Loss Of Osmolytes Research Articles

Abstract 23: Body Fluid And Sodium Loss Leads To Aldosterone Hypersecretion And Muscle Catabolism In Hepatocellular Carcinoma Rats.

Background: The number of cancer survivors coincides with cardiovascular diseases is increasing, therefore, we are promoting the concept of “Onco-Hypertension” to clarify the mechanism linking cancer and blood pressure. In this study, we evaluated body osmolyte and water imbalance in hepatocellular cancer (HCC) model rats. Methods: Wistar rats were administered diethylnitrosamine (DEN) (1.5 μg/day, p.o.), a carcinogenic drug, for 8 weeks to establish liver cancer. Three weeks after the completion of DEN administration, we investigated blood pressure, tissue osmolyte and water content, and its association with aldosterone secretion. Results: HCC rats significantly reduced blood pressure, skin sodium, potassium, and water content. In the carcass (muscle + bone), dry weight, sodium, potassium, and water content were dramatically reduced without changing bone mass in HCC rats, suggesting that HCC causes muscle wasting to supply osmolyte and water for the dehydrated organs. These osmolytes and water loss were significantly associated with increased urinary aldosterone excretion. Supplementation of 0.25% salt water to drink improved body sodium and water loss and muscle wasting in HCC rats, which were completely suppressed by treatment with spironolactone (75 mg/kg/day, p.o.), a mineralocorticoid receptor (MR) blocker. Conclusion: These findings suggest that HCC causes body osmolyte and water loss, which leads to aldosterone hypersecretion and muscle catabolism to compensate for dehydration. A relatively small amount of salt supplement ameliorates the HCC-induced dehydration and muscle wasting via aldosterone/MR-mediated sodium and water restoration.

Facilitated NaCl Uptake in the Highly Developed Bundle of the Nephron in Japanese Red Stingray Hemitrygon akajei Revealed by Comparative Anatomy and Molecular Mapping.

Batoidea (rays and skates) is a monophyletic subgroup of elasmobranchs that diverged from the common ancestor with Selachii (sharks) about 270 Mya. A larger number of batoids can adapt to low-salinity environments, in contrast to sharks, which are mostly stenohaline marine species. Among osmoregulatory organs of elasmobranchs, the kidney is known to be dedicated to urea retention in ureosmotic cartilaginous fishes. However, we know little regarding urea reabsorbing mechanisms in the kidney of batoids. Here, we performed physiological and histological investigations on the nephrons in the red stingray (Hemitrygon akajei) and two shark species. We found that the urine/plasma ratios of salt and urea concentrations in the stingray are significantly lower than those in cloudy catshark (Scyliorhinus torazame) under natural seawater, indicating that the kidney of stingray more strongly reabsorbs these osmolytes. By comparing the three-dimensional images of nephrons between stingray and banded houndshark (Triakis scyllium), we showed that the tubular bundle of stingray has a more compact configuration. In the compact tubular bundle of stingray kidney, the distal diluting tubule was highly developed and frequently coiled around the proximal and collecting tubules. Furthermore, co-expression of NKAα1 (Na+/K +-ATPase) and NKCC2 (Na+- K+-2Cl- cotransporter 2) mRNAs was prominent in the coiled diluting segment. These findings imply that NaCl reabsorption is greatly facilitated in the stingray kidney, resulting in a higher reabsorption rate of urea. Lowering the loss of osmolytes in the glomerular filtrate is likely favorable to the adaptability of batoids to a wide range of environmental salinity.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis

Alamethicin is a well-studied antimicrobial peptide (AMP) that kills Gram-positive bacteria. It forms narrow, barrel-stave pores in planar lipid bilayers. We present a detailed, time-resolved microscopy study of the sequence of events during the attack of alamethicin on individual, live Bacillus subtilis cells expressing GFP in the cytoplasm. At the minimum inhibitory concentration (MIC), the first observed symptom is the halting of growth, as judged by the plateau in measured cell length vs time. The data strongly suggest that this growth-halting event occurs prior to membrane permeabilization. Gradual degradation of the proton-motive force, inferred from a decrease in pH-dependent GFP fluorescence intensity, evidently begins minutes later and continues over about 5min. There follows an abrupt permeabilization of the cytoplasmic membrane to the DNA stain Sytox Orange and concomitant loss of small osmolytes, causing observable cell shrinkage, presumably due to decreased turgor pressure. This permeabilization of the cytoplasmic membrane occurs uniformly across the entire membrane, not locally, on a timescale of 5s or less. GFP gradually leaks out of the cell envelope, evidently impeded by the shrunken peptidoglycan layer. Disruption of the cell envelope by alamethicin occurs in stages, with larger and larger species permeating the envelope as time evolves over tens of minutes. Some of the observed symptoms are consistent with the formation of barrel-stave pores, but the data do not rule out “chaotic pore” or “carpet” mechanisms. We contrast the effects of alamethicin and the human cathelicidin LL-37 on B. subtilis.

