Ionophore-induced Cl- transport in human erythrocyte suspensions: a multinuclear magnetic resonance study.

To investigate the effect of ionophores on Cl- distribution in human erythrocyte suspensions, we measured the membrane potential by using 19F and 31P NMR methods. Incubation of human erythrocytes with 0.005 mM of the neutral ionophores valinomycin and nonactin resulted in membrane potentials of -21.2 and -17.8 mV in the presence and absence of DIDS. However, 0.020 mM of the carboxylic ionophores lasalocid, monensin, and nigericin yielded membrane potentials similar to those measured in the absence of ionophore (-9.4 mV). In methanol, the 35Cl- NMR linewidth in the presence of valinomycin was twice as broad as those observed in the presence of carboxylic ionophores, suggesting that neutral ionophores induce Cl- efflux in part via ion pairing.     

Effect of Ethanol on γ-Aminobutyric Acid and Glycine Receptor-Coupled Cl− Fluxes in Rat Brain Synaptoneurosomes

Chloride fluxes in synaptoneurosomes in response to additions of gamma-aminobutyric acid, glycine, and ethanol were measured using a chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). The Cl- gradient was directed outward by bathing cells in a medium low in Cl- concentration. The synaptoneurosomes responded to both gamma-aminobutyric acid and glycine by outflow of Cl- ions, as judged from an increase in SPQ fluorescence. These effects were inhibited by picrotoxin and strychnine, respectively. Ethanol also produced an outflow of Cl- ions from the synaptoneurosomes. Both picrotoxin and strychnine inhibited this effect. When the antagonists were used together, the inhibiting effect was additive. These results indicate that ethanol affects both gamma-aminobutyric acid and glycine receptor-linked chloride fluxes in the rat brain.     

Effects of chloride deficiency on the pancreatic B-cell response to acetylcholine

Muscarinic stimulation of pancreatic B-cells markedly amplifies insulin secretion through complex mechanisms which involve changes in membrane potential and ionic fluxes. In this study, normal mouse islets were used to evaluate the role of Cl- ions in these effects of acetylcholine (ACh). Whatever the concentration of glucose, the rate of 36Cl- efflux from islet cells was unaffected by ACh. Replacement of Cl- by impermeant isethionate in a medium containing 15 mM glucose did not affect, or only slightly decreased, the ability of ACh to depolarize the B-cell membrane and increase electrical activity, to accelerate 45Ca2+ and 86Rb+ efflux from islet cells, and to amplify insulin release. In the absence of extracellular Ca2+, a high concentration of ACh (100 microM) mobilized intracellular Ca2+ and caused a transient release of insulin and a sustained acceleration of 86Rb+ efflux. None of these effects was affected by Cl- omission or by addition of furosemide, a blocker of the Na+, K+, 2Cl- cotransport. Isethionate substitution for Cl- in a medium containing a nonstimulatory concentration of glucose (3 mM) barely reduced the depolarization of B-cells by ACh, but inhibited the concomitant increase in 86Rb+ efflux. We have no explanation for the latter effect that was not mimicked by furosemide. In conclusion, ACh stimulation of pancreatic B-cells, unlike that of exocrine acinar cells, is largely independent of Cl- and is insensitive to furosemide. The acceleration of ionic fluxes produced by ACh does not involve the Na+, K+, 2Cl- cotransport system.     

Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line.

NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.     

The Na+/K+/Cl- cotransport in C6 glioma cells. Properties and role in volume regulation

The role of the Na+/K+/Cl- cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide greater than bumetanide greater than piretanide greater than furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide-sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl- cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl- cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso-osmotic medium deprived of either Na+, K+ or Cl- also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl- cotransport together with a cell swelling back to the normal volume were observed upon returning ion-deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl- cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.     

The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers

The intracellular dialysis technique was used to measure unidirectional Cl- fluxes and net acid extrusion by single muscle fibers from the giant barnacle. Decreasing pHi below normal levels of 7.35 stimulated both Cl- efflux and influx. These increases of Cl- fluxes were blocked by disulfonic acid stilbene derivatives such as SITS and DIDS. The SITS-sensitive Cl- efflux was sharply dependent upon pHi, increasing approximately 20-fold as pHi was decreased from 7.35 to 6.7. Under conditions of normal intracellular Mg2+ concentration, the apparent pKa for the activation of Cl- efflux was 7.0. We found that raising [Mg2+]i, but not [Mg2+]o, had a pronounced inhibitory effect on both SITS-sensitive unidirectional Cl- fluxes as well as on SITS-sensitive net acid extrusion. Increasing [Mg2+]i shifted the apparent pKa of Cl- efflux to a more acid value without affecting the maximal flux that could be attained. This relation between pHi and [Mg2+]i on SITS-sensitive Cl- efflux is consistent with a competition between H ions and Mg ions. We conclude that the SITS-inhibitable Cl- fluxes are mediated by the pHi-regulatory transport mechanism and that changes of intracellular Mg2+ levels can modify the activity of the pHi regulator/anion transporter.     

