Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl⁻ transport inhibitor DIDS and the K⁺ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl⁻ flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl⁻ fluxes rather than blocking cell shrinkage or K⁺ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Quantitative ion localization within Suaeda maritima leaf mesophyll cells

Grown under saline conditions, Suaeda maritima accumulates Na⁺ and Cl^- into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na⁺, K⁺ and Cl^- concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.Abbreviation TAEM transmission analytical electron microscopy     

Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease

The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy—a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30–90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na⁺, while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na⁺, K⁺ and Cl^? channels failed to prevent apoptosis or AVD.     

The relationship between sodium,potassium, and chloride in amphibian muscle

The Na,⁺ Cl^-, and K⁺ content of toad plasma and the sartorius muscle has been determined. Although the Na⁺ and Cl^- level of the muscles in the living animal varied greatly (0 to 38.0 m.eq. per kg., and 0 to 31.8 m.eq. per kg. respectively) the K⁺ level was subject to a smaller variation (76.5 to 136 m.eq. per kg.). There was a direct relationship between Na⁺ and Cl^-, which was independent of the K⁺ level. There is a closely related gain of Na⁺ and Cl^- when muscle is soaked in normal Ringer. These gains are not related to the K⁺ loss, frequently found on soaking. The relationship between the three ions was studied in a large series of 124 muscles in normal Ringer. As found in vivo, there was a correlation between Na⁺ and Cl.^- This correlation was independent of K⁺ content, except when this was abnormally low. Alteration of the external NaCl level produced concomitant changes in the internal levels of these ions. Alteration of the external KCl level produced an increase in internal Cl^- similar to that found with high NaCl solutions, but the amount of K⁺ entering the cell was approximately one-third of the external increase. Removal of K⁺ from the external solution did not result in a loss of K⁺ from the cell, although there was an adequate amount of Cl^- present to accompany it. The results cannot be reconciled with either a Donnan concept for the accumulation of K⁺, or a linked carrier system. A theory is proposed to account for the ionic differentiation within the cell. The K⁺ is assumed to be adsorbed onto an ordered intracellular phase. The normal metabolic functioning of the cell is necessary to maintain the specificity of the adsorption sites. There is another intracellular phase, which lacks the structural specificity for K⁺, and which contains Na⁺, Cl^-, and K⁺ in equilibrium with the external solution. The dimensions of the free intracellular phase will vary from cell to cell, but it will be smaller in the intact animal, and will increase on soaking in normal Ringer, until it is approximately one-third of the total cellular volume. The increase in this phase may be ascribed to a decrease in the energy available to maintain the ordered phase.     

Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease

Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C₆-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K⁺ from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K⁺ concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K⁺ concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane. water movement; major intrinsic protein; channel; enzyme     

Elongation of Outer Transmembrane Domain Alters Function of Miniature K+ Channel Kcv

Apoptosis is an essential process in organ development, tissue homeostasis, somatic cell turnover, and the pathogenesis of degenerative diseases. Apoptotic cell death occurs in response to a variety of stimuli in physiological and pathological circumstances. Efflux of K⁺ and Cl⁻ leads to apoptotic volume decrease (AVD) of the cell. Both mitochondrion-mediated intrinsic, and death receptor-mediated extrinsic, apoptotic stimuli have been reported to rapidly activate Cl⁻ conductances in a large variety of cell types. In epithelial cells and cardiomyocytes, the AVD-inducing anion channel was recently determined to be the volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel which is usually activated by swelling under non-apoptotic conditions. Blocking the VSOR Cl⁻ channel prevented cell death in not only epithelial and cardiac cells, but also other cell types, by inhibiting the induction of AVD and subsequent apoptotic events. Ischemia-reperfusion-induced apoptotic death in cardiomyocytes and brain neurons was also prevented by Cl⁻ channel blockers. Furthermore, cancer cell apoptosis induced by the anti-cancer drug cisplatin was recently found to be associated with augmented activity of the VSOR Cl⁻ channel and to be inhibited by a Cl⁻ channel blocker. The apoptosis-inducing VSOR Cl⁻ channel is distinct from ClC-3 and its molecular identity remains to be determined.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Ion composition of unicellular marine and fresh-water algae,with special reference to Platymonas subcordiformis cultivated in media with different osmotic strengths

