodium through multiple pathways; Na-K-2Cl cotransport accounted for greater than 50% of the influx, and approximately 20% was via Na-H exchange, which led to a net alkalinization of the cells. Ionomycin stimulated similar fluxes through these two pathways, but also promoted sodium influx through an additional pathway which was nearly equivalent in magnitude to the combined uptake through the other two pathways. The carbachol-induced increase in Nai and decrease in Ki stimulated the activity of the sodium pump, measured by the ouabain-sensitive rate of oxygen consumption, to nearly maximal levels. In the absence of extracellular calcium or in cells loaded with the calcium chelator BAPTA (bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) the magnitudes of agonist- or ionomycin-stimulated ion fluxes were greatly reduced. The parotid cells displayed a marked desensitization to substance P; within 10 min the elevation of [Ca]i and alterations in Ki, Nai, and cell volume spontaneously returned to near baseline levels. In addition to quantitating the activation of various ion flux pathways in the rat parotid acinar cell, these results demonstrate that the activation of ion transport systems responsible for fluid secretion in this tissue is closely linked to the elevation of [Ca]i.     

Intracellular mediators of granulysin-induced cell death

Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.     

Amphotericin B-induced apoptosis and cytotoxicity is prevented by the Na+, K+, 2Cl(-)-cotransport blocker bumetanide

Amphotericin B is the most commonly used antifungal drug although it exhibits poor effectiveness and considerable toxicity during treatment. It acts as a ionophore inducing cellular potassium efflux. The efflux of potassium, which is necessary for cell shrinkage during apoptosis, is counteracted by increased inward pumping of potassium ions. Modulation of potassium pump activity could therefore interact with programmed cell death depending on the nature of the disruption of cellular potassium homeostasis and subsequently affect the cytotoxicity of various drugs. We explored the role of apoptosis in amphotericin B-induced cytotoxicity in a mesothelioma cell line (P31) and investigated the role of K+ influx inhibitors of Na+, K+, ATPase and Na+, K+, 2Cl(-)-cotransport in these processes. Clone formation was used to determine the cytotoxicity of amphotericin B, ouabain (Na+, K+, ATPase blocker), and bumetanide (Na+, K+, 2Cl(-)-cotransport blocker), alone or in combination. Apoptosis was estimated by quantifying free nucleosomes. Amphotericin B (3.2 micromol/L, 3 mg/L) per se reduced the percentage of surviving clones to 64% and increased the number of nucleosomes by 31% compared to untreated control. When ouabain (100 micromol/L) was added to amphotericin B a less than additive effect on clone formation was seen but no reduction of nucleosomes was noted. Bumetanide (100 micromol/L) per se was not cytotoxic but increased cellular nucleosome expression. Bumetanide eradicated amphotericin B-induced reduction of formed clones and generated nucleosomes. In conclusion, the induction of apoptosis seems to be of significant importance in amphotericin B-induced cytotoxicity. Amphotericin B-induced cytotoxicity and apoptosis was eradicated by the Na+, K+, 2Cl(-)-cotransport inhibitor bumetanide. The changes of cellular K+ fluxes induced by bumetanide combined with amphotericin B needs further elucidation. Bumetanide could possibly be used in antifungal therapy to increase amphotericin B effectiveness doses without increasing its adverse effects.     

Cell volume regulation in frog urinary bladder

We have studied the problem of cell volume homeostasis in toad and frog urinary bladder by using electrophysiological measurements and an optical measure of cell volume. After osmotically induced swelling, urinary bladder cells spontaneously regulate their volume through a net loss of potassium, chloride, and water. During inhibition of sodium transport by amiloride the cells swell to the same extent as controls, but the volume-regulatory process is blocked. Electrophysiological results under isosmotic conditions indicate that basolateral membrane resistance increases simultaneously with the amiloride-induced rise in apical membrane resistance during transport inhibition. These independent observations indicate that inhibition of apical membrane sodium entry results in a secondary decrease in basolateral membrane potassium permeability. When cells are exposed to calcium-free, hyposmotic Ringer's solution, cell volume regulation is blocked; subsequent addition of the calcium ionophore A23187 is ineffective in restoring the regulatory process. The ionophore does induce volume regulation, however, in amiloride-inhibited, osmotically swollen cells in the presence of external calcium. Calcium thus seems to control basolateral membrane potassium permeability and may be the intracellular mediator of apical and basolateral membrane interactions.     

Evidence for chloride dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost,Opsanus tau

Summary Red blood cells of the marine teleost,Opsanus tau (oyster toadfish), were characterized as to their normal hemoglobin, ion and water contents. Cells were exposed to ouabain containing, hyposmotic salt solutions (osmolarity reduced to 2/3 of normal) in which the cation or anion composition was varied. It was found that the initial cell volume expansion due to water influx was independent of the anion present. However, a secondary volume reduction was dependent on the presence of chloride or bromide anions. During volume reduction, cellular potassium and chloride ion contents fell by about equal amounts. Potassium loss was commensurate to the total amount of potassium ions detected extracellularly about 1.5h after the initial osmotic shock. No major changes were seen in the cellular sodium ion contents. When chloride ions within the cells and in the suspending medium were replaced by nitrate, iodide or thiocyanate, the cells failed to return to volumes close to those of isosmotically suspended controls, and the cellular potassium content also remained constant. In hypotonic potassium chloride the cells failed to extrude potassium chloride and water, and hence retained their expanded volume. Neither potassium loss nor volume decrease occurred in cells swollen in hypotonic sodium chloride media containing furosemide or 4,4 diisothiocyano-2,2-stilbene-disulfonic acid (DIDS). These two compounds are known inhibitors of monovalent cation cotransport and anion self exchange, respectively, in mammalian red cells. Hence toadfish red cells respond to osmotic swelling primarily by activation of an ouabain-insensitive, chloride dependent potassium transport system which is sensitive to inhibition by furosemide and DIDS.     

Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation

Differentiation of lens epithelial cells into fiber cells involves an increase in cell volume which previously was proposed to be the direct cause of the extensive cell elongation which accompanies fiber formation. In this study we have continued to investigate the mechanism underlying cell elongation by defining the minimum nutrient and ion requirements of elongating cells, measuring potassium and sodium fluxes in stimulated and unstimulated lens epithelia, and determining the effects of several pharmacological agents on elongation and ion transport. We have shown that elongation will occur in a basic salt/glucose solution with Insulin-like growth factor I/somatomedin-C stimulation. Neither sodium nor any metabolite appears to be the osmotically active species which drives the increase in cell volume. However, potassium efflux rate coefficient was 47% lower in differentiating cells than in unstimulated cells, whereas potassium uptake rates and ouabain effects were similar. Cells did not elongate in potassium-free medium nor in the presence of several drugs which prevent the accumulation of intracellular potassium or hinder osmotic water flux. Unstimulated cells elongated, however, with the application of quinidine, a drug known to block potassium channels. We propose that stimulation of lens epithelial cells with an insulin-like growth factor signals the closure of a certain population of potassium channels. As a result, potassium efflux from the differentiating cells slows while active potassium uptake continues at a constant rate. Potassium then accumulates within the cell causing water influx, an increase in cell volume, and cell elongation.