Chloride fluxes in lily pollen tubes: a critical reevaluation

Microelectrodes, made from a Cl(-)-selective liquid ion exchanger previously used to measure putative Cl- fluxes in Lilium longiflorum pollen tubes, were characterized. The electrodes were poorly selective, possessing only about 10-fold selectivity for Cl- over other anions tested. They had only 2.4-fold selectivity for Cl- over the anionic form of the H+ buffer, MES, indicating that the electrode can indirectly detect H+ gradients. Apparent anion influx was detected along the pollen tube shafts and at the grains while apparent anion efflux was detected near the tip of the tube. During oscillating growth, the peak of the oscillating apparent anion efflux at the tip occurred, on average, 7.9 sec after the peak of the growth oscillations. Consideration of the previously characterized H+ fluxes in lily pollen grains and tubes, as well as the poor anion selectivity of the Cl- electrodes, indicates that the putative Cl- fluxes are in fact changes in the anionic concentration of the buffer resulting from H+ gradients and not changes in Cl- concentration. The claim of a central role for Cl- in lily pollen tube growth is further undermined by the fact that these tubes grow at the same rate if the Cl- content of the growth medium is reduced to trace levels (< or =31 microM), and that the grains have only small reserves of Cl-. These results lead to the conclusion that Cl- fluxes are not a significant component of pollen tube growth and Cl- itself is not required for growth.     

Net uptake of chloride across the posterior gills of the Chinese crab (Eriocheir sinensis)

Two methods are commonly used for the determination of transbranchial net fluxes of Na+ and Cl-: direct analysis of changes in ion concentrations in the external medium using flame spectrophotometry or titration (net flux method), and measurement of unidirectional ion fluxes by means of radioactive tracers (tracer method). When we applied both methods in the same preparation, the isolated perfused posterior gill of freshwater-acclimated Eriocheir sinensis, to determine net fluxes of Cl-, the results differed substantially. In artificial fresh water (AFW) containing NaCl, the net flux method yielded a net uptake, but the tracer method showed a net efflux of Cl-. The net uptake of Cl- was abolished in Na(+)-free AFW indicating that Cl- uptake is coupled with the uptake of Na+. Applying the tracer method, net efflux of Cl- remained almost unchanged in Na(+)-free AFW. This suggests the opposite mechanism, i.e. uncoupled uptake of Na+ and Cl-. The discrepancy in the results obviously depends on the method employed. Since the data obtained with the net flux method explain the osmoregulatory performance of crabs living in fresh water, we consider this method as appropriate for determining net transbranchial ion fluxes.     

Effect of furosemide on unidirectional fluxes of sodium and chloride across the skin of the frog, Rana pipiens.

1. The diuretic furosemide, when added to the outside solution at a concentration of 5-10-4 M, increases the electrical potential difference (PD) across the isolated frog skin, but the short-circuit current (Isc) is unchanged. Lower concentrations had no significant effect on these electrical parameters. 2. When SO42- or NO3- are substituted for Cl- in the Ringer's solution furosemide has no effect on the PD or Isc. 3. Simultaneous unidirectional fluxes of Na+ and Cl- show that furosemide (5-10-4 M outside) reduces both the influx and outflux of Cl-, while the Na+ fluxes are not altered. 4. Furosemide (5-10-4 M) on the corium side of the frog skin had no significant effect on either PD, Isc or undirectional fluxes of Cl-. 5. It is suggested that furosemide reduces passive Cl- transfer, possibly by interacting with the Cl-/Cl- exchange diffusion mechanism which has been observed in this tissue. These observations further suggest that perhaps the diuretic action of furosemide may be mediated by such an effect on passive Cl- permeability which is linked to the active Cl- transport mechanism in the renal tubule.     