Summary Ion compositions (K⁺, Na⁺ Mg²⁺, Ca²⁺, Cl^-, phosphate) of the euryhaline algae, Platymonas subcordiformis, Chlorella salina, grown in media with a salinity range from 0.1 to 0.6 M NaCl and of the fresh-water algae, Ankistrodesmus braunii and Scenedesmus obliquus, were compared. Enhancement of ion concentrations with increasing salinity in Platymonas was attributed largely to decreasing cell volume. In both the euryhaline algae, Na⁺ and — partially — Cl^- content per cell increased significantly with rising salinity. The contents per cell of the other ions were not affected. Considering the relevance of ions and mannitol (Platymonas) and proline (Chlorella) as osmotically active particles, it was found that the ions alone maintained osmotic balance with low external salinity. With increasing salinity the organic compounds contributed up to 20–30% of the cellular solute potential. The main cation, K⁺, was the main contributor to the osmotic balance; the accumulation of organic compounds as well as of Na⁺ and Cl^- contributes further to the ability of the algae to adapt to high salinity. The results confirm the hypothesis of low Cl^- concentrations in nonvacuolate cells in comparison to vacuolate cells.     

渗透胁迫和离子毒害对转Bt基因棉幼苗生长及离子分布的影响

研究了渗透和盐胁迫处理对转Bt基因抗虫棉(Gossypium hirsutum) 99B种子的萌发和幼苗生长的影响,以及幼苗不同器官离子吸收和分配的差异。结果表明:渗透和盐胁迫均对转Bt基因抗虫棉幼苗的生长有抑制作用,其中PEG的抑制作用最强,而3种盐的抑制程度以CaCl₂>NaCl>Na₂SO₄,且在Na⁺含量相同时,Cl^-的毒害大于SO₄^2-。渗透胁迫下使根、茎和叶中的Na⁺和Cl^-含量提高,K⁺、Ca²⁺、SO₄^2-含量和K⁺/Na⁺、Ca²⁺/Na⁺和SO₄^2-/Cl^-比值降低,且地上部的变化幅度大于地下部的,其中以PEG的影响最为显著,其次是CaCl₂,Na₂SO₄处理最弱。这些说明,转Bt基因抗虫棉99B的耐盐性较弱。     

Patterns of ion distribution in selected NaCl tolerant and normal lines of four grass species

Selected NaCl tolerant and unselected control lines ofHolcus lanatus L.,Lolium perenne L.,Dactylis glomerata L., andFestuca rubra L. were grown in sand culture at 0, 100, 200, 250, and/or 300 ml m^-3NaCl for seven weeks. The tolerant lines of all four species produced significantly greater both shoot and root dry matter at all NaCl treatments compared with the unselected control lines. Na⁺, K⁺, Cl^-, Ca²⁺, and Mg²⁺ contents of leaf, stalk, and roots of each species were determined. The tolerant lines ofH. lanatus contained less Na⁺ and less Ca²⁺ but higher K⁺ in shoots, compared with the unselected line. By contrast theL. perenne tolerant line had higher Na⁺ and Cl^- contents at 250, and 300 mol m^-3 NaCl in shoots than the unselected line suggesting a halophytic nature of the tolerant line.D. glomerata accumulated greater quantities of ions compared with the other species examined. The tolerant line contained significantly less Cl^- but more K⁺ in its shoots than the unselected line. Na⁺, Cl^-, and K⁺ contents in the shoots of the tolerant line ofF.rubra were higher than in the unselected line shoots. Therefore selection for NaCl tolerance may provide useful material for examining the basis of tolerance.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

