A component of fluid absorption linked to passive ion flows in the superficial pars recta

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.     

Anion permeation in an apical membrane chloride channel of a secretory epithelial cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

Cation and anion sequences in dark-adapted Balanus photoreceptor.

Anion and cation permeabilities in dark-adapted Balanus photoreceptors were determined by comparing changes in the membrane potential in response to replacement of the dominant anion (Cl-) or cation (Na+) by test anions or cations in the superfusing solution. The anion permeability sequence obtained was PI greater than PSO4 greater than PBr greater than PCl greater than Pisethionate greater than Pmethanesulfonate. Gluconate, glucuronate, and glutamate generally appeared more permeable and propionate less permeable than Cl-. The alkali-metal cation permeability sequence obtained was PK greater than PRb greater than PCx greater than PNa approximately PLi. This corresponds to Eisenman's IV which is the same sequencethat has been obtained for other classes of nerve cells in the resting state. The values obtained for the permeability ratios of the alkali-metal cations are considered to be minimal. The membrane conductance measured by passing inward current pulses in the different test cations followed the sequence, GK greater than GRb greater than GCs greater than GNa greater than GLi. The conductance ratios obtained for a full substitution of the test cation agreed quite well with permeability ratios for all the alkali-metal cations except K+ which was generally higher.     

Chloride reabsorption by renal proximal tubules of necturus

Summary Movement of Cl from the lumen ofNecturus proximal tubule into the cells is mediated and dependent on the presence of luminal Na. Intracellular Cl activity was monitored with ion selective microelectrodes. In Cl Ringer's perfused kidneys, cell Cl activity was 24.5±1.1mm, 2 to 3 times higher than that predicted for passive distribution. When luminal NaCl was partially replaced by mannitol (capillaries perfused with Cl Ringer's) cell Cl decreased showing a sigmoidal dependence on luminal NaCl. Peritubular membrane potential was unaltered. Sulfate Ringer's perfusion of the kidneys washed out all cell Cl but did not alter peritubular membrane potential. Chloride did not enter the cell when the tubule lumen was perfused with 100mm KCl, LiCl, or tetramethylammonium Cl. Luminal perfusion of NaCl caused cell Cl to rise rapidly to the same value as the controls in the Cl Ringer's experiments. Perfusion of the tubule lumen with mixtures of NaCl and Na₂SO₄, while the capillaries contained sulfate Ringer's yielded a sigmoidal dependence of cell Cl on luminal NaCl activity. Chloride movement from the lumen into the proximal tubule cells required approximately equal concentrations of Na and Cl. Current clamp experiments indicated that intracellular chloride activity was insensitive to alterations in liminal membrane potential, suggesting that chloride entry was electrically neutral. The transcellular chloride flux was calculated to constitute about one half of the normal chloride reabsorption rate. We conclude that the cell Cl activity is primarily determined by the NaCl concentration in the tubule lumen and that Cl entry across the luminal membrane is mediated.     

Mechanisms of Anion and Cation Permeations in the Resting Membrane of a Barnacle Muscle Fiber

The resting membrane of a barnacle muscle fiber is mostly permeable to cations in a solution of pH 7.7 whereas it becomes primarily permeable to anions if the pH is below 4.0. Mechanisms of ion permeation for various monovalent cations and anions were investigated at pH 7.7 and 3.9, respectively. Permeability ratios were obtained from the relationship between the membrane potential and the concentration of the test ions, and ionic conductances from current-voltage relations of the membrane. The permeability sequence for anions (SCN > I > NO₃ > Br > ClO₃ > Cl > BrO₃ > IO₃) was different from the conductance sequence for anions (Br, Cl > ClO₃, NO₃ > SCN). In contrast, the permeability and conductance sequences were identical for cations (K > Rb > Cs > Na > Li). The results suggest that anion permeation is governed by membrane charges while cation permeation is via some electrically neutral mechanism.     

