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

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

Apparent anion imbalance in the fresh water adapted eel

Summary On adapting brackish waterAnguilla anguilla to fresh water it was noted that, while the plasma sodium, magnesium,pCO₂ and pH were held reasonably constant, there was a substantial fall in chloride concentration (–33 mEq). The gradient of the linear correlations between plasma sodium and chloride also fell (brackish water gradient=0.92, fresh water gradient=0.21) indicating that a new pattern of plasma ion interrelationships was being established. Comparison with plasma Na/Cl ion ratios from other fishes suggested that this phenomenon was peculiar toA. anguilla. Corresponding with the very low plasma chloride levels plasma bicarbonate was four to five times that found in other fishes, and this was thought related to the finding that the haematocrit value almost doubled during adaptation to fresh water. In fresh water adapted fish a fall in plasma chloride was associated with a rise in plasma bicarbonate, however the charge compensation effect of this response was only partial as summing the common plasma cations and anions left an anion deficit of about 34 mEq to be accounted for.     

SODIUM AND CHLORIDE FLUXES IN SYNAPTOSOMES IN VITRO

Synaptosomes incubated in a physiological saline extrude sodium and take up potassium. As would be expected this process is completely blocked by metabolic inhibitors such as cyanide and iodoacetate. However, when metabolic inhibitors are replaced by ouabain (100 μM) there is an increase in the steady state intrasynaptosomal sodium and chloride content even though there is no change in the potassium content. The increases are prevented when synaptosomes are incubated with metabolic inhibitors in addition to ouabain. There is therefore a ouabain-insensitive process that transports sodium, chloride and concomitantly water into synaptosomes. It appears not to function when the supply of metabolic energy is inhibited. The diuretic furosemide (1 mM) in the presence of ouabain inhibits the entry of sodium and chloride without affecting the intrasynaptosomal potassium concentration. Ethacrynic acid (1 mM) has a somewhat similar effect but in addition appears to damage the synaptosome membrane. Kinetic measurements were made of the uptake of sodium, potassium and chloride under conditions of metabolic inhibition and the permeability constants of the membrane determined. Values of 0.068, 0.117 and 0.032 × 10^-6 (cm s^-1) were found for the permeability constants of the membrane to (respectively) sodium, potassium and chloride. Measurements of the rate of uptake in the presence of ouabain revealed an inwardly directed sodium and chloride flux of 5-20 pmol cm^-2 s^-1. Calculation of the fluxes from the steady state ion concentrations also reveals an inwardly directed sodium and chloride flux, though of lesser magnitude. The influx of water is less than would be expected to preserve osmotic equality suggesting that the translocation of sodium and chloride is the primary event. Although its function remains uncertain the flux has a considerable effect on the ion content of synaptosomes.     

Cation-Anion Balance during Potassium and Sodium Absorption by Barley Roots

Steady-state rates of potassium ion and sodium ion absorption by excised barley roots accompanied by various anions were compared with the rates of anion absorption and the concomitant H⁺ and base release by the roots. The cation absorption rates were found to be independent of the identities, concentrations, and rates of absorption of the anions of the external solution, including bicarbonate. Absorption of the anion of the salt plus bicarbonate could not account for the cation absorption. H⁺ is released during cation absorption and base during anion absorption. The magnitude by which one or the other predominates depends on the relative rates of anion and cation absorption under various conditions of pH, cation and anion concentration, and inhibitor concentrations. The conclusion is that potassium and sodium ions are absorbed independently of the anions of the absorption solution in exchange for H⁺, while anions are exchanged for a base. The H⁺ release reflects a specificity between K⁺ and Na⁺ absorption such that it appears to be H⁺ exchanged in the specific rate-limiting reactions of the cation absorption.     