Observations on the regulation of cell volume and metabolic controlin vitro; changes in the composition and ultrastructure of liver slices under conditions of varying metabolic and transporting activity

Summary Liver slices incubated at 1 °C underwent swelling of both cellular and intercellular compartments, as judged by electronmicroscopy. The ultrastructure showed marked changes, including disorganization of the cytocavitary network and plasma membrane and alterations of mitochondria. Restoration of metabolically favorable conditions (oxygenated medium at 38 °C) caused a nearly complete recovery of ultrastructure closely associated with extrusion of water; measurements of inulin space and electronmicroscopy both indicate a recovery of cell volume, with intercellular spaces remaining somewhat expended. The fluid lost was a roughly isotonic solution of Na⁺ and Cl^–, while K⁺ was reaccumulated in exchange for Na⁺. Cyanide prevented recovery. Ouabain and oligomycin each partially prevented fluid extrusion, but had little effect on ultrastructural recovery except to induce intracellular vesicles containing particles of thorium dioxide derived from sinusoidal spaces. The vesicles were, however, markedly different in form with each inhibitor. There are, thus ouabain-sensitive and-insensitive components of volume regulation; the former appears to depend on the coupled transport of Na⁺ and K⁺ and the latter, we suggest, on a secretion of Na⁺ and Cl^– into vesicles which release their contents into the bile canaliculi by an oligomycin-sensitive mechanism. Mitochondria showed conformational changes between orthodox and condensed forms, but these could not be directly related to tissue energy states; the numbers of mitochondrial dense granules bore a closer relation to tissue ATP.     

The Ionic Permeability Changes during Acetylcholine-Induced Responses of Aplysia Ganglion Cells

ACh-induced depolarization (D response) in D cells markedly decreases as the external Na⁺ is reduced. However, when Na⁺ is completely replaced with Mg⁺⁺, the D response remains unchanged. When Na⁺ is replaced with Tris(hydroxymethyl)aminomethane, the D response completely disappears, except for a slight decrease in membrane resistance. ACh-induced hyperpolarization (H response) in H cells is markedly depressed as the external Cl^- is reduced. Frequently, the reversal of the H response; i.e., depolarization, is observed during perfusion with Cl^--free media. In cells which show both D and H responses superimposed, it was possible to separate these responses from each other by perfusing the cells with either Na⁺-free or Cl^--free Ringer's solution. High [K⁺]₀ often caused a marked hyperpolarization in either D or H cells. This is due to the primary effect of high [K⁺]₀ on the presynaptic inhibitory fibers. The removal of this inhibitory afferent interference by applying Nembutal readily disclosed the predicted K⁺ depolarization. In perfusates containing normal [Na⁺]₀, the effects of Ca⁺⁺ and Mg⁺⁺ on the activities of postsynaptic membrane were minimal, supporting the current theory that the effects of these ions on the synaptic transmission are mainly presynaptic. The possible mechanism of the hyperpolarization produced by simultaneous perfusion with both high [K⁺]₀ and ACh in certain H cells is explained quantitatively under the assumption that ACh induces exclusively an increase in Cl^- permeability of the H membrane.     

Mechanisms of Salinity Control in Sea Bass

Sea bass can regulate the concentration of Na⁺, K⁺, and Cl^-, among other ions, in their blood, skin, gills, and kidney. Therefore, the salinity of the water does not have a great influence on their metabolism, and sea bass can live in both sea and freshwater in accordance with the salt concentration. Most salinity control occurs in the gills, primarily through the control of chloride cells present there. The concentration of ions in the blood is controlled by the cotransporter Na⁺ / K⁺ / 2Cl^- (NKCC) in the chloride cell, and the subunits of Na⁺ / K⁺ ATPase (NKA) function to maintain homeostasis. The expression of NKA is regulated by subunits of the protein FXYD, allowing the sea bass to survive in compliance with the salinity. In this way, it is possible for sea bass to live in sea and freshwater by controlling the salinity of its body using functions of various channels, proteins, and genes present in the chloride cells of sea bass. In this study, we investigated recent studies of salt control mechanisms in sea bass and their application.     

