Net Ion Movement Research Articles

Pathways of ion movement in the canine tracheal epithelium

The pathways of ion movement across canine tracheal epithelium, a Cl-secreting tissue, were examined by three techniques. First, the measurement of simultaneous, unidirectional fluxes of Na or Cl and mannitol, a large hydrophilic molecule that serves as a marker of the paracellular pathway, indicated that a significant fraction of both the Na flux from submucosa to mucosa (J Na sm) and the flux of Cl from mucosa to submucosa (J Cl ms) traverse the cellular pathway. The ratio of the Na-to-Cl diffusion coefficients through the paracellular pathway was 0.23, in contrast to the free solution ratio of 0.63. Second, in voltage-clamp experiments we examined the effect of transepithelial voltage differences on the unidirectional fluxes of Na and Cl. The results agree with the previous findings, suggesting that there are voltage-independent, or transcellular, backfluxes of Na and Cl, and that the relative permeability of Na to Cl through the voltage-dependent (presumably paracellular) pathway was 0.28. Third, measurement of transepithelial diffusion potentials gave a Na-to-Cl permeability ratio of 0.31 +/- 0.02 (mean +/- SE). These results suggest that there are significant transcellular backfluxes of Na and Cl and that the paracellular pathway in the canine trachea is anion selective. An anion-selective pathway would tend to shunt the secreted Cl back through the paracellular pathway, thus minimizing the net ion and fluid movement across the tissue in the open-circuit condition.

Multiple fractions of sodium exchange in human lymphocytes.

Human lymphocytes contain a large, saturable fraction of K+ that exchanges slowly with K+ in the external medium, and a small non-saturable fraction that exchanges rapidly. We determined whether or not Na+ exchanges in a similar manner with external Na+. Cells were pre-equilibrated to ensure absence of net ion movements. Efflux was studied by loading with 22Na and transferring without washing to a non-labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na+ exchange, with half-times of 2, 14, and 120 minutes. At normal external K+, most cells Na+ exchanges rapidly, while at lower external K+ the Na+ that replaces cell K+ exchanges slowly. Parellel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous 42K and 22Na fluxes and by a quantitative analysis of the combined K+ and Na+ content and flux data over a range of external K+ and Na+ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclear exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K+ and Na+ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate-limited by adsorption onto and desorption from cellular macromolecules. The data support the association-induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.

Control of Mucus Secretion and Ion Transport in Airways

The output of secretions from the airway submucosal glands is regulated by vagal efferent nerves. Stimulation of cough receptors increases mucus output reflexly via the vagus nerves. Adrenergic agonists increase submucosal gland secretions in some species, which indicates that adrenergic receptors are present in these cells. However, evidence for adrenergic nervous pathways to the glands is limited. Irritants and drugs stimulate secretion from epithelial cells by direct effects. There is also evidence that the secretion of epithelial cells can be stimulated by parasympathetic nervous pathways in birds but not in mammals. Active ion transport of Cl- toward the lumen and of Na+ toward the submucosa results in net ion movement toward the airway lumen in unstimulated tracheal epithelia. Drugs and mediators increase the net movement of ions toward the lumen. No agents have yet been found that increase net ion movement toward the submucosa. The link between ion transport and water secretion in airway epithelia, although speculative, seems likely in view of the evidence from other epithelia. Since airway epithelium is a "tight junction" epithelium, modification of the tight junction may alter the transepithelial movement of water and ions. We suggest that the depth and consistency of the periciliary layer of airway secretions determine the ability of the cilia to propel the mucoprotein gel and thereby modify mucociliary transport. To achieve this, secretion of mucus must be controlled separately from the secretion of water. Studies are needed to determine which of the specialized functions of the epithelial cells interact to regulate the clearance of secretions from the airway. Is the sol maintained by secretion and reabsorption of fluid across the epithelium? Does the sol move with the gel by ciliary action or does it remain stationary? Do changes in the epithelial tight junctions influence net water movement and thus indirectly alter the depth of the sol layer? To answer these questions, techniques are needed to study subunits of the airway, including isolated surface cells and submucosal glands; and sensitive methods are required to analyze the very small samples of secretions for glycoprotein and electrolyte content. Intracellular measurements of electrolyte concentrations and electrical potentials may help to elucidate the mechanisms of transepithelial ion and water movement. The control system for the production and removal of respiratory tract secretions may be altered in disease. For instance, chronic stimulation of cough receptors causes reflex secretion and may be the cause of the hyperplasia of submucosal glands and of the abnormal secretions that occur in chronic bronchitis and asthma (50, 58). The abnormally viscid mucus in cystic fibrosis may be due to a defect in Cl- transport, which provides too little water for both the gel and sol layers. These speculations are intended to identify areas for further research, which hopefully will reduce the morbidity and mortality in these common lung diseases.