Characteristics of the chloride conductance in muscle fibers of the rat diaphragm

In muscle fibers from the rat diaphragm, 85% of the resting membrane ion conductance is attributable to Cl-. At 37 degree C and pH 7.0, GCl averages 2.11 mmho/cm2 while residual conductance largely due to K+ averages 0.34 mmho/cm2. The resting GCl exhibits a biphasic temperature dependence with a Q10 of 1.6 between 6 degree C and 25 degree C and a Q10 of nearly 1 between 25 degree C and 40 degree C. Decreasing external pH reversibly reduced GCl; the apparent pK for groups mediating this decrease is 5.5. Increasing pH up to 10.0 had no effect on GCl. Anion conductance sequence and permeability sequence were both determined to be Cl-greater than Br-greater than or equal to I-greater than CH3SO4-. Lowering the pH below 5.5 reduced the magnitude of the measured conductance to all anions but did not alter the conductance sequence. The permeability sequence was likewise unchanged at low pH. Experiments with varying molar ratios of Cl- and I- indicated a marked interaction between these ions in their transmembrane movement. Similar but less striking interaction was seen between Cl- and Br-. Current-voltage relationships for GCl measured at early time-points in the presence of Rb+ were linear, but showed marked rectification with longer hyperpolarizing pulses (greater than 50ms) due to a slow time-and voltage-dependent change in membrane conductance to Cl-. This nonlinear behavior appeared to depend on the concentration of Cl- present but cannot be attributed to tubular ion accumulation. Tubular disruption with glycerol lowers apparent GCl but not GK, suggesting that the transverse tubule (T-tubule) system is permeable to Cl- in this species. Quantitative estimates indicate that up to 80% of GCl may be associated with the T tubules.     

Mechanism of inhibition of the proximal tubular isotonic fluid absorption by polylysine and other cationic polyamino acids.

The present study was initiated with the hope of clarifying the role of negative charges in the luminal brush border membrane in the overall process of trans-epithelial isotonic sodium and water absorption. Using micropuncture techniques, cationic polyamino acids such as polylysine (mol wt 100,000, 17,000 and 1,500-5,000, 1 mg/ml), tetralysine, polyornithine (mol wt 100,000, 1mg/ml), polyethyleneimine (2 mg/ml), polymyxin B (2 mg/ml), protamine sulfate (25 mg/ml) and histone (0.5 mg/ml) were perfused through the segments of rat kidney proximal tubule for 30 sec to 2 min. The rate of isotonic fluid absorption was measured before and after each perfusion with the Gertz's split drop method using Ringer's solution as a shrinking drop. Polylysine 100,000 and 17,000 and polyornithine were the most potent, inhibiting isotonic reabsorption by 93%. The sequence of inhibitory effect was: polylysine 100,000 congruent to polyornithine 100,000 congruent to polylysine 17,000 greater than polyethyleneimine greater than polylysine 1,500-5,000 congruent to polymyxin B greater than protamine sulfate congruent to histone. In contrast, tetralysine (2 mg/ml) showed no inhibitory effect. Electrical potential difference (p.d.) of the proximal tubular cells was destroyed within 10 sec of luminal perfusion with polylysine 100,000 (1 mg/ml). Simultaneously with the drop in p.d., electrical resistance of the luminal brush border membrane was nearly totally eliminated, whereas transepithelial input resistance remained unaltered. Furthermore, trypan blue dye was taken up by polylysine 100,000-perfused tubular cells but not by normal cells. Expanding drop analysis (mannitol solution as a split drop) was performed as a screening test to examine if the permeability for water and sodium in the lateral paracellular pathway is altered by polylysine 100,000. No significant difference was observed in the velocity of split drop expansion between untreated and polylysine-perfused tubules. A lower concentration of polylysine 100,000 (0.1 mg/ml) showed a much less inhibitory effect on fluid absorption and on cell p.d. These observations indicate that the strong inhibition on proximal tubular fluid absorption exerted by polylysine and perhaps also by other cationic polyamino acids is due not to modification of membrane negative charges but to the lysis of tubular cells by these polycations.     

