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When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15–30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na+K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4–8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.

Platinum-195 spectra are reported for [Ptn(CO)2n]2- (n = 3, 6, 9, 12 and 15) and carbon-13 spectra are reported for n = 6, 9 and 12 over a range of temperatures. The spectra provide evidence for (a) intramolecular rotation of the Pt3-triangles about the principal three-fold axis, (b) inter-exchange of Pt3-triangles, (c) lack of terminal/edge carbonyl exchange within the Pt3(CO)3(μ-CO)3 group. Evidence is also presented for the formation of [Ni3Pt3(CO)12]2- on mixing [Pt6(CO)12]2- [Ni6(CO)12]2-.

The patch-clamp technique for the recording of single-channel currents was used to investigate the activity of ion channels in the intact epithelium of the toad urinary bladder. High resistance seals were obtained from the apical membrane of tightly stretched tissue. Single-channel recordings revealed the activity of a variety of ion channels that could be classified in 4 groups according to their mean ion conductances, ranging from 5 to 59 pS. In particular, we observed highly selective, amiloride-sensitive Na channels with a mean conductance of 4.8 pS, channels with a similar conductance that were not Na-selective and channels with mean conductance values of 17-58 pS that were mostly seen after stimulation of the tissue with vasopressin or cAMP. When inside-out patches from the apical membrane were exposed to 110 mM fluoride, large conductances (86-490 pS) appeared.

Epithelia transport a variety of solutes and water. Study of such transport requires a determination of the driving forces responsible for transport, of the pathways through which transport occurs, and of the factors controlling such transport. Transepithelial driving forces are readily determined where the composition of the bathing media can be altered and electrical forces negated. Where substances move only through a paracellular pathway such manipulations may be adequate to define the permeability and selectivity of the pathways. For substances utilizing a cellular pathway, driving forces and permeabilities across the two dissimilar apical and basolateral cellular membranes must be determined. Where a substance can be shown to move across a membrane against its electrochemical potential gradient, the source of the energy for such movement must be assessed. This review focuses on the applicability and validity of a variety of techniques utilized for the study of epithelial transport to answer these questions. These include microelectrode techniques, chemical analyses, microprobe analysis, microscopy, and techniques for assessing the coupling of metabolism to transport.

The structure of the major zinc-containing species in an aqueous solution of 2.20 mol dm–3 ZnCl2+ 2,2′,2″-triaminotriethylamine (tren)(1 : 1) has been investigated by X-ray diffraction. The data have been analysed in terms of six models containing four-, five- or six-coordinate zinc. The preferred model involves the cation [Zn(tren)Cl]+, in which the disposition around the Zn is approximately trigonal-bipyramidal and similar to that reported in two single-crystal determinations on zinc(tren) complexes. The average Zn–N distance is 2.09(2)A and the Zn—Cl distance 2.37(3)A.

1. Thin slices (0.2-0.3 mm) of rabbit renal cortex have been incubated in isosmotic oxygenated acetate media at 25 degrees C with or without ouabain (10(-3) M), amiloride (2 x 10(-3) M) or iodoacetamide (10(-3) M). 2. Slices in normal isosmotic 146 mM-sodium-132 mM-acetate media swelled as reported previously (Cooke & Macknight, 1984). This swelling was not prevented by amiloride or by metabolic inhibition. 3. Slices in isosmotic 132 mM-choline-132 mM-acetate media gained much less water and were little affected by ouabain, amiloride or metabolic inhibition. Choline was able to substitute neither for sodium nor for potassium in activating preparations of renal cortical Na+-K+-ATPase in chloride or in acetate media. 4. Slices in isosmotic 20 mM-sodium-132 mM-acetate medium swelled nearly as much as did slices in normal sodium acetate medium. However, this swelling was impaired markedly by amiloride, by ouabain and by metabolic inhibition. 5. There was a direct correlation between medium sodium concentration and slice water content as sodium was increased from 1.25 to 30 mM in 132 mM-acetate media. However, up to a sodium concentration of 10 mM, amiloride (2 x 10(-3) M) completely prevented this increase in water content. 6. Increasing medium amiloride concentration from 10(-5) to 10(-3) M progressively inhibited cellular swelling in 10 mM-sodium-132 mM-acetate medium. It is concluded that, under these experimental conditions, the dominant pathway for hydrogen ion extrusion from the cells was via amiloride-sensitive sodium-hydrogen exchange. 7. The results are discussed in terms of a model which explains cellular swelling in acetate media in terms of (a) non-ionic diffusion of acetic acid across plasma membranes impermeable to the acetate anion, (b) removal from the cells of the hydrogen ion gained with the acetate by amiloride-sensitive sodium-hydrogen counter-transport and (c) subsequent extrusion of sodium from the cell accompanied by potassium uptake via the ouabain-sensitive Na+-K+-ATPase. 8. The results provide evidence for ion movements across the luminal plasma membrane of proximal tubular cells in rabbit renal cortical slices.

