Acute ethanol effects on cell volume regulation

In this work, the effects of ethanol on cellular osmoregulation were studied in isolated proximal renal tubules of Carassius auratus (goldfish). In hypotonic solutions the tubule cells swell rapidly (osmometric phase) and subsequently shrink towards isotonic volumes (volume regulatory decrease phase, VRD). The osmometric phase depends on the water permeability of the cell membrane and the magnitude of the osmotic gradient. The VRD phase is complex and is a function of activation of osmotic transporters with net KCl efflux followed by osmotically, obligated water. At 7, 10, and 12 mM ethanol, the membrane water permeability and osmotic ionic effluxes were increased. At higher ethanol concentrations (14 and 28 mM), the osmometric phase was moderately inhibited and VRD was abolished. The stimulatory effects of ethanol (7, 10, and 12 mM) on cellular osmoregulation are probably due to enhanced membrane fluidity (water permeability) and increased membrane calcium release (activation of KCl efflux). Inhibitory effects of ethanol (14 and 28 mM) on cell volume control are due to a combined decrement in water and KCl effluxes. This dose-related inhibition is likely due to incorporation of the amphipathic ethanol molecules with decreased hydrophobicity of the microenvironment of channels and transporters owing to displacement of structural lipids. In this system the threshold for osmoregulatory inhibition is between 12 and 14 mM.     

Calcium signaling in cell volume regulation.

It is evident from the present analysis that although a role for Ca2+ in controlling hypertonic cell volume regulation and RVI mechanisms has not been shown, Ca2+ plays a central role in activating and controlling hypotonic cell volume regulation and RVD mechanisms in most cells. However, this Ca2+ dependency is highly variable among cell types and tissues. Cells can be grouped into three general categories based on the relative dependency of RVD on Ca2+: 1) cells that are highly dependent on extracellular Ca2+ and the activation of Ca2+ influx, supposedly reflecting activation of Ca2+ channels, such as observed for the renal PST cells and osteosarcoma cells; 2) cells that are not dependent on extracellular Ca2+ and Ca2+ influx but that require at least a certain basal intracellular Ca2+ level or transient release of Ca2+ from internal stores, such as observed for the Ehrlich ascites tumor cells and medullary thick ascending limb cells; and 3) cells that display little if any Ca2+ dependency, such as the lymphocytes. There is initial evidence that this variable dependency of RVD on Ca2+ may reflect, in large part, a variable Ca2+ threshold of RVD processes, although this notion has not been fully investigated. The site and mechanism of Ca2+ dependency of RVD are poorly understood. Initial studies pointed to a possible direct control of K+ and/or Cl- channels by Ca2+ to modulate KCl efflux and, hence, RVD. This view appears to be too simplistic, however, as it is increasingly evident that the ion channels involved in RVD may not be directly Ca2+ dependent and that some other regulatory process controlling the channels, perhaps a phosphorylation step, may be the Ca(2+)-dependent event. Given the added complexity of the time-dependent variability of the action of Ca2+, i.e., the Ca2+ window, coupled with the variability of the RVD mechanisms among cell and tissue types, it is likely that the RVD mechanism is a highly complex process involving events and biochemical pathways throughout the cell rather than events simply localized to the inner face of the plasma membrane. It remains for future studies to determine the exact biochemical events that underly the RVD mechanism and its control, and the Ca2+ dependency of each step, before a full understanding will be attained of the role of Ca2+ in modulating RVD.     

Immunohistochemical analysis of rat and human respiratory cilia with anti-dynein antibody: comparison between normal cilia and pathological cilia in primary ciliary dyskinesia

Wistar Imamichi rat and human respiratory cilia were examined with anti-dynein antibody (AD2), which is specific for sea urchin sperm flagellar dynein. AD2-labelled fresh-frozen normal rat and human cilia stained clearly by immunofluorescence and the peroxidase-antiperoxidase (PAP) technique. On immunoelectron microscopy, AD2 labelled the outer dynein arms of normal human cilia. Paraffin-embedded normal human cilia also stained by immunofluorescence, although not always clearly. Neither the cilia of WIC-Hyd male rats, an animal model of Kartagener's syndrome, nor human cilia from patients with primary ciliary dyskinesia (PCD) reacted positively by the immunofluorescence or PAP technique. Western blots of normal rat cilia yielded a single band of about 450 kDa. In conclusion, AD2 recognizes the outer arm dynein heavy chains of healthy cilia and may be useful in diagnosing and classifying PCD light microscopically especially when only paraffin-embedded specimens are available. This approach may be of potential use for better defining and classifying PCD.     