Cytoplasmic Domain Filter Function in the Mechanosensitive Channel of Small Conductance

Mechanosensitive channels, inner membrane proteins of bacteria, open and close in response to mechanical stimuli such as changes in membrane tension during osmotic stress. In bacteria, these channels act as safety valves preventing cell lysis upon hypoosmotic cell swelling: the channels open under membrane tension to release osmolytes along with water. The mechanosensitive channel of small conductance, MscS, consists, in addition to the transmembrane channel, of a large cytoplasmic domain (CD) that features a balloon-like, water filled chamber opening to the cytoplasm through seven side pores and a small distal pore. The CD is apparently a molecular sieve covering the channel that optimizes loss of osmolytes during osmoadaptation. We employ diffusion theory and molecular dynamics simulations to explore the transport kinetics of Glu− and K+ as representative osmolytes. We suggest that the CD indeed acts as a filter that actually balances passage of Glu− and K+, and possibly other positive and negative osmolytes, to yield a largely neutral efflux and, thereby, reduce cell depolarization in the open state and conserve to a large degree the essential metabolite Glu−.

Mathematical Model of the Regulatory Cell Volume Decrease

Renal collecting duct principal cells perform vasopressin regulated water reabsorption and form the composition of tubular fluid. The osmotic pressure of the extracellular fluid varies significantly. To maintain viability in hypotonic conditions cells have mechanisms for regulatory volume decrease (RVD). Despite its importance, very little is known about the volume regulation of renal collecting duct cells. The purpose of this study was to investigate the time course of cell volume changes in response to hypotonic shock and to create a mathematical model of the renal epithelial cells’ reaction to the change of extracellular osmolarity on the basis of the experimental data. The change of extracellular osmolarity in experiments leads to the rapid cell swelling which is followed by regulatory volume decrease (RVD) and recovery of the initial level of cell volume. After the second consequent hypotonic shock the cell behavior is close to the behavior of ideal osmometer. On the basis of foregoing experimental data the mathematical model of cell reaction to the hypotonic shock was created. This model calculates the time dependence of cell volume, transmembrane potential and intracellular amount of osmolytes such as Na+, K+, Cl- and hypothetical organic anions. The quantitative estimation of the intracellular osmolyte loss during the RVD was made along with the calculation of the contribution of different membrane transport systems (channels, cotransporters, Na/K-pump) to this process. Analysis of the model revealed essential features of RVD in collecting duct cells: 1) the increase of membrane K+ and Cl- permeability; 2) the activation of KCl-cotransporters; 3) the organic anions efflux; 4) the decrease of membrane osmotic water permeability. The simultaneous activation of all these mechanisms allows the cell to decrease the volume rapidly and saves it from the excessive swelling when the hypotonic shock is repeated.

The Role of MscS Cytoplasmic Domain As An Osmolyte Filter

Mechanosensitive (MS) channels, inner membrane proteins of bacteria, open and close in response to mechanical stimuli such as changes in membrane tension during osmotic stress. In bacteria, these channels act as safety valves thus preventing cell rupture upon hypoosmotic shock.The MS channels of small conductance, MscS, are homoheptameric and consist of a large cytoplasmic (CP) domain that features a balloon-like, water filled chamber opening to the cytoplasm through seven side pores and a small distal pore. This CP domain is considered to be a molecular sieve, which prevents a loss of essential osmolytes and metabolites at the cytoplasmic side. In bacteria, glutamate is a predominant anion that helps to maintain potassium pools at an optimum level and also is a prevalent osmoprotectant maintaining the cell turgor. We have explored, using molecular simulations, the free energy landscape characterizing the translocation and exit of a glutamate ion through one of the side pores, to illustrate the role of the CP domain in selective filtering of glutamate. The adaptive biasing force (ABF) method is applied to the glutamate molecule to determine the free energy barrier along the chosen translocation pathway. The transport kinetics of glutamate based on the measured free energy profile, suggests the presence of an entropic barrier that slows down the passage of glutamate through the pore but lets water pass quickly. A low enthalpic barrier of ∼4 kBT is sufficiently low to prevent glutamate from clogging the pore. Analysis of the electrostatic potential of the CP domain indicates that the CP interior presents an environment relatively unfavorable for anions.