Cation and anion balance in the xylem exudate of tobacco roots

Summary The sum of Na, K, Ca, Mg in the exudate of tobacco generally exceeded the sum of mineral anions. Insufficient organic acids were present to account for the differences and bicarbonate appeared to be the other anion involved. Amino acids were present in very low concentrations relative to mineral cations. When nitrate salts only were in the external solutions, the anions were mostly, but not entirely, nitrate. When chloride salts only were in the external solutions, the cations far exceeded the level of mineral anions in the exudate. It is postulated that nitrate is actively transported when nitrate salts are in the external solution regardless of the cation, but when anions other than nitrate are in the external solution, the cations are actively transported with the anions passively following. Nitrate transport was via a symplasm, but that of the other anions seemed to be different. When bicarbonate is the only anion in the external solution and when present at relatively high concentrations (5 × 10⁻³ M or higher), the volume of exudate is decreased. It appears that the organic acids which were synthesized as a result of the bicarbonate absorption were not transferred to the xylem vessels.     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Dielectric properties of synaptosomes isolated from rat brain cortex

Dielectric measurements were performed on the suspensions of synaptosomes isolated from rat brain cortex. The synaptosomes in buffered salt media showed typical dielectric dispersions caused by the presence of a thin limiting membrane of sufficiently low conductivity. An analysis of the dielectric data revealed that the electric conductivity of the synaptosome interior was about 37 % of the external medium conductivity under isotonic conditions and that the dielectric constant for the interior phase was about 35. The membrane capacitance (0.7 ΜF cm⁻²) remained constant irrespective of nature and concentration of the univalent salts examined. Significant reduction in both the conductivity and the dielectric constant of the internal phase can be explained theoretically provided that some intra-synaptosomal structure (synaptic vesicles and/or small mitochondria) of non-conducting nature occupies about 50 % of the particulate volume, the remainder being in equilibrium with the external salt medium.     

Indirect photometric ion chromatographic analysis of anions in Haematococcus pluvialis culture media

An indirect photometric ion chromatographic method for the simultaneous determination of chloride, nitrate and sulfate ions was developed and applied to the determination of anions, mainly nitrate, in the alga Haematococcus pluvialis culture media. Using phthalic acid/sodium tetraborate aqueous solution as the mobile phase, anions can be detected indirectly by a UV detector. The calibration curves for these anions gave good linearity from 1 to 1000 g ml^–1.     

Properties of an anion/H+ contransport system in L1210 cells that utilizes phthalate as a nonphysiological substrate

Summary [¹⁴C]Phthalate is transported into L1210 cells via two separate routes, an anion exchange system whose primary substrates are folate compounds, and a second less active system which is sensitive to bromosulfophthalein. When the principal uptake component was blocked by a specific irreversible inhibitor of this system, the remaining route (at pH 7.4) appeared to be saturable and was inhibited by several anions in addition to bromosulfophthalein (K _i =2 m), including 8-anilino-1-naphthalein sulfonate (K _i =25 m), unlabeled phthalate (K _i =500 m), and chloride (K _i =3500 m). A pronounced effect by pH was also observed. Influx and total uptake of phthalate both increased progressively with decreasing pH and reached values that were 20-fold higher at pH 6.0, compared with pH 7.4. This pH-dependent increase could be blocked, however, by the addition of compounds (nigericin and carbonylcyanidem-chlorophenylhydrazone) which, in combination, collapse proton gradients. Phthalate efflux was relatively insensitive to changes in extracellular pH but could be inhibited (up to 90%) by bromosulfophthalein. Several other anions also inhibited efflux, but to a lesser extent, while chloride, phthalate, lactate, glycolate and acetate enhanced efflux up to 1.8-fold. Efflux also increased at pH 6.0, but not at pH 7.5, upon addition of nigericin and carbonylcyanidem-chlorophenylhydrazone. These results suggest that phthalate is a nonphysiological substrate for a carrier system which mediates transport via an anion/H⁺ symport mechanism. This system is not the lactate/H⁺ symport carrier of L1210 cells since: (A) phthalate and lactate influx were inhibited to differing degrees by various anions; and (B) lactic anhydride inhibited the influx and efflux of lactate but had no effect on the transmembrane movement of phthalate. The specificity of this system suggests that its primary anion substrate may be chloride.     