With some 55 to 60% of body weight attributable to water and approximately two-thirds of this within cells, the constancy of body weight is testimonial to the precision with which cells regulate their water content. Understanding of how this is accomplished is still, however, largely speculative.

The content and concentration of major ions within the scraped mucosal epithelial cells of the urinary bladder of the toad Bufo marinus were determined using [ 14 C]- and [ 3 H]-inulin to correct for adherent extracellular fluid. In aldosterone treated hemi-bladders the radioactive sodium content and concentration increased significantly as compared with paired control hemi-bladders when both tissues were exposed to [ 24 Na] in the mucosal bathing medium. Changes in total non-inulin space sodium were not detected but the stimulation of transepithelial sodium transport by aldosterone in this set of observations was small, averaging only some 30% above control. The results are compatible with our hypothesis that the major action of aldosterone is to facilitate the entry of sodium from the mucosal medium across the apical permeability barrier of the transporting mucosal cells.

Recent investigations of ion transport across epithelia have placed much emphasis on sodium movements. Less attention has been given to transepithelial potassium transport, which in general contributes much less to total ion movement than does sodiumu Yet, the movement of potassium across epithelial cells and its regulation are essential for potassium homeostasis. This review discusses some of the factors which affect potassium movements across cellular membranes: and across epithelia, it summarizes our knowledge of potassium transport across different epithelia, and it looks briefly at the dependency of transepithelial sodium transport on potassium in the medium bathing the inner surface of the epithelial cells.

Publisher Summary This chapter discusses aspects of the regulation of cellular volume only under isosmotic conditions. Despite the greater knowledge of membrane transport processes, many questions fundamental to the understanding of the regulation of cellular volume remain unanswered. To study cellular volume regulation under isosmotic conditions, preparations are required in which both water and ions are easily measured in a variety of experimental situations. There is no ideal preparation. Cellular volume may be estimated directly by microscopy. Determination of absolute volumes in fixed specimens is complicated by the problem of possible alterations in volume during fixation and processing. In many studies of cellular volume, water contents rather than volumes are measured. Because 70 to 80% of the mass of most cells is water, provided that solid matter in the cells remains constant during the course of the experiment, changes in water content can be regarded as synonymous with changes in volume.

13 C n.m.r. studies on the three structurally related carbonyl clusters [Rh6(CO)16], [Rh6(CO)15]2– and [Rh7(CO)16]3– show that [Rh6(CO)16] is not fluxional at +70°, [Rh6(CO)15]2– readily undergoes intra-carbonyl exchange at –70° and [Rh7(CO)16]3– is not fluxional at –70° but undergoes partial intra-exchange at +25°.