The Effect of Heavy Metal Compounds on the Permeability of Chlorella Cells

The loss of potassium from Chlorella is investigated when cells are exposed to 12 heavy metal compounds including zinc chloride (ZnCl 2) , mercuric chloride (HgCl 2 ), cupric chloride (CUCl 2 ), methylmercuric chloride (MeHg) and phenylmercuric acetate (PhHg). Over 90% of the potassium loss occurs under both dark or light conditions; the rest appears to require energy from photosynthesis. The effects of C0 2 -free air, anaerobic conditions and the photosynthetic inhibitor 3-(3'-4'-dichlorophenyl)-1,1-dimethylurea (DCMU) are investigated. In the light, potassium loss (efflux) is not coupled to a hydrogen ion (H + ) uptake mechanism during net ion movement. The effects of zinc and mercury ions on potassium loss at sublethal concentrations appeared to be minimal, and toxicity as well as resistance cannot be explained by gross changes in internal potassium levels. The results suggest that only at high concentrations, the primary effect caused by heavy metal ions is to increase the permeability of cells. The metal induced loss of potassium is strongly correlated to the strength of the metal-sulphydryl (-SH) bond, and the implications of this in relation to permeability of cell walls and membranes are discussed.

Fast and slow fractions of K+ flux in human lymphocytes

Potassium influx and efflux were studied in human peripheral blood lymphocytes equilibrated over a wide range of external K+ levels. The absence of a net ion movement throughout the flux study was established, trapped space was measured with polyethylene glycol, and cells were separated from incubation media without exposure to any washing solution. There are both rapid and slow cellular fractions of 42K influx and efflux, and half-times of exchange of around 2 minutes, and 400 minutes, respectively. The rapid component is identical in magnitude to the smaller non-saturable component of cell K+, while the slow component is identified with the larger, sigmoidal, saturable component of cell K+ that was previously shown to follow a cooperative adsorption isotherm. These results support the association-induction hypothesis, which predicts (a) a rapid fraction of K+ flux due to equilibration of ion within cell water existing in a state of polarized multilayers, and (b) a slower component of K+ flux limited by adsorption onto, or desorption from, fixed anionic sites existing throughout the cell. K+ influx, as a function of external K+, showed a triphasic relation with a peak around 1 mM K+ex, then a trough around 4mM K+ex, and then a gradual rise. This relation was readily explained, in terms of the association-induction hypothesis, by the cooperative interaction between, and ion occupancy of, fixed anionic sites that adsorb K+ or Na+.

Validation of the use of a tube with a proximal occlusive balloon for measurement of intestinal absorption in man.

Because of its simplicity, a tube with a proximal occlusive balloon is widely used to measure segmental intestinal absorption in man. Yet, the possibility that the presence of an inflated balloon might alter the absorptive capacity of the underlying segment of intestine has never been tested. In the present work the jejunal absorption of water and ions has been measured in 13 healthy subjects with the proximally occlusive tube and with the triple lumen nonocclusive method; precautions were taken to achieve similar flow rate and ionic composition of the fluid entering the test segment with both techniques in each subject. Under these conditions net water and ion movements measured by the two methods were identical. This finding validates the use of the proximal occlusive tube to measure intestinal absorption in man.

