The effect of hypertension and its treatment on cerebral cell volume regulation during hypernatremic dehydration

Since hypertension may compromise the ability to withstand hypernatremic dehydration, we investigated the impact of two experimental models of hypertension and pharmacologic normalization of blood pressure on the tolerance to chronic hypernatremic dehydration. In DOCA-salt hypertensive animals and the spontaneously-hypertensive rat (SHR), there was increased mortality and cerebral cell shrinkage during hypernatremic dehydration, compared to control Sprague-Dawley or Wistar-Kyoto rats. These findings were paralleled by significant differences in the brain intracellular water compartment size (ml/100 g dry weight), i.e. 233 +/- 6, Sprague-Dawley vs 189 +/- 8, DOCA-salt, P less than 0.01; 246 +/- 3, Wistar-Kyoto vs 194 +/- 6, SHR, P less than 0.01. Normalization of the blood pressure in the SHR with captopril restored 48% of the cerebral cell volume regulatory capacity observed in normotensive Wistar-Kyoto rats. We conclude that sustained hypertension increases the risk of hypernatremic dehydration in select circumstances. Correction of the elevated blood pressure promotes partial recovery of normal cerebral cell volume regulation.     

The cytoskeleton and cell volume regulation

Although the precise mechanisms have yet to be elucidated, early events in osmotic signal transduction may involve the clustering of cell surface receptors, initiating downstream signaling events such as assembly of focal adhesion complexes, and activation of, e.g. Rho family GTPases, phospholipases, lipid kinases, and tyrosine- and serine/threonine protein kinases. In the present paper, we briefly review recent evidence regarding the possible relation between such signaling events, the F-actin cytoskeleton, and volume-regulatory membrane transporters, focusing primarily on our own work in Ehrlich ascites tumer cells (EATC). In EATC, cell shrinkage is associated with an increase, and cell swelling with a decrease in F-actin content, respectively. The role of the F-actin cytoskeleton in cell volume regulation in various cell types has largely been investigated using cytochalasins to disrupt F-actin and highly varying effects have been reported. Findings in EATC show that the effect of cytochalasin treatment cannot always be assumed to be F-actin depolymerization, and that, moreover, there is no well-defined correlation between effects of cytochalasins on F-actin content and their effects on F-actin organization and cell morphology. At a concentration verified to depolymerize F-actin, cytochalasin B (CB), but not cytochalasin D (CD), inhibited the regulatory volume decrease (RVD) and regulatory volume increase (RVI) processes in EATC. This suggests that the effect of CB is related to an effect other than F-actin depolymerization, possibly its F-actin severing activity.     

On the role of G-protein coupled receptors in cell volume regulation.

Cell volume is determined genetically for each cell lineage, but it is not a static feature of the cell. Intracellular volume is continuously challenged by metabolic reactions, uptake of nutrients, intracellular displacement of molecules and organelles and generation of ionic gradients. Moreover, recent evidence raises the intriguing possibility that changes in cell volume act as signals for basic cell functions such as proliferation, migration, secretion and apoptosis. Cells adapt to volume increase by a complex, dynamic process resulting from the concerted action of volume sensing mechanisms and intricate signaling chains, directed to initiate the multiple adaptations demanded by a change in cell volume, among others adhesion reactions, membrane and cytoskeleton remodeling, and activation of the osmolyte pathways leading to reestablish the water balance between extracellular/intracellular or intracellular/intracellular compartments. In multicellular organisms, a continuous interaction with the external milieu is fundamental for the dynamics of the cell. It is in this sense that the recent surge of interest about the influence on cell volume control by the most extended family of signaling elements, the G proteins, acquires particular importance. As here reviewed, a large variety of G-protein coupled receptors (GPCRs) are involved in this interplay with cell volume regulatory mechanisms, which amplifies and diversifies the volume-elicited signaling chains, providing a variety of routes towards the multiple effectors related to cell volume changes.     

The role of system A for neutral amino acid transport in the regulation of cell volume

System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,K-ATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.     

Principles of cell volume regulation

Cell volume is determined by the content of osmotically active solute (cell osmoles) and the osmolarity of the extracellular fluid. Cell osmoles consist of non-diffusible and diffusible solutes. A large fraction of the diffusible cation content balances negative charges on the non-diffusible solutes. The content of diffusible solutes is determined by the electrochemical gradients driving them across the plasma membrane and the availability and activity of transport pathways in the membrane. The classical view that the sodium pump offsets passive leaks must be modified to accommodate the contributions of a number of secondary active transport processes, as well as to allow for changes in cell nondiffusible osmoles and in their net negative charge. The behaviour of cells in anisosmotic media is often different from that predicted for a perfect osmometer. In many cases this is a consequence of changes in cell osmole content. However, caution is required in extrapolating from in vitro responses of isolated cells to large, acutely induced changes in medium osmolality to the responses of tissues in vivo to more subtle changes in extracellular osmolality.     

Energy-dependent volume regulation in primary cultured cerebral astrocytes

Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo.     

Epithelial cell volume modulation and regulation

Summary Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.     

The role of atypical ubiquitination in cell regulation

Ubiquitination is a type of intracellular proteins post-translational modification (PTM) characterized by covalent attachment of ubiquitin molecules to target proteins. This includes monoubiquitination (attachment of one ubiquitin molecule), multiple monoubiquitination also known as multiubiquitination (attachment of several monomeric ubiquitin molecules to a target protein), and polyubiquitination (attachment of ubiquitin chains consisting of several, most frequently four ubiquitin monomers to a target protein). In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-linked polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins (not necessarily) targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes including immune response, genome stability, signal transduction, etc. Alterations of ubiquitination machinery is crucial for development of serious diseases.     