Purinergic activation of anion conductance and osmolyte efflux in cultured rat hippocampal neurons

The majority of mammalian cells demonstrate regulatory volume decrease (RVD) following swelling caused by hyposmotic exposure. A critical signal initiating RVD is activation of nucleotide receptors by ATP. Elevated extracellular ATP in response to cytotoxic cell swelling during pathological conditions also may initiate loss of taurine and other intracellular osmolytes via anion channels. This study characterizes neuronal ATP-activated anion current and explores its role in net loss of amino acid osmolytes. To isolate anion currents, we used CsCl as the major electrolyte in patch electrode and bath solutions and blocked residual cation currents with NiCl(2) and tetraethylammonium. Anion currents were activated by extracellular ATP with a K(m) of 70 microM and increased over fourfold during several minutes of ATP exposure, reaching a maximum after 9.0 min (SD 4.2). The currents were blocked by inhibitors of nucleotide receptors and volume-regulated anion channels (VRAC). Currents showed outward rectification and inactivation at highly depolarizing membrane potentials, characteristics of swelling-activated anion currents. P2X agonists failed to activate the anion current, and an inhibitor of P2X receptors did not block the effect of ATP. Furthermore, current activation was observed with extracellular ADP and 2-(methylthio)adenosine 5'-diphosphate, a P2Y(1) receptor-specific agonist. Much less current activation was observed with extracellular UTP, suggesting the response is mediated predominantly by P2Y(1) receptors. ATP caused a dose-dependent loss of taurine and alanine that could be blocked by inhibitors of VRAC. ATP did not inhibit the taurine uptake transporter. Thus extracellular ATP triggers a loss of intracellular organic osmolytes via activation of anion channels. This mechanism may facilitate neuronal volume homeostasis during cytotoxic edema.

The tonicity of murine epididymal spermatozoa and their permeability towards common cryoprotectants and epididymal osmolytes

The permeability of murine cauda epididymidal spermatozoa was determined from the swelling caused by penetrating agents at isotonicity, which lies between 422 and 530 mmol/kg. Spermatozoa were permeable to a range of solutes with size <200 Da. Relative entry rates of cryoprotective agents (CPAs) were ethylene glycol approximately DMSO>propane-1,2-diol>glycerol>propane-1,3-diol. More polar compounds including major epididymal secretions were impermeant. None of the compounds entered spermatozoa through quinine-sensitive channels; rather, quinine increased the size of solute-swollen spermatozoa, suggesting that regulatory volume decrease and osmolyte loss occurred under these conditions. Volume responses to lowered osmolality revealed a greater volume-regulating ability of spermatozoa from the B6D2F1 strain than the C57BL6 strain. As the former strain displays better post-thaw fertility, their spermatozoa may have greater osmolyte loads enabling them to cope better with osmotic stress. Inadequate volume regulation, due to CPA-induced osmolyte loss, may affect post-thaw fertility. Knowing the permeability towards cryoprotectants will help to make a better choice of CPAs that are less damaging to sperm during cryopreservation.

Characterisation of three pathways for osmolyte efflux in human erythroleukemia cells

Cell volume decrease is a key step during differentiation of erythroid cells. This could arise from membrane transporter activation leading to a loss of cell osmolytes; however, the pathways involved are poorly understood. We have characterised Cl −-independent K + and 3H-taurine efflux from the erythroleukemia cell line, K562. K + efflux (measured using 86Rb +) from pre-loaded cells subjected to hypo-osmotic challenge demonstrated two phases, a rapid increase in K + efflux followed by a smaller slower increase. Swelling-activated taurine efflux only demonstrated a single phase. Both phases of K + efflux were significantly ( P < 0.05) blocked by anion channel inhibitor 5-nitro-2-(3-phenypropylamino)-benzoic acid (NPPB). However the antiestrogen, tamoxifen, only inhibited the slow late phase. The initial rapid phase had a higher IC 50 for NPPB inhibition than the slow phase, and was insensitive to protein kinases inhibitors KN-62, wortmannin and PD98059. For the slow K + efflux phase, the IC 50 for NPPB inhibition and the inhibition by KN-62, wortmannin, genistein or PD98059, were very similar to those measured for the hypo-osmotically-activated taurine efflux. With NPPB (100 μM) present, the slow K + efflux phase was further significantly decreased by the Ca 2+ chelator BAPTA-AM or by the Ca 2+-activated K + channel blockers clotrimazole and charybdotoxin but not by apamin. Thus, at least 3 Cl −-independent pathways are involved: (a) a tamoxifen-sensitive and taurine-permeable anion channel; (b) a tamoxifen-insensitive and taurine-impermeable K + efflux pathway; and (c) a subtype of Ca 2+-activated K + channel. Any or all of these could be involved in the cell volume decrease associated with differentiation in K562 cells.