Summary. When serosal medium potassium was decreased from the usual concentration of 3.5 raM, the short-circuit current (SCC) of hemibladders in chambers immediately and transiently increased. The maximum SCC attained was greater the greater the decrease in serosal potassium, and was twice the initial SCC when the final serosal medium was potassium-free. The SCC then fell to its previous level for final serosal potassium concentrations greater than 2 mM and to less than its previous level for those less than 2 mM, being lowest (15% of previous level) in potassium-free sodium Ringer's. When serosal medium potassium was increased from 3.5 mM by substituting potassium for sodium, SCC transiently decreased and then recovered to its previous level. Steady SCC was the same in serosal media of 2-116 rnM potassium; conductance increased and p.d. decreased after incubation in 50-116 mM potassium serosal media. Short-circuit current and p.d. transiently increased (decreased) whenever serosal medium potassium was decreased (increased); conductance increased with any change in serosal potassium. Changing mucosal medium potassium concentration between 0 and 50 rnM did not affect SCC. The initial transient increase and subsequent decrease in SCC on removing serosal potassium were partially prevented by 3.5 mM rubidium or caesium, or by 116 nag choline in the serosal medium. The transient changes in SCC were due partly to changes in transepithelial sodium transport. The effects of medium potassium on transepithelial sodium transport remain poorly understood. Many epithelia seem to require the presence of potassium, in a concentration similar to that of the extracellular fluid, in the media bathing the surface towards which sodium is being transported (frog skin, Koefoed-Johnsen & Ussing, 1958; toad bladder, Bentley, 1959; Hays & Leaf, 1961; fish gallbladder, Diamond, 1962). However, the mechanism by which medium potassium supports transepithelial sodium transport remains controversial. One possibility, first advanced by Koefoed-Johnsen and Ussing (1958) and Ussing (1960) is that there is an obligatory requirement for potassium to exchange for sodium across

Tissue composition, membrane potentials and cellular activity of potassium, sodium and chloride have been measured in innervated and denervated rat skeletal muscles incubated in vitro. After denervation for 3 days, tissue water, sodium and chloride were increased but cellular potassium content and measured activity were little affected, despite a decrease of 16 mV in resting membrane potential which would have necessitated a decrease in cellular potassium activity of almost 50% were potassium distributed at electrochemical equilibrium. These findings, therefore, preclude a decreased electrochemical potential gradient for potassium as the cause of the membrane depolarization characteristic of denervated muscle fibers. Analysis of the data excludes an important contribution of rheogenic sodium transport to the resting potential of innervated muscles. These results strongly support the hypothesis that the decreased membrane potential in denervated fibers reflects a relative increase in the membrane permeability to sodium.

Publisher Summary This chapter provides a framework within which volume regulation in epithelia can be considered and focuses on the techniques applied to determine cell volume and cell water and ions, and finish by discussing some of the preparations that have been used to study the regulation of cell volume in epithelia. Cell volume can be examined directly using a wide range of microscopy techniques applied to fixed tissues, or, more recently, to living preparations. In alternative method for monitoring rapid changes, epithelial cells of the gallbladder of Necturus are loaded with tetramethylammonium by exposure of the apical surface to a high concentration of this substance in the presence of the polyene antibiotic nystatin. After a short time, the nystatin is removed and intracellular tetramethylammonium is found, using an ion-selective electrode, to be between 2 and 15 mM, and to remain unchanged for several hours. Changes in cellular volume could then be monitored by determining the changes in intracellular tetramethylammonium concentration following changes in osmolality of the mucosa bathing solution.

The structures of the hydrated zinc(II) and nitrate ions in 4.72 mol dm–3 aqueous Zn(NO3)2 have been determined by X-ray diffraction. Octahedral coordination in Zn2+aq is confirmed and the average Zn—Owater distance is 2.17 (4)A; there is no evidence for coordination by NO–3. Approximately three water molecules are hydrogen bonded to each nitrate O-atom.