Kinetics of Sodium and Lithium Movements Across the Blood-Brain Barrier of An Insect

The electrical responses of axons were used to monitor the time-course of a change in the concentration of an ion species in the fluid bathing the axons in connectives of isolated cockroach nerve cords. Initial exposure of the connectives to sodium-deficient Ringer resulted in a depletion of extra-axonal sodium which was much slower than the restoration observed on return of the sodium Ringer. It is suggested that this asymmetry could result from a sodium reservoir which delays the initial decline. Subsequent net inward and outward movements of sodium ions were rapid and symmetrical. Unlike sodium ions, lithium ions were apparently unable to reach the axon surfaces following sodium depletion. In view of the similar properties of sodium and lithium ions in many biological systems it is therefore unlikely that the sodium movements were passive. Instead, the results support the idea of net sodium transport by the perineurial and/or glial elements.

Light-regulated permeability of rhodopsin:egg phosphatidylcholine recombinant membranes.

Purified rhodopsin was incorporated into phospholipid bilayers of egg phosphatidylcholine to give recombinant membrane vesicles, which were examined by proton and phosphorus nuclear magnetic resonance spectroscopy. Increased rhodopsin content in the membranes appears to progressively inhibit the molecular motions of the methyl, methylene, and phosphate groups of the phospholipid molecules. This indicates that regions of the rhodopsin molecule interact in a manner that affects the phospholipids from the aqueous interface to the bilayer midline. In the dark, the recombinant vesicles were sealed to europium, manganese, or cobalt ions. Light exposure allowed rapid equilibration of Mn2+ and Co2+, and somewhat slower equilibration of Eu3+ across the membrane. Light changed the membrane permeability, and the gradient in chemical potential resulted in a net ion movement across the rhodopsin:phospholipid recombinant membrane. The results suggest rhodopsin is a transmembrane protein.

Glass electrode measurement of net Na+ and K+ fluxes in erythrocytes of the spontaneously hypertensive rat.

The exchange of Na+ and K+ in rat red cells at 40% hematocrit incubated in a physiological salt solution containing 1 MM ouabain was monitored with ion-specific glass electrodes. Because this system measures a change in the activity of an ion as a logarithmic function of its preexisting level, delta[K+]o is more accurately measured than delta[Na+]o; within this limitation, the downhill ion movements were close to 1:1 over the temperature span from 37 to 3 degrees C. These transmembrane movements are reasonably well expressed in terms of first-order kinetics and an Arrhenius plot constructed for K+ as the accurate indicator of the exchange yields an activation energy of 12 kcal/mol. A well-defined reversal of the usual direct relation of temperature to exchange rate was observed between 6 and 3 degrees C. The net ion movements were distinctly faster at low temperature in cells from rats with spontaneous hypertension (SHR-Okamoto) than in cells from matched controls (Woodlyn and WKY).

Functional roles of luminal sodium and potassium in water transport across desert scorpion ileum.

IN terrestrial insects the rectum is a major site of fluid absorption and is, therefore, important in the physiological control of water balance. Water absorption in the insect rectum occurs against osmotic gradients greater than 300 mosmol 1−1 and apparently in the absence of net ion movements from lumen to haemolymph1,2. Evidence suggests that net water flow from the rectal lumen to haemolymph results from increases in intercellular space osmotic pressure due to the transport across lateral cell membranes of recyclable intracellular solutes3,4. Little is known about the mechanism of water transport in the insect gut anterior to the rectum nor about gastrointestinal processes involved in water regulation in other terrestrial arthropods. Scorpions are ideal organisms for measurements of arthropod deal transport because this part of the gut extends nearly the entire length of the tail and can be isolated for in vitro perfusion. We present here preliminary information on a sodium-dependent, potassium-inhibited water transport mechanism in the ileum of the desert scorpion Hadrurus arizonensis.

The functional response of the dog ileum to one hour's ischaemia.

1. The function and structure of dog ileal mucosa was examined immediately after arterial ischaemia for 1 h and 1, 3 or 7 days after the trauma. 2. Immediately after the ischaemia, there was a net movement of water and ions into a luminal perfusate in vivo. One day later, there was no net movement of water and electrolytes but net absorption returned on the third day. 3. Active transport of phenylalanine or beta-methylglucoside by mucosal strips in vitro was abolished immediately after the trauma but was largely restored 1 day later. There was a close correlation between glucose absorption in vivo and beta-methylglucoside uptake in vitro. 4. Morphological study revealed pronounced desquamation of enterocytes immediately after the trauma. The regenerating mucosa had shorter villi and crypts, and lower enzyme activities assessed histochemically 1 day later. Most parameters had returned to normal by the third post-operative day.