Carbonic anhydrase inhibition and cell volume regulation in Necturus gallbladder

Gallbladder epithelial cells transport salt and water isotonically as the renal proximal tubule. The cells also have the property of regulating their cell volume in response to osmotic stress (Fisher et al. 1981, Persson & Spring 1982, Fisher & Spring 1984, Foskett & Spring 1985). The volume-regulating phenomenon is the result of a balance between cell uptake of salt and water at the luminal membrane and exit at the basolateral membrane. Different properties regarding volume regulatory increase and decrease have been found (Eriksson & Spring 1982 and Larson & Spring 1983). The present study links fluid transport and volume regulatory increase of the cell. First we concluded from histological techniques that carbonic anhydrase is present in the cell membrane or in the vicinity of the epithelial cells. Then we measured a decreased net fluid transport in the presence of increasing concentrations of the carbonic anhydrase inhibitor acetazolamide. We showed that the volume regulatory increase is substantially slowed down and that the steady-state volume of the cells changed when carbonic anhydrase was inhibited. Our conclusion is that the rate of CO2 hydration was a limiting step, at carbonic anhydrase inhibition, in both the net transfer of salt and water and also in the ability of the cells to efficiently regulate their volume.     

Cell volume regulation: physiology and pathophysiology

Cell volume perturbation initiates a wide array of intracellular signalling cascades, leading to protective and adaptive events and, in most cases, activation of volume-regulatory osmolyte transport, water loss, and hence restoration of cell volume and cellular function. Cell volume is challenged not only under physiological conditions, e.g. following accumulation of nutrients, during epithelial absorption/secretion processes, following hormonal/autocrine stimulation, and during induction of apoptosis, but also under pathophysiological conditions, e.g. hypoxia, ischaemia and hyponatremia/hypernatremia. On the other hand, it has recently become clear that an increase or reduction in cell volume can also serve as a specific signal in the regulation of physiological processes such as transepithelial transport, cell migration, proliferation and death. Although the mechanisms by which cell volume perturbations are sensed are still far from clear, significant progress has been made with respect to the nature of the sensors, transducers and effectors that convert a change in cell volume into a physiological response. In the present review, we summarize recent major developments in the field, and emphasize the relationship between cell volume regulation and organism physiology/pathophysiology.     

The role of the neutral amino acid transporter SNAT2 in cell volume regulation

Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.     

Basolateral membrane chloride permeability of A6 cells: implication in cell volume regulation

The permeability to Cl^– of the basolateral membrane (blm) was investigated in renal (A6) epithelial cells, assessing their role in transepithelial ion transport under steady-state conditions (isoosmotic) and following a hypoosmotic shock (i.e. in a regulatory volume decrease, RVD). Three different complementary studies were made by measuring: (1) the Cl^– transport rates (F/F _o · s^–1 (× 10^–3)), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and F _o the maximal fluorescence (×10^–3) of both membranes by following the intracellular Cl^–3 activities (a _iCl^–, measured with MQAE) after extracellular Cl^– substitution (2) the blm ⁸⁶Rb and ³⁶Cl uptakes and (3) the cellular potential and Cl^– current using the wholecell patch-clamp technique to differentiate between the different Cl^– transport mechanisms. The permeability of the blm to Cl^– was found to be much greater than that of the apical membranes under resting conditions: a _iCl^– changes were 5.3±0.7 mM and 25.5±1.05 mM (n=79) when Cl^– was substituted by NO₃ ^– in the media bathing apical and basolateral membranes. The Cl^– transport rate of the blm was blocked by bumetanide (100 M) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 M) but not by N-phenylanthranilic acid (DPC, 100 M). ⁸⁶Rb and ³⁶C1 uptake experiments confirmed the presence of a bumetanide- and a NPPB-sensitive Cl^– pathway, the latter being approximately three times more important than the former (Na/K/2Cl cotransporter). Application of a hypoosmotic medium to the serosal side of the cell increased F/F _o · s^–1 (×10^–3) after extracellular Cl^–3 substitution (1.03±0.10 and 2.45±0.17 arbitrary fluorescent units·s^–1 for isoosmotic and hypoosmotic conditions respectively, n=11); this F/F _o·s^–1 (×10^–3) increase was totally blocked by serosal NPPB application; on the other hand, cotransporter activity was decreased by the hypoosmotic shock. Cellular Ca²⁺ depletion had no effect on F/F _o·s^–1 (×10^–3) under isoosmotic conditions, but blocked the F/F _o·s^–1 (×10^–3) increase induced by a hypoosmotic stress. Under isotonic conditions the measured cellular potential at rest was –37.2±4.0 mV but reached a maximal and transient depolarization of –25.1±3.7 mV (n=9) under hypoosmotic conditions. The cellular current at a patch-clamping cellular potential of –85 mV (close to the Nernst equilibrium potential for K⁺) was blocked by NPPB and transiently increased by hypoosmotic shock ( 50% maximum increase). This study demonstrates that the major component of Cl^– transport through the blm of the A6 monolayer is a conductive pathway (NPPB-sensitive Cl^– channels) and not a Na/K/2Cl cotransporter. These channels could play a role in transepithelial Cl^– absorption and cell volume regulation. The increase in the blm Cl^– conductance, inducing a depolarization of these membranes, is proposed as one of the early events responsible for the stimulation of the ⁸⁶Rb efflux involved in cell volume regulation.     