CD95 activation in the liver: Ion fluxes and oxidative signaling

Apoptosis is characterized by typical features as cell shrinkage, nuclear condensation, DNA fragmentation, and apoptotic body formation. Whereas some signs of apoptosis are cell type-and death signal-dependent, apoptotic cell volume decrease is an early and ubiquitous event and little is known about the signalling events, which are localized upstream of the plasma membrane transport steps leading to apoptotic cell volume decrease and the proapoptotic events, which are induced by osmolyte loss and cell shrinkage. Ion fluxes and oxidative signaling were recently shown to play an important role in signal transduction with respect to apoptotic cell death within the liver, as a ceramide-dependent activation of the NADPH oxidase was identified as the source of reactive oxygen species generation in rat hepatocytes upon treatment with CD95 ligand, hydrophobic bile salts or hyperosmolarity. The NADPH oxidase-derived ROS signal then allows via Yes, JNK, and EGFR activation for CD95 tyrosine phosphorylation as a prerequisite for CD95 targeting to the plasma membrane and formation of the death inducing signalling complex. Other covalent modifications such as CD95-tyrosine-nitration or CD95-serine/threonine-phosphorylation can interfere with the CD95 activation process. The findings not only provide a mechanistic explanation for the high susceptibility of dehydrated cells for apoptosis, but also give insight into the role of ion fluxes and oxidative signaling with respect to apoptotic cell death within the liver.

Brain Volume Regulation in Response to Hypo-osmolality and Its Correction

Hyponatremia exerts most of its clinical effects on the brain. An acute onset (usually in <24 hours) of hyponatremia causes severe, and sometimes fatal, cerebral edema. Given time, the brain adapts to hyponatremia, permitting survival despite extraordinarily low serum sodium concentrations. Adaptation to severe hyponatremia is critically dependent on the loss of organic osmolytes from brain cells. These intracellular, osmotically active solutes contribute substantially to the osmolality of cell water and do not adversely affect cell functions when their concentration changes. The adaptation that permits survival in patients with severe, chronic (>48 hours’ duration) hyponatremia also makes the brain vulnerable to injury (osmotic demyelination) if the electrolyte disturbance is corrected too rapidly. The reuptake of organic osmolytes after correction of hyponatremia is slower than the loss of organic osmolytes during the adaptation to hyponatremia. Areas of the brain that remain most depleted of organic osmolytes are the most severely injured by rapid correction. The brain’s reuptake of myoinositol, one of the most abundant osmolytes, occurs much more rapidly in a uremic environment, and patients with uremia are less susceptible to osmotic demyelination. In an experimental model of chronic hyponatremia, exogenous administration of myoinositol speeds the brain’s reuptake of the osmolyte and reduces osmotic demyelination and mortality caused by rapid correction.

Influence of Hypoxia and Sex on Hyponatremic Encephalopathy

Over the past 20 years it has become increasingly apparent that hyponatremic encephalopathy is a major cause of inhospital morbidity and mortality, particularly in postoperative patients. The factors that may lead to death or permanent brain damage and the susceptible patient groups have been gradually elucidated. Hyponatremic encephalopathy most commonly leads to brain damage in young women and in prepubescent children. The causes of brain damage include brain edema, cerebral hypoxemia, decreased brain blood flow, increased intracranial pressure, and improper therapy. Cerebral hypoxia occurs through a combination of impaired brain adaptation and cerebral vasoconstriction. Brain adaptation consists largely of brain cell loss of sodium and potassium by means of the Na-K adenosine triphosphatase (ATPase) system. There is also loss of organic osmolytes. The brain Na-K ATPase system is impaired by a combination of vasopressin plus estrogen and is stimulated by testosterone. Similarly, vasopressin plus estrogen leads to cerebral vasoconstriction, resulting in a decrement of brain oxygen utilization and cerebral blood flow. Vasopressin also directly decreases brain production of ATP. The combination leads to hypoxic brain damage, which appears to be the major cause of brain damage associated with hyponatremic encephalopathy. Measurement of arterial P o 2 in patients with symptomatic hyponatremia usually demonstrates a P o 2 <50 mm Hg. Improper therapy is another possible cause of brain damage in patients with hyponatremic encephalopathy. The type and distribution of such lesions are similar to those found in patients with hyponatremic encephalopathy who have severe hypoxia. Current scientific knowledge indicates that patient survival can be improved through aggressive treatment of hypoxia associated with hyponatremic encephalopathy, particularly in young women.

