Influence of lithium upon the intracellular potential of frog skin epithelium

Summary The effect of Li upon the intracellular potential of frog skin (Rana esculenta) was investigated. In the range between 1 and 25mm Li in the epithelial bathing solution, a semilogarithmic linear relationship between [Li] and intracellular potential under short circuit conditions was obtained. The intracellular potential at all [Li] is quantitatively sufficient to explain the previously reported accumulation of Li in the intracellular space of the frog skin epithelium (Leblanc, G. 1972.Pfluegers Arch. 337:1) on the basis of a passive entrance step at the outer border. A reduction of the intracellular potential by Li is also observed in the presence of 6mm Na in the epithelial bathing solution. Consequences regarding the mechanism of uptake of Na across the outer border of the frog skin are discussed.     

Sodium transport and distribution of electrolytes in frog skin

The objective of this study on frog skin was to examine correlations between transepidermal active Na-transport and intracellular [Na]c, [K]c, [Cl]c homeostasis. Isolated, whole skins, and "split skins" were used in measurements of short-circuit current (SCC) and open skin potential (PD). Water and ion contents were estimated on split skins. Absolute [Na]c and [K]c varied over the range of 18 to 46, and 113 to 80 mM, respectively (Figure 7), but a complementary relationship existed between Na and K, such that [Na]c + [K]c remained approximately equal to 129 mM. Average values for [Na]c and [K]c were approximately equal to 31 and approximately equal to 96 mM, respectively. [Cl]c remained constant at approximately equal to 38 mM. This complementary relationship does not seem to be an artifact, caused by collagenase, used in the preparation of split skins. Whole skins and split skins in Ringer's solution, when treated with fluoroacetate (FAc), ouabain (Ou), or vanadate (Va) over wide ranges of concentrations, showed that FAc greatly depressed the SCC and the PD, without changing [Na]c, [K]c, [Cl]c. FAc acted only from the corium side of the skin. The decreasing SCC remained a Na-current, as in control skins. By comparison, such a separation of cellular functions could not be established with Ou, or Va. These inhibitors either affected SCC, PD, and cellular ion concentration, or they had no effect on any of these parameters. The complementary relationship between [Na]c and [K]c, with [Cl]c remaining again at approximately equal to 38 mM, was also found in tissues exposed to inhibitors. These results indicate that transcellular active Na transport and electrolyte homeostasis are not always rigidly coupled, suggesting that these processes may not be uniformly distributed within the epithelial cells, or among the interconnected cell layers of the frog skin epidermis.     

Interaction between the Effects of Inside and Outside Na and K on Bullfrog Skin Potential

The composition of the solution bathing one border of the isolated frog skin affects the response of the potential across the skin to changes in the composition of the solution bathing the opposite border. Increasing the K concentration of the inside (corium) bathing solution decreased the sensitivity of the potential to a change in outside Na concentration. Decreasing the outside Na concentration decreased the sensitivity of the potential to a change in inside K concentration. Increasing the total ionic strength of the outside bathing solution or of both bathing solutions decreased the sensitivity of the potential to a change in outside Na concentration.     

The Effects of Alkali Metal Cations and Common Anions on the Frog Skin Potential

The effects on the potential difference across isolated frog skin (R. catesbeiana, R. pipiens) of changing the ionic composition of the bathing solutions have been examined. Estimates of mean values and precision are presented for the potential changes produced by substituting other alkali metal cations for Na at the outside border and for K at the inside border. In terms of ability to mimic Na at the outside border of bullfrog skin, the selectivity order is Li > Rb, K, Cs; at the outside border of leopard frog skin, Li > Cs, K, Rb. In terms of ability to mimic K at the inside border of bullfrog and leopard frog skin: Rb > Cs > Li > Na. Orders of anion selectivity in terms of sensitivity of the potential for the outside border of bullfrog skin are Br > Cl > NO₃ > I > SO₄, isethionate and of leopard frog skin are Br, Cl > I, NO₃, SO₄. An effect of the solution composition (ionic strength?) on the apparent Na-K selectivity of the outside border is described. The results of the investigation have been interpreted and discussed in terms of the application of the constant field equation to the Koefoed-Johnsen-Ussing frog skin model. These observations may be useful in constructing and testing models of biological ionic selectivity.     