Water and electrolyte balance in protoscoleces of Echinococcus granulosus incubated in vitro: general procedures for the determination of water,sodium, potassium and chloride in protoscoleces

Reisin I.L. and Rotunno C.A. 1981. Water and electrolyte balance in protoscoleces of Echimcoccus granulosus incubated in vitro: General procedures for the determination of water, sodium, potassium and chloride in protoscoleces. International Journal for Parasitology11: 399–404. Protoscoleces of E. granulosus (sheep strain) were incubated in vitro at 37°C in Ringer Krebs solution (RKS) for up to 3 h. When they were briefly washed in sucrose 0.3 M at 4°C, the water and electrolyte contents were: 1.768 ± 0.034 mlg^?1 d.w. for water content and 123 ± 2, 209 ± 2 and 78 ± 2 μmolg^?1 d.w. for Na⁺, K⁺ and Cl^? respectively. When protoscoleces were not washed in sucrose solution but were spun down from RKS, the K⁺ content suffered a very small change but larger values for Na⁺ and Cl⁺ contents were obtained. These higher Na⁺ and Cl^? contents are attributed to the RKS ions retained in the trapping space. The steady state distribution of Na⁺ and K⁺ in the protoscoleces incubated at 37°C indicates the activity of an active transport mechanism.     

Fluxes and compartmentation of K+, Na+ and Cl−, and action of auxins in suspension-cultured Petroselinum cells

Transport of ⁸⁶Rb⁺/K⁺, ²²Na⁺, ³⁶Cl^?, and [³H]indole acetic acid (IAA) has been studied on suspension-cultured cells of the parsley, Petroselinum crispum (Mill) Nym. By compartmental analysis two intracellular compartments of K⁺, Na⁺, and Cl^? have been identified and ascribed to the cytoplasm and vacuole; half-times of exchange were around 200 s and 5 h, respectively. According to the Ussing-Teorell flux equation, active transport is required for the influx into the cytoplasm at the plasmalemma (K⁺, Cl^?) and the tonoplast (K⁺, Na⁺, Cl^?). The plasmalemma permeability pattern, P_K:P_Na:P_Cl=1.00:0.24:0.38, features an increased chloride permeability compared with cells from higher plant tissues. IAA uptake showed an exponential timecourse, was half-maximal after 10 min, and a linear function of the IAA concentration from 10^?9 to 10^?5 M. IAA and 2,4-dichlorophenoxy acetic acid reduce the apparent influx of K⁺, Na⁺, Cl^? during the initial 30 min after addition and subsequently accelerate both in- and efflux of these ions. We discuss that auxins could affect the ion fluxes in a complex way, e.g. by protonophorous activity and by control of the hypothetical proton pump.     

Ecophysiologic investigations in the family of the Mesembryanthemaceae

Summary Overnight accumulation of malate, citrate, and isocitrate in a large number of species of Mesembryanthemaceae grown under identical environmental conditions was studied. Of the 27 species investigated, 24 showed malate accumulation, which in 3 cases was accompanied by considerable overnight accumulation of citrate. In the leaves of the same plants, the Na⁺, K⁺, Cl^-, SO₄ ^2-, and PO₄ ^3- contents were determined. Although the plants were not exposed to substrates of high NaCl content, they exhibited extraordinarily high levels of Na⁺ and Cl^-. All plants accumulated, much more Na⁺ than K⁺. No readily discernible correlation between the amount of any particular ion and the extend of CAM was found. It is concluded that halophilism and CAM are widespread phenomena in the family of Mesembryanthemaceae that possess ecologic significance.Abbreviations CAM Crassulacean Acid Metabolism - PEP Phosphoenolpyruvate On leave from Canberra     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.