Phorbol esters and gene expression: the role of rapid changes in K+ transport in the induction of ornithine decarboxylase by 12-O- tetradecanoylphorbol-13-acetate in BALB/c 3T3 cells and a mutant cell line defective in Na+K+Cl- cotransport

A BALB/c 3T3 preadipose cell line defective in Na+K+Cl- cotransport (3T3-E12a cells) has been used to study the relationship between phorbol ester-induced rapid changes in cation fluxes and changes in expression of a gene known to be modulated by this agent. In contrast to its effect on parental 3T3 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) did not inhibit either furosemide-sensitive 86Rb+ influx or the rate of 86Rb+ efflux from preloaded mutant cells. TPA-induced changes in intracellular K+ content were diminished in 3T3-E12a cells as compared with parental cells. Thus, mutation of the Na+K+Cl- cotransport system renders overall potassium transport in mutant cells largely insensitive to modulation by TPA. The morphological and functional responses of 3T3 and 3T3-E12a cells to TPA were also compared. In contrast to the extensive and long-lasting changes in morphology of 3T3 cells after 0.16 microM TPA addition, only slight and shorter-lived morphological effects of TPA were observed in 3T3-E12a cells. The transport properties of mutant cells were not totally unresponsive to TPA since hexose transport (2-deoxyglucose uptake) could be stimulated in both cell types. To establish a possible link between early changes in cation fluxes and activation of gene expression by TPA, the induction of the enzyme ornithine decarboxylase (ODC) was studied in detail. Addition of fresh medium containing serum or exposure to hypoosmotic conditions resulted in the induction of ODC in both 3T3 and 3T3-E12a cells. However, TPA failed to cause an increase in ODC activity in mutant cells, although a substantial induction of the enzyme was seen in parental cells. These results suggest that rapid changes in ion fluxes mediated by the Na+K+Cl- cotransport system are necessary for at least one of the phorbol ester-induced changes in gene expression in responsive cells.     

Anion-dependent cation transport in erythrocytes

A selective survey of the literature reveals at least three major anion-dependent cation transport systems, defined as Na+ + Cl-, K+ + Cl- and Na+ + K+ + Cl- respectively. In human red cells, kinetic data on the fraction of K+ and Na+ influx inhibitable by bumetanide are presented to indicate an Na+:K+ stoichiometry of 1:2. For LK sheep red cells the large Cl- -dependent K+ leak induced by swelling is shown to share many characteristics with that induced by N-ethylmaleimide (NEM) treatment. NEM has complex effects, both inhibiting and then activating Cl- -dependent K+ fluxes dependent on NEM concentration. The alloantibody anti-L can prevent the action of NEM. In human red cells NEM induces a large Cl- -dependent specific K+ flux, which shows saturation kinetics. Its anion preference is Cl- greater than Br- greater than SCN- greater than I- greater than NO3- greater than MeSO4-. This transport pathway is not inhibited by oligomycin or SITS, although phloretin and high concentrations of furosemide and bumetanide (over 0.3 mM) do inhibit. Quinine (0.5 mM) is also an inhibitor. It is concluded that at least two distinct Cl- -dependent transport pathways for K+ are inducible in mammalian red cells, although the evidence for their separation is not absolute.     

ATP-dependent and DCCD-insensitive Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions

ATP-dependent Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions was not affected by the addition of 40 mM of K+, Na+ or HCO3- to the assay medium. Na+ and K+ did not alter the uptake even in the presence of a K+ ionophore, valinomycin (10 microM), or a H+/K+ exchanger, nigericin (10 microM), whereas in the presence of both of these ionophores, K+, but not Na+, reduced the Cl- uptake. Inhibitors of proton pump activity, N,N'-dicyclohexylcarbodiimide (1 mM) and 5-(N,N-hexamethylene)amiloride (40 microM), however, did not affect the Cl- uptake. These findings suggest the presence of a primary Cl- transport system probably associated with passive H+ flux in the brain plasma membranes.     

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.     

Action of exogenous potassium and calcium ions on in vitro metacyclogenesis in Trypanosoma cruzi.

The cations Ca2+ and K+ and the anions Cl-, HCO3-, and PO4- were studied for their contribution to metacyclic trypomastigote formation of Trypanosoma cruzi in starvation media consisting of phosphate-buffered saline (PBS) + 10 mM proline + 10 mM sodium acetate as well as one of the following salts: 0.035% NaHCO3 (PBSNPA), 0.035% K2CO3 (PBSKPA) or 0.035% K2HPO4 (PBSPPA). Isolates CL and DM28c were activated to transform with 5% CO2 and the percent metacyclogenesis determined after incubation for 96 h in PBS starvation media. Maximal metacyclogenesis was found with CaCl2 and KCl. In the presence of K+, the percent transformation was highest with the phosphate salt, followed by the carbonate and the chloride salts. Cells incubated in PBSNPA and the cationic ionophores A23187 (5 x 10(-6) M), lasalocid (5 x 10(-6) M), and valinomycin (10(-8) M) do not survive; addition of 2 mM CaCl2 or 17 mM KCl to DM28c cells, reversed the lethal action of the ionophores permitting differentiation into metacyclic forms. The addition of CaCl2 to CL cells incubated in ionophores abrogated the lethal effect of the ionophores but transformation was significantly different than in control preparations. Adding KCl to ionophore incubated cells resulted in normal levels of transformation except in the case of valinomycin. DM28c and CL cells incubated in PBSKPA show significantly greater metacyclogenesis in the presence of 5 mM EGTA. These results indicate that exogenous concentrations of several cations and anions significantly influence T. cruzi metacyclogenesis and that the degree of response by the parasite to free ion levels may be strain dependent.     

