Short-term changes in natripheric and hydrosmotic water fluxes across the skin and in urine production due to increases in the osmolarity of the external environment in the toad

1. Sudden decreases in the osmotic gradient across the skin due to the replacement of water of the bath by 115 mM NaCl had no effect on water uptake of intact or hypophysectomized toads. 2. A concomitant decrease in the urine production was observed in intact but not in hypophysectomized animals. 3. Addition of amiloride chlorydrate (0.25 mM) to the 115 mM NaCl bath induced a significant decrease in water uptake both in intact and in hypophysectomized toads. 4. The osmotic permeability coefficient (LPD) increased significantly during the osmotic gradient reduction with 115 mM NaCl plus 0.25 mM amiloride or 230 mM sucrose in both groups. 5. No changes in the plasmatic osmolarity were detected during the development of these responses to the osmotic gradient reduction. 6. These results are consistent with the hypothesis of short-term changes in the natripheric and hydrosmotic fluxes of water across the skin and in urine production triggered by the osmotic gradient reduction. The possible participation of arginine vasotocin in these responses is discussed.     

Potassium secretion by the cortical collecting tubule

The isolated perfused rabbit cortical collecting tubule has been shown to actively transport K from bath to lumen. The first step in this process is active uptake of K across the basolateral membrane via and Na:K exchange pump as evidenced by: 1) basolateral localization and Na:K exchange properties of the ouabain-sensitive Na,K-ATPase, 2) ouabain sensitivity of the Na and K fluxes, 3) interdependence of the Na and K fluxes, and 4) ouabain-sensitivity of 42K uptake into the cell across the basolateral membrane. At the luminal border, a significant K permeability of the apical cell membrane has been identified using electrophysiological techniques. This K permeability is insensitive to the diuretic amiloride, and, thus, differs from the pathway for Na entry, which is highly amiloride sensitive. A significant K permeability of the paracellular pathway is not apparent. It is concluded that K secretion by the rabbit cortical collecting tubule occurs via a two-step process: active uptake of K across the basolateral membrane via the Na:K exchange pump, followed by passive efflux of K across the apical membrane via an amiloride-insensitive K conductive pathway.     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

Effect of aldosterone and insulin on mannitol, Na+ and Cl- fluxes in cultured epithelia of renal origin (A6): evidence for increased permeability in the paracellular pathway

Transepithelial fluxes of mannitol, Na+ and Cl- were measured under open circuit conditions in cultured epithelia derived from toad kidney (A6). Both aldosterone and aldosterone plus insulin produced significant increases in the apparent permeability to mannitol (40 and 83%, respectively). Na+ permeabilities calculated from basolateral to apical Na+ fluxes showed approximately the same percentage increases in response to aldosterone and aldosterone plus insulin. Cl- permeabilities calculated from basolateral to apical Cl- fluxes did not show the same percentage increases. The flux ratios for Cl- were significantly lower than would be predicted for simple electrochemical diffusion in both control and hormone-treated epithelia. In aldosterone-treated epithelia, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) caused Cl- flux ratios to approach predicted values. The unidirectional Cl- fluxes may have significant contributions from both the transcellular and paracellular pathways, with the direction of departure from predicted values being consistent with the presence of Cl- exchange diffusion. In aldosterone plus insulin-treated epithelia, amiloride significantly reduced both the mannitol and Na+ permeabilities. These findings are consistent with aldosterone- and aldosterone plus insulin-induced increases in paracellular pathway permeability which may be secondary to the change in active Na+ transport rather than a primary effect.     

Ion selectivity of the apical membrane Na channel in the toad urinary bladder

The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H greater than Li greater than Na much greater than K. K permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

Regulation of the permeability of the medaka fish embryo chorion by exogeneous sodium and calcium ions