Hyperosmotic activation of the CD95 death receptor system

Apoptosis is characterized by cell shrinkage, nuclear condensation, DNA fragmentation and apoptotic body formation. These features distinguish apoptosis from other types of cell death, such as necrosis. Whereas some signs of apoptosis, such as externalization of phosphatidylserine, altered mitochondrial function or activation of caspases are cell type- and death signal-dependent, apoptotic cell volume decrease (AVD) is an early and ubiquitous event and little is known about the signalling events, which are localized upstream of the plasma membrane transport steps leading to AVD and the proapoptotic events, which are induced by osmolyte loss and cell shrinkage. In hepatocytes hyperosmotic shrinkage sensitizes the cells towards CD95 ligand-induced apoptosis by activating the CD95 system. This complex process with a NADPH oxidase-derived reactive oxygen species signal as an important upstream event, allows via Yes, JNK and epidermal growth factor-receptor activation for CD95 tyrosine phosphorylation as a prerequisite for CD95 targeting to the plasma membrane and formation of the death inducing signalling complex. Other covalent modifications such as CD95-tyrosine-nitration or CD95-serine/threonine-phosphorylation can interfere with the CD95 activation process. The findings not only provide a mechanistic explanation for the high susceptibility of dehydrated cells for apoptosis, but also give insight into the role of AVD.

Phylogeny of anion exchangers: Could trout AE1 conductive properties be shared by other members of the gene family?

A phylogenetic tree of anion exchangers (AE) was performed in order to better understand relationships between the different known AE and how they arose. Indeed, the different known AE1 from mammals or fish do not exhibit the same transport features: all studied anion exchangers 1 (AE1) catalyse an electroneutral Cl −/HCO 3 − exchange through the plasma membrane; however, trout AE1 (tAE1) is able to spontaneously form an anion conductive pathway permeable to some inorganic cations (Na + and K +) as well as to organic osmolytes such as taurine. Therefore, it has been proposed that this major erythrocyte membrane protein could play a key role for the cell volume regulation of trout red cells. By analogy, it was envisioned that other fish anion exchangers could play a similar role in osmolyte loss induced by erythrocyte swelling. We have cloned AE1 from Raja erinacea and Danio rerio and studied their properties after expression in Xenopus laevis oocytes. In this study, we show that none of them is able to induce any conductive pathway or taurine permeability in Xenopus oocytes. Our phylogenetic analyses show that, first, all present AE1 genes have a common ancestor distinct from that of AE2 and AE3 and second, tAE1 is a true AE1 ortholog. The question of whether tAE1 conductive properties are a derived character in the trout lineage within Euteleostei or whether other AE1 members can share these properties is then discussed.

Thymocyte K+, Na+ and Water Balance During Dexamethasone- and Etoposide-Induced Apoptosis

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant density of the thymocytes in a continuous Percoll gradient. The K⁺ and Na⁺ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 µM Dex for 4-5.5 h or 50 µM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K⁺ content, which exceeded the simultaneous increase in cellular Na⁺ content. Cellular loss of K⁺ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K⁺ concentration was less than one third. Due to gain of Na⁺ and loss of cell water the cytosolic Na⁺ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.

Is isosmotic MgSO 4 isotonic with Condylactis gigantea tentacles? Comparison of their ionosmotic response to H 2O–diluted seawater before and after exposure to isosmotic MgSO 4–sodium seawater mixtures