Intracellular voltage of isolated epithelia of frog skin: apical and basolateral cell punctures

Isolated epithelia of frog skin were prepared with collagenase, and the cells were punctured with intracellular microelectrodes across their apical (outer) and basolateral (inner) surfaces. Regardless of the route of cell puncture, the intracellular voltage (Vosc) in short- circuited isolated epithelia was markedly negative, averaging -70.4 mV for apical punctures and -91.6 mV for basolateral punctures. As in intact epithelia, amiloride outside caused the Vosc to become more negative (means of -96.7 and -101.8 mV), with a concomitant increase in the resistance of the apical barrier. Increasing the [K)i of the basolateral solution from 2.4 to 8.0 or 14.4 mM caused rapid step depolarization (5-10 s) of the Vosc under transepithelial Na transporting and amiloride-inhibited conditions of Na transport, with the delta Vosc ranging between 23.9 and 68.3 mV per decade change of [K]i. The finding that the Vosc of isolated epithelia of frog skin is independent of the route of cell penetration is consistent with the notion that the cells of the stratified epithelium are electrically coupled (functional syncitium). Moreover, the isolated epithelium can serve as a useful preparation, especially in studies designed to investigate the properties of the basolateral surfaces of cells.     

The effect of ethanol on ion transport in frog skin.

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of 22Na+ influx. Ethanol did not alter sodium outflux when bathin the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na+ and C1- were increased. The movement of C1- was evaluated by comparison of C1- flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect of Na+ transport.     

Intracellular electrolyte concentrations in the frog skin epithelium: Effect of vasopressin and dependence on the Na concentration in the bathing media

Summary The intracellular electrolyte concentrations of the frog skin epithelium have been determined in thin freeze-dried cryosections using the technique of electron microprobe analysis. Stimulation of the transepithelial Na transport by arginine vasopressin (AVP) resulted in a marked increase in the Na concentration and a reciprocal drop in the K concentration in all epithelial cell layers. The effects of AVP were cancelled by addition of amiloride. It is concluded from these results that the primary mechanism by which AVP stimulates transepithelial Na transport is an increase in the Na permeability of the apical membrane. However, also some evidence has been obtained for an additional stimulatory effect of AVP on the Na pump. In mitochondria-rich cells and in gland cells no significant concentration changes were detected, supporting the view that these cells do not share in transepithelial Na transport. Furthermore, the dependence of the intracellular electrolyte concentrations upon the Na concentration in the outer and inner bathing solution was evaluated. Both in control and AVP-stimulated skins the intracellular Na concentration showed saturation already at low external Na concentrations, indicating that the self-inhibition of transepithelial Na transport is due to a reduction of the permeability of the apical membrane. After lowering the Na concentration in the internal bath frequently a Na increase in the outermost and a drop in the deeper epithelial layers was observed. It is concluded that partial uncoupling of the transport syncytium occurs, which may explain the inhibition of the transepithelial Na transport and blunting of the AVP response under this condition.     

Intracellular gradients of ion activities in the epithelial cells of theNecturus gallbladder recorded with ion-selective microelectrodes

In Necturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from -28 mV in the mucosal end to about -50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39 mM to between 8 and 19 mM. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5 X 10(-4) M) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5 degrees C, the Cl- activity varied from about 55 mM in the mucosal end to 28 mM in the serosal end, and the K+ activity from 50 mM to between 95 and 131 mM. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about -5 mV. The Cl- activity became constant in 65 mM, and the K+ activity became constant at 109 mM, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1 mM. The ratio of cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl-. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assess from the net solute absorption. Animals kept at 15 degrees C had lower intracellular Na+ activities, higher Cl- and K+ activities, and higher rates of absorption than animals stored at 5 degrees C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.     