We questioned if the optically transparent noncellular chorion, or egg envelope, which encapsulates the entire medaka fish (Oryzias latipes) embryo might in some way constitute a permeability barrier to high concentrations of the diuretic called amiloride. More specifically, we questioned if removal of cations from the exogenous environment of the medaka embryo might make the chorion more permeable to amiloride and thereby make the fish embryos more sensitive to the inhibitory and lethal effects of this drug. To test this question, chorion-encapsulated medaka embryos were exposed to: deionized-distilled water, to Yamamoto-Ringer's (Y-R) solution, to Yamamoto-Ringer's containing choline chloride as a substitute for NaCl, and to isotonic NaCl solution in the presence of and in the absence of amiloride. Briefly, the prediction that the medaka embryos would be most sensitive to amiloride's inhibitory effects in distilled water was confirmed. Further studies showed that the presence of Na+ or of Ca2+ alone in the culture solution gave partial protection against the lethal effects of the amiloride. Electron probe X-ray microanalysis studies indicated that addition of Ca2+ and other cations to the culture solution caused the concentrations of cations to increase in the chorion, and that increase was correlated to a visible decrease in the permeability of the chorion to the amiloride. This decreased permeability of the chorion apparently protected the embryo from the amiloride. The decreased permeability of the chorion to amiloride, which occurred in the presence of the cations present in Y-R solution, was found to be reversible once the cations were washed from the chorion. Key words medaka, chorion, Na+, Ca2+ permeability, x-ray microanalysis, Oryzias latipes, egg envelope.     

The ATP4- receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.

Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. I. Sodium fluxes

The nature of Na+ fluxes in resting and in chemotactic factor-activated human neutrophils was investigated. In resting cells, ouabain-insensitive unidirectional 22Na+ in- and effluxes represented passive electrodiffusional fluxes through ion channels: they were nonsaturable and voltage-dependent (PNa = 4.3 X 10(-9) cm/s). Amiloride (1 mM) had little effect on resting 22Na+ influx (approximately 0.8 meq/liter X min), thereby suggesting a minor contribution of Na+/H+ exchange and a lack of amiloride-sensitive Na+ channels. When neutrophils were exposed to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM), 22Na+ influx was stimulated approximately 30-fold (initial rate approximately 22 meq/liter X min). The FMLP-induced 22Na+ influx was saturable with respect to external Na+ (Km 26-35 mM, Vmax approximately 28 meq/liter X min), was electroneutral, and could be competitively inhibited by amiloride (Ki 10.6 microM). From a resting value of approximately 30 meq/liter of cell water, internal Na+ in FMLP-stimulated cells rose exponentially to reach a concentration of approximately 60 meq/liter by 10-15 min. This uptake was blocked by amiloride. FMLP also stimulated the efflux of 22Na+ which followed a single exponential time course (rate coefficient approximately 0.16 min-1). The FMLP-induced 22Na+ fluxes were similar to those observed with 10 microM monensin, a known Na+/H+ exchanging ionophore. The data indicate that FMLP activates an otherwise quiescent, amiloride-sensitive Na+/H+ exchange. Furthermore, all of the FMLP-induced 22Na+ fluxes can be satisfactorily accounted for by transport through the exchanger, leaving little room for an appreciable increase in Na+ conductance.     

Mode of action of amiloride in toad urinary bladder

Summary The effect of amiloride on the sensitivity to Na of the mucosal border of toad urinary bladder was investigated by recording Na concentration-dependent transepithelial potential difference (V _t ) and the intracellular potential. When mucosal Na concentration was normal, amiloride added to the mucosal solution at 10^–4 m markedly reduced the mucosal membrane potential (V _m ) and altered the potential profile from a two-step type to a well type. Similar changes were observed when Na was totally eliminated from the mucosal medium. The serosal membrane potential was insensitive to amiloride and elimination of mucosal Na. In the absence of amiloride, theV _t could be described by the Goldman-Hodgkin-Katz equation in the range of mucosal Na concentration from 0 to 16mm, and amiloride extended this concentration range. By using the Goldman-Hodgkin-Katz equation, Na permeability was calculated from the data ofV _t 's obtained in the allowed ranges of Na concentration and compared before and after the addition of amiloride. The results show that Na permeability decreases to 1/600 of control when the maximum dose of amiloride (10^–4 m) is applied. The relationship between Na permeability and amiloride concentration is well explained on the basis of assumptions that amiloride binds to the Na site of the mucosal border in one-to-one fashion and in a competitive manner with Na and that Na permeability reduces in proportion to increase in number of the sites bound with amiloride.     