Role of synaptosomal Na-accumulation in transmitter release

The mechanism whereby Na⁺, K⁺-ATPase inhibitors such as ouabain trigger transmitter release in a calcium-independent manner remains obscure. We have examined the possible role of intra-synaptosomal sodium ion accumulation in ouabain-induced acetylcholine (ACh) release by: 1) Measuring²²Na accumulation in cat cortical synaptosomes in the presence of ouabain, A23187, veratridine, or strophanthidin over the same time course in which we previously determined their effects on ACh release; and 2) measuring synaptosomal²²Na accumulation and ACh-release in the presence of ouabain plus tetrodotoxin in normal or calcium-free buffer. Our results indicate that tetrodotoxin-dependent²²Na accumulation is at least partially responsible for ouabain-induced ACh release in normal and calcium-free media, but that this ion-accumulation per se is not sufficient to elicit release with other secretogogues.Dedicated to Henry McIlwain.     

Plant cation-anion balance as affected by the ionic composition of the growing medium

Plants that remove an excess of cations over anions may cause soil acidification. The acidification potential of plants has been evaluated using solution culture techniques, but the influence of ionic composition of the medium on the plant cation-anion balance remains unclear. Our objective was to determine how electrolyte concentration and salt type affect the cation- anion balance of two test plants [barley (Hordeum vulgare L.) and kochia (Kochia scoparia L. Schrad.)]. Seedlings were grown in sand culture and irrigated with nutrient solution (Hoagland’s solution), which was adjusted to a range of electrolyte concentrations (target electrical conductivity of 7.5, 17.5 and 27.5 dS m⁻¹) using either chloride or sulphate salts. Increase in electrolyte concentration reduced yield of kochia, a salt-tolerant plant, by up to 38%. Total cation (Ca + Mg + K + Na) equivalents in kochia exceeded those of anions (Cl + S + P + NO₃) by 250 to 280 cmol_c kg⁻¹ of dry matter. Electrolyte concentration had no effect on the cation-anion balance of kochia, but excess cation values were significantly greater in the sulphate than in the chloride system. Kochia had a large content of water-soluble oxalate (194 to 226 cmol_c kg⁻¹), which was linearly related to the excess cation content. Growth of barley was severely restricted at the intermediate and high electrolyte concentrations. Cations exceeded anions by 21 to 59 cmol_c kg⁻¹ of barley dry matter. Excess cation content was greater in the sulphate than in the chloride medium, but electrolyte concentration did not have a consistent effect on the cation-anion balance. The small amounts of oxalate found in barley (0.9 to 2.6 cmol_c kg⁻¹) were insufficient to balance the cation excess.     

Calcium-dependent anion channel in the water mold,Blastocladiella emersonii

Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr²⁺ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr²⁺. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.     

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.     

Perturbation of Anion Balance during Inhibition of Growth of Escherichia coli by Weak Acids

During inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm. This study was undertaken to determine the impact of anion accumulation on cellular pools. At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K⁺ and Na⁺ levels. At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion. However, at high osmolarity, glutamate compensated for over half of the accumulated acetate. Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.     