Anions permeate and gate GCAC1, a voltage-dependent guard cell anion channel

GCAC1 is a strongly voltage-dependent anion channel in the guard-cell plasma membrane of Vicia faba . In patch–clamp experiments, we have investigated the permeation and gating properties of GCAC1 with respect to its anion dependence in the whole-cell and excised-patch configuration. The relative permeability followed the order SCN^– > NO₃^– > Br^– > Cl^–, while the single-channel conductances in symmetrical anionic solutions exhibited a nearly inverse sequence. The Cl^– dependence of inward currents (Cl^– release) is characterized by a maximum single-channel conductance of 89 pS half-saturating at 87 mM cytoplasmic chloride. In addition to this substrate saturation, anion release was also dependent on the external Cl^– activity ( K _m = 16 mM). In the presence of SCN^– and Cl^–, the single-channel conductance exhibited an anomalous mole-fraction dependence, identifying GCAC1 as a multi-ion single-file pore. Using anions with increasing ionic size, a minimum pore diameter of 0.5 nm was assumed from their relative permeabilities. In line with an anion-selective channel, a tenfold increase in the extracellular anion activity shifted the reversal potential by –59.8 mV. Simultaneously, the half-activation potential shifted negatively by about 23 mV. A further analysis of the anion dependence revealed that extracellular rather than cytosolic anions affect the gating process of GCAC1. From anion substitution experiments, we conclude that anion concentration and species determines both permeation and gating of the plant anion channel GCAC1.     

Characterization of GABAA receptor-mediated 36chloride uptake in rat brain synaptoneurosomes

gamma-Aminobutyric acid (GABA) receptor-mediated 36chloride (36Cl-) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated 36Cl- uptake in a concentration-dependent manner with the following order of potency: Muscimol greater than GABA greater than piperidine-4-sulfonic acid (P4S)greater than 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) = 3-aminopropanesulfonic acid (3APS) much greater than taurine. Both P4S and 3APS behaved as partial agonists, while the GABAB agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated 36Cl- uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated 36Cl- uptake was also dependent on the anion present in the media. The muscimol response varied in media containing the following anions: Br- greater than Cl- greater than or equal to NO3- greater than I- greater than or equal to SCN- much greater than C3H5OO- greater than or equal to ClO4- greater than F-, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl- channel.     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

Lyotropic anions. Na channel gating and Ca electrode response

The effects of external anions on gating of Na channels of frog skeletal muscle were studied under voltage clamp. Anions reversibly shift the voltage dependence of peak sodium permeability and of steady state sodium inactivation towards more negative potentials in the sequence: methanesulfonate less than or equal to Cl- less than or equal to acetate less than Br- less than or equal to NO-3 less than or equal to SO2-4 less than benzenesulfonate less than SCN- less than ClO-4; approximately the lyotropic sequence. Voltage shifts are graded with mole fraction in mixtures and are roughly additive to calcium shifts. The peak PNa is not greatly affected. Except for SO2-4, these anions did not change the Ca++ activity of the solutions as measured with the dye murexide. Shifts of gating can be explained as the electrostatic effect of anion adsorption to the Na channel or to nearby lipid. Such adsorption is expected to follow the lyotropic series. Anions also interfere significantly with the response of a Ca-sensitive membrane electrode following the same sequence of effectiveness as the shifts of gating. The lyotropic anions decrease the Ca++ sensitivity and cause anomalously negative responses of the Ca electrode because these anions are somewhat permeant in the hydrophobic detector membrane.     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

The localization of transport properties in the frog lens.

The selectivity of fiber-cell membranes and surface-cell membranes in the frog lens is examined using a combination of ion substitutions and impedance studies. We replace bath sodium and chloride, one at a time, with less permeant substitute ions and we increase bath potassium at the expense of sodium. We then record the time course and steady-state value of the intracellular potential. Once a new steady state has been reached, we perform a small signal-frequency-domain impedance study. The impedance study allows us to separately determine the values of inner fiber-cell membrane conductance and surface-cell membrane conductance. If a membrane is permeable to a particular ion, we presume that the conductance of that membrane will change with the concentration of the permeant ion. Thus, the impedance studies allow us to localize the site of permeability to inner or surface membranes. Similarly, the time course of the change in intracellular potential will be rapid if surface membranes are the site of permeation whereas it will be slow if the new solution has to diffuse into the intercellular space to cause voltage changes. Lastly, the value of steady-state voltage change provides an estimate of the lens' permeability, at least for chloride and potassium. The results for sodium are complex and not well understood. From the above studies we conclude: (a) surface membranes are dominated by potassium permeability; (b) inner fiber-cell membranes are permeable to sodium and chloride, in approximately equal amounts; and (c) inner fiber-cell membranes have a rather small permeability to potassium.     

