Hans H. Ussing

The alkali metal ions in isolated systems and tissues

More than thirty years have passed since the appearance of Hober’s treatise1 on the alkali metal ions in this handbook. Since then the literature on the biological functions and effects of these ions has encreased enormously. The mere bulk of material has forced the authors to abandon the vain attempt to cover all the papers with a bearing on the field. Instead they have tried to choose from the abundance primarily what they feel can be organized into a coherent picture of the biological role of the alkali metal ions.

ANOMALOUS TRANSPORT OF ELECTROLYTES AND SUCROSE THROUGH THE ISOLATED FROG SKIN INDUCED BY HYPERTONICITY OF THE OUTSIDE BATHING SOLUTION

Some years ago we proposed that the frog skin potential arises as the resultant of an active inward transport of sodium and the shunting effect of passively diffusing ions like chloride (Ussing, 1949: Ussing & Zerahn, 195 1 ) . An anatomical localization of the “sodium pump” was not attempted but it was assumed that a single cell layer in the stratum germinativum was responsible for the active sodium transport (compare also Koefoed-Johnsen & Ussing, 1958). Recently it has turned out, however, that the more distal cells of the epithelium are also involved in the transport (for a detailed discussion, see Ussing, 196%). Thus Koefoed-Johnsen (1966) has shown that even the outermost living cells of the epithelium readily exchange their potassium with the inside bathing solution but not with that on the outside, indicating that the barrier which permits sodium to enter, but stops potassium must be located near the outer surface of the skin. Measurements of the intracellular potentials with microelectrodes also showed that the first potential step is recorded when the tip passes from the outer bathing solution into the first living cell layer and then increases stepwise until the full skin potential is reached as the tip pierces the basement membrane (Ussing & Windhager, 1964). The electric responses to short-circuiting of the intracellular potentials show that the cells of adjacent layers are electrically connected. Based on these and other pieces of evidence the following hypothesis was advanced (Ussing & Windhager, 1964; Ussing, 1965a) : Sodium enters the outermost layer of living cells by passive diffusion through a sodium-selective membrane. From the first cell layer, sodium can be either actively transported into the interspaces or pass on to the next layer of cells through intercellular bridges until all the sodium ions have undergone active transport into the interspaces. From there, they diffuse onward through the basement membrane via gaps between the basal cells. This model in many respects agrees with one which was proposed independently by Farquhar and Palade ( 1964) based upon electron-microscopic observations. The shunt path for passive ions might be either through the cells, or through the interspaces, or both pathways might contribute to the shunt. According to Farquhar and Palade (1964), the interspaces seem to be closed towards the outside solution by tight seals. This observation clearly is in favor of a cellular shunt, but, on the other hand, if just a small fraction of the seals were faulty, it might suffice for a considerable extracellular leak. Now, the shunt can be drastically but reversibly increased by making the outside hypertonic (Ussing & Andersen, 1955; Ussing & Windhager, 1964). It was therefore decided to do a careful study of the active and passive transports through the skin with solutions on the outside which were made hypertonic by addition of urea in order to find out whether an extracellular shunt path really exists, at least under these conditions. The first approach was based on the following argument: According to Farquhar and Palate (1964) the ATPase of the epithelium cells is located at the cell membranes facing the interspaces and not at the basal end of the stratum

Ion transport by mitochondria-rich cells in toad skin

SummaryThe optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9±1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to −100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at −100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (Ki) to 0.5mm, but was independent of whether Ki was 2.4, 5, or 10mm. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%. Using this value and the change in cell volume in response to bilateral Cl-free solutions, we calculated an intracellular steady-state Cl concentration of 19.8±1.7mm (n=6) of the short-circuited cell.

Volume regulation and basolateral co-transport of sodium, potassium, and chloride ions in frog skin epithelium

Frog skin epithelium, which is normally almost tight to chloride, acquires a basolateral leakiness to chloride during osmotic swelling. By measuring the epithelial thickness (volume) after equilibration first with half thiocyanate Ringer, and then full thiocyanate Ringer, one obtains the chloride-free volume. Partial or full recovery of the volume and cellular chloride concentration occurs only when the inside of the skin is exposed to solutions containing K as well as Na and Cl. This recovery process is totally inhibited by low concentrations of bumetanide. The data suggest a basolateral NaKCl2 co-transport.

Localization of chloride conductance to mitochondria-rich cells in frog skin epithelium

SummaryCell volume determinations and electrophysiological measurements have been made in an attempt to determine if mitochondria-rich (MR) cells are localized pathways for conductive movements of Cl across frog skin epithelium. Determinations of cell volume with video microscope techniques during transepithelial passage of current showed that most MR cells swell when the tissue is voltage clamped to serosa-positive voltages. Voltage-induced cell swelling was eliminated when Cl was removed from the mucosal bath solution. Using a modified vibrating probe technique, it was possible to electrically localize a conductance specifically to some MR cells in some tissues. These data are evidence supporting the idea that MR cells are pathways for conductive movements of Cl through frog skin epithelium.