1. Rat, rabbit and guinea-pig liver slices were incubated in media at 37° C to study effects of ouabain and of metabolic inhibition on tissue composition. 2. Slices initially incubated in oxygenated balanced medium did not swell when exposed to either balanced media or potassium-free media containing ouabain in concentrations ranging from 0·1 to 10 m M though they lost potassium and gained a comparable amount of sodium. 3. Without prior incubation in oxygenated medium slices initially swelled when incubated with ouabain but then maintained their new volume. 4. Slices whose metabolism was inhibited by incubation with nitrogen and 1 or 10 m M iodoacetamide lost as much potassium as slices exposed to 10 m M ouabain but became swollen with the uptake of sodium, chloride and water. 5. The results show that liver cells, like renal cortical cells, possess a metabolically dependent, ouabain-insensitive, potassium-independent mechanism concerned in the regulation of cellular volume.

Cell volume is determined by the content of osmotically active solute (cell osmoles) and the osmolarity of the extracellular fluid. Cell osmoles consist of non-diffusible and diffusible solutes. A large fraction of the diffusible cation content balances negative charges on the non-diffusible solutes. The content of diffusible solutes is determined by the electrochemical gradients driving them across the plasma membrane and the availability and activity of transport pathways in the membrane. The classical view that the sodium pump offsets passive leaks must be modified to accomodate the contributions of a number of secondary active transport processes, as well as to allow for changes in cell nondiffusible osmoles and in their net negative charge. The behaviour of cells in anisosmotic media is often different from that predicted for a perfect osmometer. In many cases this is a consequence of changes in cell osmole content. However, caution is required in extrapolating from in vitro responses of isolated cells to large, acutely induced changes in medium osmolality to the responses of tissues in vivo to more subtle changes in extracellular osmolality.

Cellular volume can be varied substantially by replacing medium Cl- isosmotically by other univalent anions. Since K+ content changes in parallel, cellular K+ concentration is well maintained. Gluconate behaves as an impermeant anion so cells shrink. Acetate enters cells apparently by non-ionic diffusion causing marked cellular swelling. These changes in volume are fully reversed when Cl- is again restored to the medium. However, ouabain (10(-3) M) largely prevents this reversal when Cl- replaces acetate, arguing against a ouabain-insensitive volume regulating mechanism. In toad urinary bladder, serosal gluconate inhibits transepithelial Na+ transport and cells shrink. Analysis suggests that cell shrinkage results in a loss of Ba2+-sensitive highly selective basolateral membrane K+ conductance channels.

1. Renal cortical slices from rat, rabbit, and guinea-pig were incubated in media in which choline, caesium or rubidium replaced sodium.2. Slices of rabbit and guinea-pig renal cortex incubated in oxygenated choline Ringer decreased in volume initially and did not swell over 3 hr at 25 degrees C. There was a steady loss of potassium. Inhibition of metabolism (N(2) + 1 mM iodoacetamide) caused some swelling. Ouabain, 10 mM, in choline Ringer affected neither loss of potassium nor tissue water content.3. Slices of rat renal cortex similarly incubated in choline Ringer swelled over 3 hr at 25 degrees C whether or not metabolism was inhibited; ouabain (15 mM) affected neither tissue potassium loss nor tissue water content.4. Incubation in choline Ringer containing either 0.2 mMp-chloromercuribenzoic acid, or 1 mM ethacrynic acid increased the tissue water content of guinea-pig renal cortical slices.5. Depletion of cellular potassium (by preliminary incubation in oxygenated potassium-free sodium Ringer with 10 mM ouabain at 30 degrees C) resulted in increased tissue water content when rabbit renal cortical slices were subsequently incubated in oxygenated choline Ringer at 25 degrees C for 3 hr.6. There was no evidence of energy-dependent extrusion of water or ions from either equilibrated rat or rabbit renal cortical slices leached at 0.5 degrees C and then reincubated at 25 degrees C in choline Ringer.7. Rat and guinea-pig renal cortical slices leached at 0.5 degrees C and reincubated at 25 degrees C swelled in rubidium Ringer and in caesium Ringer. There was no evidence of energy-dependent water or ion extrusion when metabolism was restored after leaching in either of these media. Metabolizing rat slices but not guinea-pig slices swelled faster than slices whose metabolism was inhibited.8. These results lend no support to the mechano-chemical hypothesis which ascribes cellular volume regulation to a contractile mechanism squeezing isotonic extracellular fluid from the cells. Instead it is suggested that cellular water content in these experiments reflects the balance between the rate of loss of potassium (and chloride) from the cells and the rate of uptake of extracellular cation (and chloride) into the cells - these rates reflecting both the electrochemical potential gradients of the ions and membrane permeability to them. The implications in relation to the hypothesis of ouabain-insensitive cellular volume regulation are discussed.