Mechanisms of acid disposal in canine duodenum

Distal duodenal pouches chronically excluded from the intestinal stream were perfused in seven awake dogs by a constant-circulation technique using isomolar solutions containing 60-120 mM HCl. The rate of disappearance of hydrogen ions (H+) and net ion movements was measured. In all experiments there was a loss of H+ from the perfusate accompanied by a gain of sodium (Na+), potassium (K+) and chloride (Cl-), together with an increase in volume and a decrease in osmolality. The magnitude of this H+ loss was a function of instillate [H+]. The H+ lost by neutralization was separated from that lost by diffusion by calculations based on the assumption that the observed changes in somolality were the result of neutralization by bicarbonate (HCO3-). The bulk of the acid (67%) was lost by neutralization, the remainder back-diffusing in a one-to-one exchange for diffusable Na+. This ability to dispose of acid decreased with increasing age of the pouches due to a diminished volume of HCO3- secretion, the magnitude lost by backdiffusion remaining unchanged.

A furosemide-sensitive cotransport of sodium plus potassium in the human red cell.

The influxes of Na(+) and K(+) into the human red cell appear to be interrelated. This relationship was investigated under conditions in which either Na(+) or K(+) concentration outside the cell was varied or one cation was replaced by Mg(2+), choline(+), or Li(+). The effects of furosemide on Na(+) and K(+) movements were studied in the presence of ouabain. When ouabain was present, Na(+) influx was higher with K(+) ions externally than with other cations externally. Furosemide inhibited this K(+)-stimulated Na(+) influx, but it had little effect when K(+) was absent. Ouabain-insensitive K(+) influx was stimulated two-fold by external Na(+) compared with other cations. Furosemide also inhibited this stimulation, but it had little effect when Mg(2+) or choline(+) replaced external Na(+). Thus it was confirmed that synergism exists between the ouabain-insensitive influxes of Na(+) and K(+) and it was demostrated that furosemide inhibits this cooperative effect. The ouabain-insensitive influx of both K(+) and Na(+) showed a hyperbolic "saturating" dependence on the external concentration of the transported cation. Furosemide therefore eliminates a saturable component of influx of each cation. The net uptake of Na(+) in the presence of ouabain was stimulated by K(+) ions. A similar effect was observed with red cells, in which Li(+) replaced nearly all the internal Na(+) plus K(+) ions. In these cells, net Na(+) uptake was stimulated by external K(+), and net K(+) uptake was stimulated by external Na(+). Furosemide inhibited this mutual stimulation of net cation entries. The inhibitory action of furosemide was not limited to inward flux and net movement of Na(+) and K(+). Furosemide also inhibited the efflux of Na(+) into Na(+)-free media and the efflux of K(+) into K(+)-free media. It appeared, therefore, that the action of furosemide was not explained by inhibition of exchange diffusion. These data are consistent with an ouabain-insensitive transport process that facilitates the inward cotransport of Na(+) plus K(+)-ions, and that can produce a net movement of both ions. Although this process under some conditions mediates an equal bidirectional flux of both Na(+) and K(+), it cannot be defined as exchange diffusion. The contransport process is inhibited by furosemide.

Effects of pentagastrin and cholecystikinin on intestinal transport of ions and water in the rat.

SummaryPentagastrin and CCK cause no significant changes in the net movement of ions and water in the jejunum and ileum of anesthetized rats.I thank Mrs. Mary Denton for her skilled technical assistance, and Dr. T. Robitschek of Ayerst Laboratories for the gift of pentagastrin. This work was supported in part by U.S. Public Health Service Grants AM 09022 and AM 5390 from the National Institute of Arthritis and Metabolic Disease.