External anions regulate stilbene-sensitive proton transport in placental brush border vesicles

The mechanism for HCO3-(-)independent proton permeability in microvillus membrane vesicles (MVV) isolated from human placenta was examined by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Proton fluxes (JH) across MVV were determined in response to induced pH and anion gradients from the time course of 6CF fluorescence, the MVV buffer capacity, and the 6CF vs. pH calibration. In the absence of anions, JH was 12 +/- 2 nequiv s-1 (mg of protein)-1 (pHin 7.4, pHout 6.0, MVV voltage-clamped with K+/valinomycin, 23 degrees C), corresponding to a proton permeability coefficient of 0.02 cm/s, with an activation energy of 9.1 +/- 0.3 kcal/mol. JH was inhibited 20% by dihydro-4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (H2DIDS) with KI = 8 microM [( Cl-]out = 0 mM). For a 0.5-unit pH gradient JH increased from 1.5 to 4.6 nequiv s-1 (mg of protein)-1 as the internal MVV pH was increased (5.5-7.5). External Cl-, Br-, and I- (but not SO4(2-) and PO4-) increased JH 1.3-2.5-fold for both inwardly and outwardly directed pH gradients with KD = 1.0 +/- 0.4 mM (Br-) and greater than 100 mM (Cl-). This increase was blocked by 100 microM H2DIDS but not by amiloride or furosemide. Internal Cl- did not alter JH induced by pH gradients nor were proton fluxes induced by anion gradients in the absence of a pH gradient. Experiments in which JH was driven by membrane potentials (induced by valinomycin and K+ gradients) indicated that proton transport was voltage-sensitive. These experiments demonstrate a stilbene-sensitive electrogenic proton transport mechanism in MVV that is regulated allosterically by anions at an external binding site.     

Ion selectivity of the apical membrane Na channel in the toad urinary bladder

Summary The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H>Li>NaK. K, permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.     

Sodium-hydrogen exchange system in brush border membranes from cortical and medullary regions of the proximal tubule

The Na+/H+ exchange system was studied in brush border membrane vesicles isolated from cortical and medullary regions of the proximal tubule of rabbit kidney. The activity of the exchanger was assessed by measuring hydrogen influx (monitored by acridine orange fluorescence), 22 Na influx and the sensitivity of these fluxes to amiloride and its analogue ethylisopropyl amiloride. In contrast to previously published data (indicating the absence of pH-gradient driven and amiloride sensitive 22Na-influx in medullary site vesicles (13, 15], Na+/H+ exchange activity could be detected in both membrane preparations by sodium tracer and fluorescence detection of hydrogen influx. Amiloride inhibition of 22Na influx was more effectively protected by increasing sodium concentration in cortical than in medullary vesicles, suggesting differences in the action of amiloride in these preparations.     

Amiloride, protein synthesis, and activation of quiescent cells

Amiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.     

Cellular Inhomogeneity in Dog Red Cells As Revealed by Sodium Flux

Unidirectional ²⁴Na fluxes across the dog red blood cell membrane were measured. The kinetics were incompatible with a single time constant but could be accounted for in terms of a two-series compartment cell model, with approximately 1% of cell Na in the outer compartment. Dog red blood cells are known to be inhomogeneous in their Na and K permeabilities. Theoretical analysis showed that such cellular inhomogeneity in the Na permeability coefficient might in principle account for the flux data. In order to evaluate the inhomogeneity effect, a technique based on the differential response of cells suspended in isosmolar high K buffers was devised to measure the variations in Na permeability in the cell population. A variation in the Na permeability coefficient of approximately 30% was found. This inhomogeneity is insufficient to account for the flux data.     

Effect of physicochemical parameters on skin permeability of antihypertensive

Studies were carried out to establish a correlation of skin permeability with physicochemical parameters using five antihypertensive drugs. In vitro skin permeation was carried out in vertical type diffusion cells. When steady-state fluxes of the drugs were correlated with physicochemical properties, good correlation was obtained with the reciprocal of melting point. Weak correlation was obtained with partition coefficient, molecular weight and solubility. However skin permeability versus solubility profiles revealed an interesting trend. The initial permeation rates of the poorly water soluble drugs, prazosin hydrochloride and reserpine were higher than their steady-state fluxes and moderately water soluble drug atenolol showed more or less similar permeation throughout the entire span of the study. This trend changed gradually and reversed completely in the highly water soluble drug diltiazem hydrochloride. The study suggests that drug derivatives of low melting point and good aqueous solubility could be favorable candidates for transdermal delivery.     