Tentacles of Condylactis gigantea were exposed successively to H 2O–NaASW dilutions, isosmotic 1 mol/l MgSO 4–NaASW mixtures and returned to H 2O–NaASW dilutions to explore the possible isotonicity of isosmotic MgSO 4 and the reversibility of the effects of this solution on the ionosmotic characteristics of the tentacles. Total tissue water responded as predicted by van't Hoff's osmotic relation to H 2O–diluted seawater but was not clearly affected by the admixture of seawater with isosmotic MgSO 4, suggesting that this solution may act as an isotonic medium. However, total tissue water, potassium, chloride and sodium contents, as well chloride and sodium spaces were lower in MgSO 4–NaASW mixtures than in H 2O–diluted seawater as a consequence of the loss of these electrolytes and presumably other osmolytes accompanied by an osmotic water loss. The loss of osmolytes did not appear to be compensated by MgSO 4 uptake by the tentacles with a consequent reduction of the cell compartment. Therefore, in MgSO 4–NaASW mixtures, inulin space approached total tissue water, resulting in considerable uncertainty in the estimation cell volume. When the tentacles were re-exposed to H 2O–diluted seawater, tissue ionic contents and ionic and inulin spaces did not revert completely to the values obtained previous to exposure to isosmotic MgSO 4. Therefore, although 1 mol/l MgSO 4 solution was isosmotic with artificial seawater, it could not efficiently replace the latter as an isotonic medium.

Regulation of the volume-sensitive taurine efflux pathway in NIH3T3 mouse fibroblasts.

Osmotically swollen NIH3T3 fibroblasts regulate their cell volume towards their initial value just like most mammalian cells1. This back regulation, designated regulatory volume decrease (RVD), is the result of loss of KCl and organic osmolytes1,2. It is estimated that the loss of cellular amino acids constitutes 20% of the total loss of osmolytes following exposure to a medium with half osmolarity2. The swelling-induced K+ efflux is partly via a K+ selective pathway and partly via a K+, Cl- cotransporter1,3, whereas the swelling-induced Cl- efflux is Ca2+ independent and via a non-selective anion pathway, that exhibits moderate outward rectification as well as time dependent inactivation1,3. Taurine is an important organic osmolyte in NIH3T3 cells and the swelling-induced taurine release from NIH3T3 cells is Na+-independent and mediated via a leak pathway that accepts a variety of neutral amino acids2. The volume-sensitive organic osmolyte channel (VSOAC), that accepts Cl- as well as a broad range of organic osmolytes, has been proposed as the volume-sensitive taurine efflux pathway in mammalian cells4. However, the time courses for the swelling-induced taurine efflux and Cl- efflux in NIH3T3 cells differ with respect to the time point for maximal activation and time point for inactivation2. Furthermore, the swelling-induced Cl- loss and the swelling-induced taurine loss from NIH3T3 cells diverge with respect to their sensitivity to anion channel blockers2, kinase inhibitors3 and expression of constitutive Rho3.KeywordsFocal Adhesion KinaseNIH3T3 CellProtein Tyrosine PhosphataseRegulatory Volume DecreaseEhrlich Ascites Tumor CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl −/HCO 3 − exchanger. Replacement of Cl − by gluconate, and complexation of cupric ions with histidine abolish rapid Cu 2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe 2+ to Cu 2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na +/H + exchange and residual Na + transport causing net Na + accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na + accumulation is accompanied by K + loss occurring mainly via K +–Cl − cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na + uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.

Progressive tissue injury in infantile hydrocephalus and prevention/reversal with shunt treatment

Infantile hydrocephalus, despite shunt treatment, can leave children with a variety of persistent neurological deficits. A rat strain (H-Tx) with inherited fetal-onset hydrocephalus, is a natural model for the study of progressive tissue changes resulting from hydrocephalus and the effects of shunt placement. The cerebral cortex of rat pups has been studied at post-natal day 4 (P4), early stage hydrocephalus and equivalent to a third trimester human fetus, at Pi 1, intermediate stage hydrocephalus and equivalent to a newborn human infant, and at P2I at advanced stage hydrocephalus. At P4, there is interstitial edema (increased water, sodium and chloride) and a non-reversible change in membrane lipids, particularly the phosphomonoesters. By PI I, there are additional, non-reversible, changes in intracellular potassium and energy metabolites (ATP and phosphocreatine). At P2I, the cells are severely damaged and further intracellular changes include a decrease in N-acetylaspartate (NAA) and loss of amino acids and many organic osmolytes. The interstitial edema is ~75% reversed after shunt treatment. The loss of energy metabolites, NAA and osmolytes can be prevented by early shunt treatment at P4, but the subsequent potassium loss is not prevented. Shunt at PI 1 does not prevent loss of NAA or aspartate, but osmolytes are normalized. It is concluded that persistent tissue damage is initiated by changes in cell membrane components leading to a decrease in energy metabolism and loss of cell homeostasis. A more complete understanding of the mechanisms involved could lead to new approaches for therapy. [Neurol Res 2000; 22: 89-96]