Active Na + and Cl − transport by the isolated chick chorioallantoic membrane

Unidirectional Na + and Cl − fluxes across 15–18-day chick chorioallantoic membranes were determined in vitro using the short-circuit technique of Ussing and Zerahn 6. In the absence of an electrochemical gradient across the membrane, the observation of any net ion movement indicates the existence of an active transport process. In the normal chorioallantoic membrane both Na + and Cl − were found to be actively transported from ectoderm to endoderm. The observed short-circuit current could be fully accounted for by their algebraic sum. Upon removal of the acellular inner shell membrane from the ectodermal side of the chorioallantoic membrane, the direction of net Na + transport reversed and net Cl − transport was abolished. The short-circuit current of these stripped membranes was nearly 3 times greater than the normal membranes and could not be entirely accounted for by the observed active Na + transport. It is suggested that the active Cl − transport site resides in the ectodermal cells and that there exist two opposing active Na + mechanisms presumably located in the ectodermal and endodermal layers, respectively.

Interrelationships of chloride, bicarbonate, sodium, and hydrogen transport in the human ileum.

Using a triple-lumen constant perfusion system, the following observations were made in normal subjects. First, chloride, bicarbonate, and sodium were found to exhibit net movement across ileal mucosa against electrochemical gradients. Second, during perfusion with a balanced electrolyte solution simulating plasma, the ileum generally absorbed, but sometimes secreted fluid. A reciprocal net movement of chloride and bicarbonate was noted when sodium movement was zero. Increasing rates of sodium absorption were associated with decreasing bicarbonate secretion rates and finally bicarbonate absorption. Even when bicarbonate was absorbed ileal contents were alkalinized (by contraction of luminal volume). Third, net chloride movement was found to be sensitive to bicarbonate concentration in ileal fluid. For instance, chloride was absorbed from solutions containing 14 or 44 mEq/liter of bicarbonate, but was secreted when ileal fluid contained 87 mEq/liter of bicarbonate. Fourth, when chloridefree (sulfate) solutions were infused, the ileum absorbed sodium bicarbonate and the ileal contents were acidified. Fifth, when plasma-like solutions were infused, the potential difference (PD) between skin and ileal lumen was near zero and did not change when chloride was replaced by sulfate in the perfusion solution. These results suggest that ileal electrolyte transport occurs via a simultaneous double exchange, Cl/HCO2 and Na/H. In this model neither the anion nor the cation exchange causes net ion movement; net movement results from the chemical reaction between hydrogen and bicarbonate. No other unitary model explains all of the following observations: (a) human ileal transport in vivo is essentially nonelectrogenic even though Na, Cl, and HCO3 are transported against electrochemical gradients, (b) the ileum can secrete as well as absorb, (c) ileal contents are alkalinized during absorption of or during secretion into a plasma-like solution, and (d) the ileum acidifies its contents when sulfate replaces chloride. Data obtained with a carbonic anhydrase inhibitor support the proposed model.

Discreteness of conductance change in bimolecular lipid membranes in the presence of certain antibiotics.

NET ion movements across biological or synthetic lipid membranes may take place by various mechanisms, underlying all of which there is a rather ill-defined and small ion leakage or background conductance. Most ions permeate by means of pathways involving either permanent or transient modifications of the basic structure of the membrane. If permanent pathways are involved, a given membrane conductance may be accounted for by routes which are either numerous and of low conductance or few and of high conductance. For transient pathways, duration must also be considered. Thus, if a carrier is invoked, the duration will be the time the carrier, complexed with an ion, spends shuttling across the membrane. For a pore, the duration is the time for which it remains open to ions. At present little is known concerning the number, conductance and duration of the ionic pathways in any membrane of the types mentioned. Limited information is available for the nerve membrane, although this is rather imprecise and indirect1.

EFFECTS OF COCAINE AND ADRENALINE ON CONTRACTURES AND DOWNHILL ION MOVEMENTS INDUCED BY INHIBITORS OF MEMBRANE ATPASE IN RAT UTERI.