Anomalous Transport of Sucrose and Urea in Toad Skin

1. Increased permeability to sucrose and anomalous transport of this substance in the inward direction are elicited in toad skin when the outside bathing solution is made hyperosmotic by addition of 200 millimoles of urea per liter. Influx and efflux of sucrose are measured on paired halves of skins from the belly of the toad, using 14C-sucrose as a tracer. 2. When the hydrostatic pressure of the inside solution is raised 25 cm H2O over that of the outside solution, the flux asymmetry for sucrose is substantially reduced. 3. Similar experiments with 14C-urea as the ‘driven species’ and inactive urea as the ‘driving species’ gave similar results: Increased permeability and anomalous inward transport. The effect of a hydrostatic pressure difference on the flux ratio was, however, less apparent than in the case of sucrose. 4. At low urea concentrations there seems to be a slow inward active transport of urea between identical solutions. The phenomenon is absent in frog skin. 5. The anomalous transports of sucrose and urea are discussed in the light of a model involving ‘anomalous solvent drag’.

The flux ratio equation under nonstationary conditions.

SummaryThe time dependent (i.e., nonstationary) unidirectional fluxes through a multilayered system consisting of sandwiched layers of arbitrary composition and exhibiting arbitrary potential and resistance profiles have been calculated, assuming that the flux is governed by the Smoluchowski equation (i.e., a flux resulting from a diffusion process superimposed upon a migration and/or a convection process, where part of the latter may arise from an active transport process). It is shown that during the building up of the concentration profile of the isotope inside the system towards the stationary value the ratio between the two oppositely directed, time-dependent unidirectional fluxes is, from the very first appearance of the isotope in the surrounding solutions, equal to the value of the stationary flux ratio. The practical implications of this result are discussed.

Epithelial Cell Volume Regulation Illustrated by Experiments in Frog Skin

The volume control of the syncytium of principal cells (as opposed to the mitochondria-rich cells) is largely confined to the movement of ions and water through the basolateral membrane. The apical membrane is nearly tight to water and ions except sodium. The basolateral membrane is normally tight to chloride, but its chloride channels open if the cells swell osmotically or if the membrane is depolarized. If the epithelium has lost KCl during osmotic swelling, it is recovered by a basolateral cotransport of KNaCl2.

The volume of mitochondria-rich cells of frog skin epithelium

SummaryThe pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog,Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.

Sodium Recirculation and Isotonic Transport in Toad Small Intestine

Abstract. Isolated small intestine of toad (Bufo bufo) was mounted on glass tubes for perfusion studies with oxygenated amphibian Ringers solution containing glucose and acetate. Under open-circuit conditions (Vt=−3.9 ± 1.8 mV, N= 14) the preparation generated a net influx of 134Cs+. The time course of unidirectional 134Cs+-fluxes was mono-exponential with similar rate constants for influx and outflux when measured in the same preparation. The flux-ratio was time invariant from the beginning of appearance of the tracers to steady state was achieved. Thus, just a single pathway, the paracellular pathway, is available for transepithelial transport of Cs+. From the ratio of unidirectional Cs+-fluxes the paracellular force was calculated to be, 18.2 ± 1.5 mV (N= 6), which is directed against the small transepithelial potential difference. The paracellular netflux of cesium ions, therefore, is caused by solvent drag. The flux of 134Cs+ entering and trapped by the cells was of a magnitude similar to that passing the paracellular route. Therefore, independent of the convective flux of 134Cs+, every second 134Cs+ ion flowing into the lateral space was pumped into the cells rather than proceeding, via the low resistance pathway, to the serosal bath. It is thus indicated that the paracellular convective flow of 134Cs+ is driven by lateral Na+/K+-pumps. Transepithelial unidirectional 42K+ fluxes did not reach steady state within an observation period of 70 min, indicating that components of the fluxes in both directions pass the large cellular pool of potassium ions. The ratio of unidirectional 24Na+ fluxes was time-variant and declined from an initial value of 3.66 ± 0.34 to a significantly smaller steady-state value of 2.57 ± 0.26 (P < 0.001, N= 5 paired observations), indicating that sodium ions pass the epithelium both via the paracellular and the cellular pathway. Quantitatively, the larger ratio of paracellular Na+ fluxes, as compared to that of paracellular Cs+ fluxes, is compatible with convective flow of the two alkali metal ions through the same population of water-filled pores. With a new set of equations, the fraction of the sodium flux passing the basement membrane barrier of the lateral space that is recirculated through the cellular compartment is estimated. This fraction was, on average, 0.72 ± 0.03 (N= 5). It is concluded that isotonicity of the transportate can be maintained by producing a hypertonic fluid emerging from the lateral space combined with reuptake of salt via the cells.