Electrochemical potentials in frog skin: inferences for electrical and mechanistic models

To evaluate possible mechanisms of transport at apical and basolateral barriers of Na transporting cells of epithelia, it is necessary to know the difference of electrochemical potentials at each barrier. A reevaluation in light of new data of intracellular voltages of frog skin leads to fundamental questions concerning the origin of the voltages at both inner and outer barriers of this tissue. Whereas the inner barrier is highly selective for K, confirming the observations of Koefoed-Johnsen and Ussing, the voltage across the inner barrier, Vi, especially in the absence of transepithelial Na transport, may be greater than the Nernst equilibrium potential for K estimated from the maximum values of intracellular [K] reported in the literature. Consequently, it is proposed that the Na:K pumps may, under some conditions, behave not only as a Na:K exchange pump but also as a cation extrusion pump for K especially when intracellular [Na] falls to low levels. In order to explain the relationship between Na entry and the voltage at the outer barrier, it is proposed that the conductance of the outer barrier is voltage dependent, in line with previous observations of the nonlinear electrical behavior of the apical barrier of Na transporting cells. Thus, the outer barrier may behave as a simple voltage independent resistor with a Thévenin electromotive force of zero at negative intracellular voltages despite the existence of a chemical potential for Na at this barrier.     

Intracellular chloride activity and membrane potential in stripped frog skin (Rana temporaria)

The regulation of cell chloride activity in frog skin was investigated using double barrelled Cl--microelectrodes to measure cell membrane potentials and chloride activity in the isolated frog epidermis. Experiments were done under short-circuit conditions, impaling cells from the serosal side. The basic electrophysiological parameters of the isolated skin were similar to those reported in the literature for whole preparations. Intracellular chloride activity was on average 21.9 mM and membrane potential was about 57 mV, implying that chloride was distributed away from its electrochemical equilibrium (i.e., concentrated inside the cells). Chloride activity decreased after removal of either Cl- or Na+ from the serosal bathing solution, with no change in membrane potential. The chloride permeability of the serosal membrane was calculated to be 2.6 X 10(-6) cm X s-1 which represents about 1/4 of the total conductance of the serosal membrane. We suggest that an electrically silent sodium-dependent uphill transport of chloride is present at the basolateral membrane of the frog skin, which accounts for the non-passive distribution of chloride.     

Intracellular Electrical Potentials in Frog Skin

The influence of changes in ionic composition of the bathing solutions on intracellular electrical potentials in frog skin has been examined. When the skin bathed in SO₄ Ringer''s solution is penetrated with a microelectrode two approximately equal potential jumps were frequently observed and most experiments were carried out with the electrode located between these steps. Substitution of Cl for SO₄ in the bathing solutions caused a decrease in PD across both the "outer" and "inner" barriers. When the skin was short-circuited an average intracellular potential of -18 mv was found with both Cl and SO₄ Ringer''s. With the skin in SO4 Ringer''s, decrease in Na concentration of the outside solution caused a decrease in PD between the microelectrode and the outside solution which was approximately the same as the decrease in total skin PD. With SO₄ Ringer''s, an increase in K concentration in the inside solution caused a marked decrease in total skin PD. However, only 50 per cent of this change occurred at the inner barrier, between the microelectrode and the inside solution. The remainder of the change occurred at the outer barrier. This observation does not appear to be consistent with the model of the skin proposed by Koefoed-Johnson and Ussing (Acta Physiol. Scand., 1958, 42, 298).     

Cellular Li+ opens paracellular path in toad skin: Amiloride blockable effect

The presence of Li in the solution bathing the outer surface of toad skin under short-circuit condition promotes an unspecific permeability increase characterized by a delayed and progressive increase in the effluxes of 24Na, 42K and 14C sucrose. The effect of Li upon sucrose permeability might indicate an increased permeability of the paracellular pathway. The Li effect is mediated by an intracellular action since blockade of Li entrance into the cell compartment by amiloride prevents the increase in Na, K and sucrose permeability. A possible mechanism of this effect is discussed in terms of a disturbance in the cellular Ca++ balance leading to an increase in cytosolic Ca++ concentration which perturbs the organization of the cytoskeleton and the interplay between cytoskeleton and tight junctions.     