Inhibition by trimethyl-tin,probenecid and phenylglyoxal of depolarization-induced acetylcholine release in Torpedo synaptosomes

The effects of trimethyl-tin (anion-hydroxyde ionophore, inhibiting oxydative phosphorylation and H⁺-ATPase) probenecid (inhibitor of anion transport in neural cells) and phenylglyoxal (arginine-specific reagent, inhibiting chloride exchanges in erythrocytes) were examined in Torpedo synaptosomes prepared from electric organ. All drugs significantly reduced the stimulated release of acetylcholine triggered by depolarization of nerve endings with high-K⁺ and/or gramicidin D. In contrast, trimethyl-tin, probenecid and phenylglyoxal did not affect the ionophore A23187-induced release of acetylcholine from the synaptosomes. The inhibitory potency of the compound trimethyl-tin was found to be similar to that of probenecid and phenylglyoxal on depolarization-induced acetylcholine release. This leads us to suggest that a relationship exists between modification of anion distribution during depolarization and acetylcholine release process. Moreover, since the release of ACh by calcium-ionophore A23187 was unaffected by trimethyl-tin, probenecid or phenylglyoxal, such compounds may also have an action on voltage-dependent Ca²⁺ flux across presynaptic membrane.     

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.     

Ion permeability of maize root membrane vesicles: Studies with light scattering

A possible modulation of permeabilities of membrane vesicles to anions and cations was explored by light scattering techniques, evaluated by measuring the capacity of the vesicles to shrink and swell in response to changes of the osmolarity of the incubation medium. Membrane fractions were obtained by phase partition. Purity was evaluated by detection and quantification of membrane enzyme markers: vanadate-sensitive ATPase for the plasma membrane, nitrate-sensitive ATPase for the tonoplast and azide-sensitive ATPase for mitochondria. Membrane vesicles (250 g protein) were exposed to hypertonic solutions of salts (0.6 osmolar). Kinetics of the changes in apparent absorbance at 546 nm were observed by the addition of potassium, nitrate and chloride salts. The diffusion of ions into vesicles was induced by an osmotic gradient across the membrane and brought about volume changes of vesicles. Upon addition of vesicles to the different solutions the following ion permselectivity sequences were observed: PNO ₃ ^– >PCl ^–>PSO ₄ ^2– and PK ⁺PNa ⁺>PNH ₄ ⁺.Abbreviations ATP adenosine 5-triphosphate - EDTA ethylene diaminetetraacetic acid - Tris-Mes (Tris[hydroxymethyl]aminomethane, Mes-(2-[N-Morpholino]ethanesulfonic acid) - PEG polyethylene glycol     

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature-dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent K_m value of 0.9μM and a V_max value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na⁺ with choline chloride in the medium. Ca²⁺ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co²- or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15-min incubation period was released under the same conditions. [³H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl-evoked release mechanisms were found to be at least partially Ca²⁺-dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca²⁺-independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.     

Outward-rectifying chloride channels in cultured adult and fetal human nasal epithelial cells

Summary The patch-clamp technique was used to characterize ion channels in the apical membranes of cultured human nasal epithelial cells, dissociated from fetal nasal mucosa and from adult nasal polyps. Outward-rectifying chloride channels were found in 4.3% of the cell-attached patches from fetal cells (n=258) and in 3.1% of the patches from adult cells (n=320). After exeision the number of patches containing active chloride channels increased threefold to 13% of the patches from the fetal cells and 10% from adult cells. The single-channel conductance at 0 mV in symmetrical 150mm NaCl solutions was 24.3 ±0.9 pS (n=28) and 26.0 ± 1.2 pS (n=30), respectively, in adult and fetal cells and showed outward rectification in the potential range from –80 to +80 mV. In fetal cells as well as in adult cells the channels were anion selective, and were almost impermeable for larger anions and monovalent cations. In cell-free patches the channels were Ca²⁺ independent. In most of the channels the open probability was voltage independent and high (±0.86); in 20% of the channels, however, the open probability increased with depolarization. In conclusion, fetal nasal epithelial cells contain chloride channels in their apical membranes with singlechannel properties and regulatory mechanisms similar to those found in cells from adults.     

How pH regulates a pH regulator: a regulatory hot spot in the N-terminal cytoplasmic domain of the AE2 anion exchanger

Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na⁺-independent chloride-bicarbonate exchanger genes and multiple Na⁺-bicarbonate cotransporter and Na⁺-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na⁺-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.