A bicarbonate-dependent process inhibitable by disulfonic stilbenes and a Na+/H+ exchange mediate 22Na+ uptake into cultured bovine corneal endothelium

22Na+ uptake into confluent monolayers of cultured bovine corneal endothelial cells was studied in the presence of ouabain (10(-4)M) to inhibit active sodium extrusion. In bicarbonate saline, uptake was reduced to a similar degree either by amiloride (10(-3)M) or by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-3)M). A further reduction was obtained with SITS-pretreated cells in the presence of amiloride. SITS-sensitive uptake was further characterized in saline containing both ouabain (10(-4)M) and amiloride (10(-3)M). It was absolutely dependent on bicarbonate, which could not be substituted by other plasma membrane permeable buffers (50 mM acetate or 25 mM glycodiazine). It was a saturable function of both bicarbonate and sodium concentration. Half-maximal fluxes occurred between 3 and 7 mM HCO3 (at 151 mM Na) and between 35 and 60 mM Na (at 28 mM HCO3). Uptake into sodium-depleted cells was reduced as opposed to sodium-rich cells, and SITS-sensitive 22Na+ efflux out of 22Na+-loaded cells into sodium-free medium was less than efflux into sodium saline, indicating trans-stimulation by sodium. The amiloride-sensitive pathway was studied in the absence of bicarbonate to inhibit uptake via the SITS-sensitive pathway. 22Na+ uptake into sodium-depleted cells increased steeply with extracellular pH in the range between pH 6 and 8 and could be largely blocked by 10(-3), but not by 10(-5) M amiloride. It is concluded that bovine corneal endothelial cells possess at least two distinct pathways for sodium uptake, amiloride sensitive 22Na+ fluxes being mediated by a Na+/H+ antiport, while the SITS-sensitive process is probably identical to a bicarbonate-sodium cotransport system postulated earlier from electrophysiological studies.     

Intracellular binding of spin-labeled amiloride: an alternative explanation for amiloride's effects at high concentration

Amiloride, an important inhibitor of Na+ transport and Na+/H+ exchange, has been used in nontransporting tissues to investigate the relationship between ionic fluxes or intracellular pH change and proliferative or synthetic events. Reports that amiloride is permeant and had direct effects on intracellular processes have led us to investigate the possibility that amiloride binds intracellularly to nuclei, mitochondria, and to purified nucleic acids. Using a nitroxide spin-labeled derivative of amiloride (ASp) and electron paramagnetic resonance (EPR) spectroscopy, we have demonstrated that nuclei and mitochondria isolated from trout liver bind significant amounts of ASp especially at the high amiloride concentrations (approximately mM) commonly used to inhibit proliferative events. While the chemical component responsible for ASp binding in these organelles was not identified, native DNA binds significant amounts of ASp whereas single stranded DNA and RNA bind much less. When these observations are taken together with reports of amiloride's direct action on cellular processes, they support the possibility that some of the effects attributed to inhibition of a transport event are caused by amiloride directly.     

OZONE-INDUCED MEMBRANE PERMEABILITY CHANGES

The effect of 0.5 ppm ozone for 0.5-1 hr on plant cell membrane permeability was ascertained. Permeabilities to both water and solutes were estimated by measuring leaf disc weight changes and following tritiated water and ⁸⁶Rb fluxes. Measurements were made immediately after ozone exposure and 24 hr after exposure. The reflection coefficient, σ, an index of solute permeability, decreased in ozone-treated primary leaves of pinto bean (Phaseolus vulgaris). The latter indicates an increase in membrane solute permeability or internal solute leakage. Water and THO flux estimates both indicated a decrease in membrane permeability to water; both the hydraulic conductivity (L_p) and the water diffusional coefficient (L_D) apparently decreased, an anomaly which is discussed. These data indicate that ozone has a direct effect on membrane function by altering permeability characteristics. We assume from these data that cell membranes are primary target sites for ozone injury.