The contractures induced by ouabain (10–3 M), potassium-free solutions, and NaF (10–2 M) in estrogen-dominated rat uterine muscle were prevented or delayed by 0.1% cocaine. In contrast to contractures from ouabain or potassium-free solutions, contractures from NaF were delayed or partly reversed but not prevented. The accompanying downhill ion movements (potassium loss and sodium gain) were partly prevented except when produced by ouabain. Cocaine itself sometimes caused contracture at this concentration but without inducing downhill ion movements. In myometrium from pregnant cats, which required less ouabain (10–6 to 10–5 M) to initiate contracture, 0.03 to 0.07% cocaine opposed both the contractures and the downhill ion movements. Higher cocaine concentrations could not be used as they provoked sustained contractures which were not accompanied by net ion movements. In myometrium from estrogendominated rabbits, both cocaine and procaine in concentrations much lower than 0.1% caused sustained contracture. In concentrations of 0.025 and 0.1%, cocaine caused no downhill ion movements in rabbit myometrium and prevented neither the contractures nor the downhill ion movements initiated by 10–6 to 10–5 M ouabain.Adrenaline (1–10 μg/ml) prevented or reversed contractures of rat uteri from ouabain and potassium-free media and this effect was prevented by nethalide, a beta receptor blocking agent with little direct action. Similar concentrations of adrenaline were less effective against acetylcholine and ATP contractions. 3′5′-Cyclic AMP could not produce relaxation. Sodium fluoride contractures were not prevented or reversed by adrenaline. Adrenaline did not prevent the downhill ion movements accompanying contractures and tended to increase them. Nethalide prevented this effect. The differing actions of adrenaline on sodium and potassium movements and contracture suggested that the systems for sodium and calcium transport were partly independent.Reserpinization of rats did not alter the high threshold of uteri to ouabain, indicating that the cause of the high threshold was not related to endogenous adrenaline or noradrenaline.Neither cocaine nor adrenaline affected the downhill electrolyte shifts induced by cooling to 7 °C, but adrenaline prevented the cold contracture. Neither relaxant affected the electrolyte shifts induced by iodoacetate, but both had slight effects on dinitrophenol electrolyte shifts. There was evidence suggesting that dinitrophenol caused shortening of the unloaded muscles used to study electrolyte shifts.Cupric ion (10–5 to 10–4 M) was found to cause contractures of rat uteri. Cupric ion (10–5 M), after a short exposure of uterine horns, potentiated the subsequent responses of these horns to ouabain, potassium-free solutions, and NaF. Potassium-free solutions and glucose removal tended to potentiate the responses to cupric ion. Cupric ion (10–5 to 10–4 M) did not cause increased potassium loss and in fact diminished the loss of this ion from rat uterine cells; it did appear to increase the uptake of sodium into cells. Its effects on contracture and electrolyte movements were opposed by cocaine. The lack of effect of adrenaline may have been partly related to the very rapid oxidation of this substance in the presence of cupric ion. It was concluded that cupric ion acts similarly but not identically to fluoride and affects Mg- and Na+K-activated membrane ATPase in such a manner as to demonstrate the dissociation of sodium extrusion from potassium uptake.

SODIUM MOVEMENT ACROSS SINGLE PERFUSED PROXIMAL TUBULES OF RAT KIDNEYS.

Using perfusion techniques in single proximal tubule segments of rat kidney, the relationship between net sodium movement and active transport of ions, as measured by the short-circuit method, has been studied. In addition, the role of the colloid-osmotic pressure gradient in proximal transtubular fluid and sodium movement has been considered. Furthermore, the limiting concentration gradient against which sodium movement can occur and the relationship between intratubular sodium concentration and fluid transfer have been investigated. Comparison of the short-circuit current with the reabsorptive movement of sodium ions indicates that this process is largely, perhaps exclusively, active in nature. No measurable contribution of the normally existing colloid-osmotic pressure gradient to transtubular water movement was detected. On the other hand, fluid movement across the proximal tubular epithelium is dependent upon the transtubular sodium gradient and is abolished when a mean concentration difference of 50 mEq/liter is exceeded.

THE ORIGIN OF THE SHORT-CIRCUIT CURRENT IN THE ISOLATED SKIN OF THE SOUTH AMERICAN FROG LEPTODACTYLUS OCELLATUS.

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.