Na transport across frog skin at low external Na concentrations

Isolated frog skin was bathed with a dilute solution containing 1 mm NaCl on the outside and with normal Ringer’s solution on the inner surface. Net Na flux was determined by simultaneous measurement of unidirectional fluxes with Na²² and Na²⁴ and intracellular electrical potentials were examined with microelectrodes. There was a net inward transport of Na under both open-circuit and short-circuit conditions. The short-circuit current was approximately 15% greater than the net Na flux; the discrepancy could be accounted for by a small outward flux of Cl. The electrical potential profile did not differ greatly from that observed in skins bathed on the outside with normal Ringer’s solution. Under open-circuit conditions, there were usually several potential steps and under short-circuit conditions the cells were negative relative to the bathing solutions. Estimates of epithelial Na concentrations utilizing radioactive Na suggested that if all epithelial Na were in a single compartment, an active entry step would be necessary to allow a net inward Na transport. The results could also be explained by a series arrangement of Na compartments without necessarily postulating an active Na entry. The behavior of the potential profile suggested that this latter alternative was more likely.     

Electron microprobe analysis of frog skin epithelium: Evidence for a syncytial sodium transport compartment

Summary For elucidation of the functional organization of frog skin epithelium with regard to transepithelial Na transport, electrolyte concentrations in individual epithelial cells were determined by electron microprobe analysis. The measurements were performed on 1-m thick freeze-dried cryosections by an energy-dispersive X-ray detecting system. Quantification of the electrolyte concentrations was achieved by comparing the X-ray intensities obtained in the cells with those of an internal albumin standard.The granular, spiny, and germinal cells, which constitute the various layers of the epithelium, showed an identical behavior of their Na and K concentrations under all experimental conditions. In the control, both sides of the skin bathed in frog Ringer's solution, the mean cellular concentrations (in mmole/kg wet wt) were 9 for Na and 118 for K. Almost no change in the cellular Na occurred when the inside bathing solution was replaced by a Na-free isotonic Ringer's solution, whereas replacing the outside solution by distilled water resulted in a decrease of Na to almost zero in all layers. Inhibition of the transepithelial Na transport by ouabain (10^–4 m) produced an increase in Na to 109 and a decrease in K to 16. The effect of ouabain on the cellular Na and K concentrations was completely cancelled when the Na influx from the outside was prevented, either by removing Na or adding amiloride (10^–4 m). When, after the action of ouabain, Na was removed from the outside bathing solution, the Na and K concentration in all layers returned to control values. The latter effect could be abolished by amiloride.The other cell types of the epithelium showed under some experimental conditions a different behavior. In the cornified cells and the light cells, which occurred occasionally in the stratum granulosum, the electrolyte concentrations approximated those of the outer bathing meium under all experimental conditions. In the mitochondria-rich cells, the Na influx after ouabain could not be, prevented by adding amiloride. In the gland cells, only a small change in the Na and K concentrations could be detected after ouabain.The results of the present study are consistent with a two-barrier concept of transepithelial Na transport. The Na transport compartment comprises all living epithelial layers. Therefore, with the exception of some epithelial cell types, the frog skin epithelium can be regarded as a functional syncytium for Na.     

The stimulation of Na+ uptake in frog skin by uranyl ions.

1. The Na+ uptake in the isolated from skin of Rana esculenta was measured by the short-circuit current (Isc). Uranyl ions increase at pH 5.5 the Isc up to 200% at concentrations of 10 mM. The half-maximal value for this effect is at about 1 mM uranyl salt. 2. The effect is (a) specific for the Na+-selective membrane, (b) fully reversible. No stimulation can be seen in presence of 1 mM H+ or 0.1 mM amiloride. 3. The decrease of the sodium permeability of the apical membrane (PNa), normally induced by increasing concentrations of Na+ in the mucosal solution, %Na]o, is partially prevented by uranyl ions. The apparent Michaelis constant of the saturable Na+ uptake is shifted to much higher values. 4. A comparison between the uranyl effect and similar effects of the other drugs leads to the conclusion that uranyl ions might act in a polar hydrophobic environment, possibly by combining with phosphate groups (of phospholipids), and, thus, enhancing Na+ permeability by changes in tertiary structure near each Na channel. The interaction of mucosal Na+ with their receptor, normally triggering the [Na]o-dependent decrease of PNa, is thought to be diminished by uranyl association in a neighbouring region, causing a noncompetitive stimulation of the Na+ translocation though the apical frog skin membrane.     