Transepithelial sodium transport was virtually abolished when toad urinary hemibladders, mounted in chambers and short-circuited, were exposed on their serosal surface to ouabain, 10-2 M,for 60 minutes. Epithelial cells scraped from such hemibladders gained sodium and lost an equal quantity of potassium when compared with controls not exposed to cardiac glycoside. Their total cellular cation content, chloride content and water content were unchanged. Experiments in which 24-Na, amiloride, or sodium-free mucosal solutions were used, revealed that a large, though variable, percentage of the sodium gained by cells exposed to oubain, came from the mucosal medium, a finding consistent with the model of passive sodium entry from the mucosal medium followed by active sodium extrusion to the serosa. The oubain-insensitive maintenance of cellular volume which was observed did not depend upon transepithelial sodium transport which had been virtually completely inhibited by ouabain. Neither did the maintenance of a normal cellular potassium content depend upon transepithelial sodium transport, for cellular potassium was unaffected when the mucosal medium was sodium-free or when it contained sufficient amiloride, 10-3 M, to virtually abolish such transport.

The electrolyte composition of toad urinary bladder epithelial cells has been measured using the technique of electron microprobe analysis. Portions of hemibladders, which had been mounted in chambers and bathed with a variety of media, were layered with albumin solution on their mucosal surfaces and immediately shock-frozen in liquid propane at -180 degrees C. From the frozen material 1--2 micrometer thick cryosections were cut and promptly freeze-dried for 12 hr at-80 degrees C and 10(-6) Torr. Electron microprobe analysis using a scanning electron microscope, an energy dispersive X-ray detector, and a computer programme, to distinguish between characteristic and uncharacteristic radiations, allowed quantification of cellular ionic concentrations per kg tissue wet wt by comparison of the intensities of the emitted radiations from the cells and from the albumin layer. Granular, mitochondrial-rich, and basal cells, and the basal portions of goblet cells, showed a similar composition, being high in K (about 110 mM/kg wet wt) and low in Na (about 13 mM/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K, presumably reflecting the composition of the mucus within them. With Na-Ringer's as the mucosal medium, cells gained Na and lost K, when their serosal surfaces were exposed to ouabain, 10(-2) M. Replacement of mucosal Na by choline virtually prevented these ouabain-induced changes. Cellular ion contents were unchanged when Na in the serosal medium was replaced by choline. No differences in Na and K concentrations were detected between nuclei and cytoplasm. These results provide independent support for the hypothesis the the cellular Na transport pool in toad bladder epithelial cells derives exclusively from the mucosal medium and that no important recycling of Na occurs from the serosal medium to the cells.

The structures of the hydrated zinc(II) and nitrate ions in 4.72 mol dm–3 aqueous Zn(NO3)2 have been determined by X-ray diffraction. Octahedral coordination in Zn2+aq is confirmed and the average Zn—Owater distance is 2.17 (4)A; there is no evidence for coordination by NO–3. Approximately three water molecules are hydrogen bonded to each nitrate O-atom.

13 C n.m.r. studies on the three structurally related carbonyl clusters [Rh6(CO)16], [Rh6(CO)15]2– and [Rh7(CO)16]3– show that [Rh6(CO)16] is not fluxional at +70°, [Rh6(CO)15]2– readily undergoes intra-carbonyl exchange at –70° and [Rh7(CO)16]3– is not fluxional at –70° but undergoes partial intra-exchange at +25°.