The role of far-red spectral states in the energy regulation of phycobilisomes

The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.     

A key role for KCl cotransport in cell volume regulation in human erythroleukemia cells

AimsKCl cotransport is believed to be involved in volume regulation in various erythroid cells of vertebrates, although the mechanism of activation and the role of the signaling elements involved remain uncertain. In this study, we characterized KCl cotransport activated by hypo-osmotic stress, and clarified several signaling elements involved in the regulation of this pathway within the human erythroleukemia cell line K562.Main methodsThe Cl^?-dependent K⁺ efflux (measured using ⁸⁶Rb⁺) and regulatory volume decrease (RVD) from pre-loaded K562 cells subjected to hypo-osmotic challenge were measured in cells treated with/without KCl cotransport inhibitors [(dihydroindenyl)oxy]alkanoic acid (DIOA) and Ba²⁺. This Cl^?-dependent K⁺ efflux has also been measured in cells treated with the phorbol 12-myristate 13-acetate (protein kinase C (PKC) activator), RO 31-8220 or calphostin C (PKC inhibitor), genistein (protein tyrosine kinase (PTK) inhibitor), PP2 (Src kinase inhibitor), AG18 or AG1478 (epidermal growth factor receptor (EGFR) kinase inhibitor), wortmannin or LY294002 (phosphatatidylinositol 3-kinase (PI 3-kinase) inhibitor), or PD98059 (mitogen-activated protein (MAP) kinase inhibitor).Key findingsCl^?-dependent K⁺ efflux was strongly stimulated by hypo-osmotic challenge and this increased K⁺ efflux was mediated by the DIOA- and Ba²⁺-sensitive KCl cotransport. RO 31-8220, calphostin C, genistein, PP2, AG18, AG1478, wortmannin, LY294002 and PD98059 were shown to significantly inhibit or stimulate the activity of this pathway.SignificanceOur results suggest that the hypo-osmotically-activated KCl cotransport is an important regulator of K562 cell volume, and the activity of this pathway is modulated by PKC, PTK, PI 3-kinase and/or MAP kinases.     

The role of swelling-induced anion channels during neuronal volume regulation

Regulation of cell volume is an essential function of most mammalian cells. In the cells of the central nervous system, maintenance of cell osmolarity and, hence, volume, is particularly crucial because of the restrictive nature of the skull. Cell volume regulation involves a variety of pathways, with considerable differences between cell types. One common pathway activated during hypo-osmotic stress involves chloride (Cl⁻) channels. However, hypo-osmotically stimulated anion permeability can be regulated by a diverse array of second messengers. Although neuronal swelling can occur in a number of pathological and nonpathological conditions, our understanding of neuronal volume regulation is limited. This article summarizes our current understanding of the role of anion channels during neuronal volume regulation.     

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.     

Solute transport and epithelial cell volume regulation

1. The regulation of epithelial cell volume is an essential requirement for normal tissue function and the maintenance of cellular integrity. 2. Renal papillary epithelial cells utilize an organic to compensate for the shrinkage associated with exposure to hypertonic solutions. 3. These cells synthesize the polyol, sorbitol, to increase their intracellular solute content. 4. Sorbitol is synthesized from glucose by the enzyme aldose reductase; exposure of the cells to hypertonic media causes aldose reductase synthesis and subsequent sorbitol generation over a two or three day period. 5. The intracellular signal for the formation of aldose reductase is not yet identified.     

Fundamental role of ClC-3 in volume-sensitive Cl- channel function and cell volume regulation in AGS cells

Volume regulation is essential for cell function, but it is unknown which channels are involved in a regulatory volume decrease (RVD) in human gastric epithelial cells. Exposure to a hypotonic solution caused the increase in AGS cell volume, followed by the activation of a current. The reversal potential of the swelling-induced current suggested that Cl- was the primary charge carrier. The selectivity sequence for different anions was I- > Br- > Cl- > F- > gluconate. This current was inhibited by flufenamate, DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)benzoate. Intracellular dialysis of three different anti-ClC-3 antibodies abolished or attenuated the Cl- current and disrupted RVD, whereas the current and RVD was unaltered by anti-ClC-2 antibody. Immunoblot studies demonstrated the presence of ClC-3 protein in Hela and AGS cells. RT-PCR analysis detected expression of ClC-3, MDR-1, and pICln mRNA in AGS cells. These results suggest a fundamental role of endogenous ClC-3 in the swelling-activated Cl- channels function and cell volume regulation in human gastric epithelial cells.     

The role of the cytoskeleton in volume regulation and beading transitions in PC12 neurites

We present investigations on volume regulation and beading shape transitions in PC12 neurites, conducted using a flow-chamber technique. By disrupting the cell cytoskeleton with specific drugs, we investigate the role of its individual components in the volume regulation response. We find that microtubule disruption increases both swelling rate and maximum volume attained, but does not affect the ability of the neurite to recover its initial volume. In addition, investigation of axonal beading—also known as pearling instability—provides additional clues on the mechanical state of the neurite. We conclude that volume recovery is driven by passive diffusion of osmolites, and propose that the initial swelling phase is mechanically slowed down by microtubules. Our experiments provide a framework to investigate the role of cytoskeletal mechanics in volume homeostasis.     

Human red cell volume regulation in hypotonic media

1. There is substantial evidence for a volume-sensitive KCl cotransport system in young human RBC. 2. The KCl cotransport system becomes latent on cell maturation. 3. There is a correlation between the activation of the KCl cotransporter by either pressure, NEM, ghosting or in certain anemias with disc/cup cell shape change. 4. The stretch-activated KCl transporter may be coupled to some component of the cell cytoskeleton.