GABAA receptor-controlled 36Cl- influx in cultured rat cerebellar granule cells

gamma-Aminobutyric acid (GABA)-mediated and bicuculline-sensitive 36Cl- influx and bicuculline-sensitive [3H] GABA binding were demonstrated in cultures of rat cerebellar granule cells. The addition of 10(-5) M GABA produced a two-fold increase in 36Cl-influx over the basal level and the maximal increase was observed after approximately 20 sec. Progressive occupation of GABAA receptor by [3H]-(1S-9R)-bicuculline methiodide decreased 36Cl- influx activated by 10 microM GABA. The above results suggest that primary cultures of rat cerebellar granule cells provide a new and reliable model for studying the GABA activated chloride fluxes.     

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.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular Na+, K+ and Cl- activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (Vm) and intracellular Na+, K+ and Cl- activities were measured under steady-state conditions in normal saline medium (Na+ = 154, K+ = 6, Cl- 150 mequiv./l). Membrane potential was estimated to be -23.3 +/- 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of Vm and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na+, K+ and Cl- activities are 4.6 +/- 0.5; 68.3 +/- 8.0; and 43.6 +/- 2.1 mequiv./l, respectively. The apparent activity coefficients for Na+ and K+ are 0.18 +/- 0.02 and 0.41 +/- 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl- (0.67 +/- 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl- is not different from zero. The results establish that the energy available from the Na+ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Potential dependence of unidirectional chloride fluxes across isolated frog skin

Isolated frog skins were voltage clamped at transepithelial potentials (Vt) ranging from -60 mV to 60 mV to measure transepithelial 36Cl- fluxes from the apical to the basolateral bathing solution (J13) and in the opposite direction (J31). The potential dependence of fluxes obtained in Na+-free choline Ringer's indicates the presence of conductive and nonconductive components that probably correspond to fluxes through paracellular and cellular pathways, respectively. Rectification of fluxes with reversal of the potential reflects a structural asymmetry, presumably in surface charge density. The data are consistent with a charge density of one negative charge per 280 A2 on the apical side. A new model for passive Cl- transport was developed that includes surface charge asymmetry and specifically accounts for the observed variation of conductance with potential. In normal frog Ringer's, J13 was larger than J31 at zero potential (active Cl- transport), J13 rose exponentially with increasing positive potential to reach a maximum at 40 mV (approximately open-circuit), and the predicted partial Cl- conductance exceeded the measured conductance leading to the conclusion that when J13 is largely driven by Na+ transport, much of the coupling occurs via nonconductive pathways. Theophylline stimulates Cl- transport that also occurs via nonconductive pathways as Vt becomes more positive.     

Antigen stimulates glycoprotein secretion and alters ion fluxes in sheep trachea

We studied the effects of in vitro challenge with specific antigen (Ascaris suum antigen) on glycoprotein secretion and ion fluxes in tracheal tissues from allergic sheep. We mounted tissues in Perspex chambers and measured secretion of 35S- and 3H-labeled glycoproteins and fluxes of Cl- and Na+. In tissues from allergic sheep, A. suum antigen (25 micrograms protein X ml-1) increased glycoprotein secretion. A. suum antigen initially reversed net Cl- flux, causing net absorption of Cl- and of Na+. This was followed 15-30 min later by net secretion of Cl- and of Na+. Pretreatment of tissues with cromolyn (10(-4) M) greatly reduced the effects of A. suum antigen but did not abolish them. The cromolyn-resistant effects were nonspecific, because they were similar to those of in vitro challenges with nonspecific proteins, ovalbumin and ragweed in allergic sheep, and A. suum antigen in nonallergic sheep. We conclude that challenge with A. suum antigen results in mucus hypersecretion in airways of allergic sheep, by both specific and smaller nonspecific effects. Specific effects (cromolyn sensitive) are produced by mediators which are released from airway cells in response to A. suum challenge.