Chloride extrusion in the isolated perfused teleost gill

Summary Chloride extrusion is examined in the isolated perfused gill of the pinfish,Lagodon rhomboides. In both sea water and Ringer's baths, the Cl efflux from the isolated gill is 45% that of the intact animal. The transepithelial electrical potential (TEP) across the isolated gill in sea water is equal to that in vivo, in Ringer's the gill TEP is slightly less than in vivo. Cl efflux is linearly dependent upon afferent flow of the perfusate. Furosemide, added to the perfusate inhibits 57% of the Cl efflux in gills bathed bilaterally by Ringer's. Ouabain causes a marked vasoconstriction and increase in afferent pressure. Removal of Na from the perfusate produces an inhibition of the Cl efflux that is not potential mediated. Net extrusion of Cl is inhibited in isolated gills bathed bilaterally by sodium free Ringer's.     

Organotin-mediated exchange diffusion of anions in human red cells

Organotin cations (R3Sn+) form electrically neutral ion pairs with monovalent anions. It is demonstrated that the tin derivatives induce exchange diffusion of chloride in red cells and resealed ghosts, without any detectable increase of membrane permeability to net movements of chloride ions. The obligatory anion exchange is believed to be due to the permeation of electroneural ion pairs, whereas the organic cation (R3Sn+) has an extremely low membrane permeability. Exchange fluxes of chloride increased with the lipophilicity of the substituting group (R3). At the same molar concentration of organotin, the relative potencies of the tin derivatives as anion carriers (with trimethyltin as a reference) were: methyl 1, ethyl 30, propyl = phenyl 1,00, and butyl 10,000. Tributyltin-mediated anion exchange was studied in detail. The organotin-induced anion transport increased through the sequence: F- less than Cl- less than Br- less than I- = SCN- less than OH-. Partitioning of tributyltin into red cell membranes was greater in iodide than in chloride media (partition coefficients 6.6 and 1.7 x 10(-3) cm, respectively). Bicarbonate, fluoride, nitrate, phosphate, and sulphate did not exchange with chloride in the presence of tributyltin. Chloride exchange fluxes increased linearly with tributylin concentrations up to 10(-5) M, and with chloride concentrations up to at least 0.9 M. The apparent turnover number for tributyltin-mediated chloride exchange increased from 15 to 1,350 s-1 between 0 and 38 degrees C. These figures are minimum turnover numbers, because it is not known what fraction of the organotin in the membrane exists as chloride ion pairs.     

Tributyltin-mediated exchange diffusion of halides in lipid bilayers

This paper describes the effect of tributyltin (TBT) on the inorganic anion permeability of lipid bilayers. When this compound is added in micromolar concentrations to one or both sides of a phosphatidyl ethanolamine (PE) membrane formed in 0.1 M NaCl or KCl (pH 7), there is no change in the electrical conductance. Under these circumstances, the Cl self-exchange flux measured with 36Cl (MCl) increases from a value of approximately 10(-12) mol.cm-2.s-1, to approximately 10(-8) mol.cm-2.s-1. It was further found that the relation between chloride flux and [TBT] and [Cl] can be described as: MCl = B[TBT] [Cl]. When chloride was replaced by an equimolar concentration of different univalent anions in the trans compartment, the heteroexchange flux of chloride followed the sequence: I greater than Br greater than Cl greater than F greater than NO3. Under all experimental conditions tested, the chloride flux was always more than 10(3) times the maximum flux predicted from the value of the membrane conductance, and at least 100 times higher than the expected fluxes of ion pairs (TBT-Cl) diffusing across the unstirred layers. Thus, the mechanism by which tributyltin increases anion permeability in bilayers seems to be that of an obligatory exchange diffusion, with the reaction between tributyltin and the halides occurring at the membrane surface. Measurements of interfacial potentials indicate that tributyltin chloride lowers the positive intrinsic dipole potential of PE membranes by approximately 70 mV (at a TBT concentration of 30 microM) without substantial alteration of other parameters of the bilayer. The estimated adsorption coefficient of TBT-Cl was found to be 3 x 10(-4) cm.     

Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Transport of chloride across cell membranes through exchange, cotransport, or conductive pathways is a subject of great biological importance. Current methods of measurement are restricted in their sensitivity, time resolution, and applicability. A new transport measurement technique has been developed on the basis of the fluorescence quenching by chloride of the dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). SPQ fluorescence quenching by chloride is rapid (less than 1 ms) and sensitive, with a greater than 50% decrease in fluorescence at 10 mM chloride. SPQ fluorescence is not altered by other physiological anions or by pH and can be used to measure both neutral and conductive transport processes. The high water solubility and membrane permeability properties of SPQ make it ideal for use in both membrane vesicles and cells. Chloride transport determined with SPQ was validated by measurement of erythrocyte chloride/anion exchange and membrane vesicle chloride conductance.     

The hypothetical role of potassium conductance on the genesis of R-wave amplitude increase during ischemia in the isolated rat heart.

The effect of increased potassium conductance on the genesis of R-wave amplitude increase during acute myocardial ischemia has been studied in the isolated perfused rat heart by simultaneously recording the R-wave amplitude of epicardial electrograms (VEE), heart rate (HR), coronary flow rate (CFR), left ventricular diastolic pressure (LVDP), and left ventricular systolic pressure (LVSP). The experiments were performed during basal and partial or total ischemic conditions at spontaneous or fixed HR. In some experiments, potassium conductance was increased by means of high-calcium (8 mM) or acetylcholine chloride (10(-6) M) perfusion. In the control experiments, partial ischemic perfusion produced an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP; total ischemic perfusion exaggerated these variations. High-calcium perfusion provoked an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP during basal conditions (p less than 0.01 vs. control experiment); these modifications increased progressively during partial ischemic perfusion (p less than 0.01 vs. control experiment) and during total ischemic perfusion (p less than 0.01 vs. control experiment). Perfusion with acetylcholine chloride produced variations similar to those observed in high-calcium solution except that LVDP under basal conditions remained unchanged from control. When the HR was maintained at a constant value by means of atrial pacing the results were similar to those observed in the unpaced hearts. In conclusion, in the isolated perfused rat heart, increasing potassium conductance may influence the genesis of R-wave amplitude increasing during acute myocardial ischemia.     

Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss)

Branchial epithelia of freshwater rainbow trout were cultured on permeable supports, polyethylene terephthalate membranes ("filter inserts"), starting from dispersed gill epithelial cells in primary culture. Leibowitz L-15 media plus foetal bovine serum and glutamine, with an ionic composition similar to trout extracellular fluid, was used. After 6 days of growth on the filter insert with L-15 present on both apical and basolateral surfaces, the cultured preparations exhibited stable transepithelial resistances (generally 1000-5000 ohms cm2) typical of an electrically tight epithelium. Under these symmetrical conditions, transepithelial potential was zero, and unidirectional fluxes of Na+ and Cl- across the epithelium and permeability to the paracellular marker polyethylene glycol-4000 (PEG) were equal in both directions. Na+ and Cl- fluxes were similar to one another and linearly related to conductance (inversely related to resistance) in a manner indicative of fully conductive passive transport. Upon exposure to apical fresh water, transepithelial resistance increased greatly and a basolateral-negative transepithelial potential developed. At the same time, however, PEG permeability and unidirectional effluxes of Na+ and Cl- increased. Thus, total conductance fell, and ionic fluxes and paracellular permeability per unit conductance all increased greatly, consistent with a scenario whereby transcellular conductance decreases but paracellular permeability increases upon dilution of the apical medium. In apical fresh water, there was a net loss of ions from the basolateral to apical surfaces as effluxes greatly exceeded influxes. However, application of the Ussing flux ratio criterion, in two separate series involving different methods for measuring unidirectional fluxes, revealed active influx of Cl- against the electrochemical gradient but passive movement of Na+. The finding is surprising because the cultured epithelium appears to consist entirely of pavement-type cells.