Altered cell volume regulation in ras oncogene expressing NIH fibroblasts

Expression of the Ha-ras oncogene has been reported to stimulate the dimethylamiloride sensitive Na⁺/H⁺ exchanger and Na⁺, K⁺, 2Cl^– cotransport, both transport systems which are involved in cell volume regulation. The present study has been performed to test for an influence of ras oncogene expression on cell volume regulation in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras). As controls served NIH 3T3 fibroblasts not expressing the ras oncogene (– ras). In isotonic extracellular fluid, the cell volume of + ras cells (2.70±0.08 pl) is significantly greater than the cell volume of –ras cells (2.04±0.10 pl). Both, + ras and – ras cells exhibit a regulatory cell volume increase in hypertonic extracellular fluid and a regulatory cell volume decrease in hypotonic extracellular fluid. The regulatory cell volume decrease is inhibited by 1 mmol/l quinidine and barium, the regulatory cell volume increase is inhibited in – ras and + ras cells by dimethyl-amiloride (100 mol/l) and, only in + ras cells, by furosemide (100 mol/l) and bumetanide (10 mol/l). In conclusion, expression of the ras oncogene leads to a shift of the set point for cell volume regulation to greater cell volumes, which may contribute to the activation of the Na⁺/H⁺ exchanger and Na⁺, K⁺, 2Cl^– cotransport.     

Brain volume regulation: osmolytes and aquaporin perspectives

Cerebral water control is critical to maintain neuronal excitability, and to prevent injuries derived from brain swelling or shrinkage. The influence of aquaporins (AQPs) in the balance of water distribution between intracranial compartments is getting much experimental support. The importance of AQPs in fluid clearance during vasogenic brain edema seems well established but their role in cytotoxic swelling and in brain cell shrinkage is not known in detail. The main AQPs function as water channels anticipates their influence on cell volume changes as well as on the mechanisms of volume recovery, which include notably the osmolyte translocation across the cell membrane. Osmolyte fluxes permit the reestablishment of an osmotic balance and volume recovery in anisosmotic-elicited cell volume changes, but are also causal factors per se of brain cell swelling or shrinkage in pathological situations. This review aims to inform on the so far described functional interactions between AQPs and osmolyte fluxes and their volume-sensitive pathways. It also points to the coincidence of AQPs and activation of osmolyte fluxes in physiological and pathological conditions and to the importance of finding possible functional links between these two events, thus enlarging the possibilities via AQP manipulations, to prevent the adverse consequences of cell volume changes in brain.     

Primer and interviews: Diverse connections between primary cilia and Hedgehog signaling

On the surface, the Hedgehog (Hh) pathway and primary cilia make strange bedfellows. Hh is a dynamic regulator of a myriad of developmental processes, ranging from spinal cord and limb patterning to lung branching morphogenesis. By contrast, immotile primary cilia were long considered ancestral holdovers with no known function. Considering the disparate perceptions of these two phenomena, the relatively recent discovery that there is a symbiotic‐like relationship between Hh and cilia was unexpected. This primer covers the basics of primary cilia and Hh signaling, highlighting variations in ways they are connected across species, and also discusses the evolutionary implications of these findings. Roles of cilia in signal transduction are analyzed further in an interview with Søren T. Christensen, PhD, and Andrew S. Peterson, PhD, in the A Conversation With the Experts section. Developmental Dynamics 239:1255–1262, 2010. © 2010 Wiley‐Liss, Inc.     

Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia

Cilia are important cellular structures that have been implicated in a variety of signaling cascades. In this review, we discuss the current evidence for and against a link between cilia and both the canonical Wnt/β-catenin pathway and the noncanonical Wnt/planar cell polarity (PCP) pathway. Furthermore, we address the evidence implicating a role for PCP components in ciliogenesis. Given the lack of consensus in the field, we use new data on the control of ciliary protein localization as a basis for proposing new models by which cell type-specific regulation of ciliary components via differential transport, regulated entry and exit, or diffusion barriers might generate context-dependent functions for cilia.