Effects of Alkali Metal Gations on the Potential across Toad and Bullfrog Urinary Bladder

Isolated urinary bladders of the bullfrog (R. catesbeiana) and the toad (B. marinus) were mounted in an Ussing chamber. Potential differences up to 114 mv were observed in bullfrog bladder when the mucosal surface was bathed in dilute Na₂SO₄ and the serosal surface in sulfate Ringer's. In experiments with bullfrogs, K was used to replace Na in the mucosal solution and Na was used for K in the serosal solutions. The selectivity was judged in terms of the relative effectiveness of the replacement cation in maintaining the bladder potential. In experiments with toads, K and Rb were equally poor replacements for Na at the mucosal border, while Rb was a good replacement for K at the serosal border. Li in the mucosal solution appeared to depress the potential in part irreversibly. At the serosal border, Li was a partially effective substitute for K, more so than was Na. However, both were poor replacements compared to Rb. The mucosal surface of the urinary bladder of both frog and toad appears to be Na-selective and the serosal surface appears to be K-selective, consistent with the Koefoed-Johnsen-Ussing model for frog skin.     

Independence of apical membrane Na+ and Cl- entry in Necturus gallbladder epithelium

Transepithelial fluid transport (Jv) and intracellular Na+ and Cl- activities (aNai, aCli) were measured in isolated Necturus gallbladders to establish the contribution of different proposed apical membrane entry mechanisms to transepithelial salt transport. In 10 mM HCO3- Ringer's, Jv was 13.5 +/- 1.1 microliter X cm-2 X h-1, and was significantly reduced by a low bicarbonate medium and by addition of amiloride (10(-3)M) or SITS (0.5 X 10(-3)M) to the mucosal bathing solution. Bumetanide (10(-5)M) was ineffective. Bilateral Na+ removal abolished Jv. The hypothesis of NaCl cotransport was rejected on the basis of the following results, all obtained during mucosal bathing solution changes: during Na+ removal, aNai fell 4.3 times faster than aCli; during Cl- removal, aCli fell 7.5 times faster than aNai; amiloride (10(-3) M) reduced aNai at a rate of 2.4 +/- 0.3 mM/min, whereas aCli was not changed; bumetanide (10(-5) M) had no significant effects on Jv or aCli. The hypothesis of Na-K-Cl cotransport was rejected for the same reasons; in addition, K+ removal from the mucosal bathing solution (with concomitant Ba2+ addition) did not alter aNai or aCli. The average rate of NaCl entry under normal transporting conditions, estimated from Jv, assuming that the transported fluid is an isosmotic NaCl solution, was 22.5 nmol X cm-2 X min-1. Upon sudden cessation of NaCl entry, assuming no cell volume changes, aNai and aCli should fall at an average rate of 4.8 mM/min. To compare this rate with the rates of Na+ and Cl- entry by ion exchange, the Na+ or Cl- concentration in the mucosal bathing solution was reduced rapidly to levels such that electroneutral cation or anion exchange, respectively, should cease. The rate of Na+ or Cl- entry before this maneuver was estimated from the initial rate of fall of the respective intracellular ionic activity upon the mucosal solution substitution. aNai and aCli decreased at initial rates of 3.7 +/- 0.4 and 5.9 +/- 0.8 mM/min, respectively. The rate of fall of aNai upon reduction of external [Na] was not affected by amiloride (10(-3) M), and the rate of fall of aCli upon reduction of external [Cl] was unchanged by SITS (0.5 X 10(-3) M), which indicates that net cation or anion exchange was, in fact, abolished by the changes in Na+ and Cl- gradients, respectively. I conclude that double exchange (Na+/H+ and Cl-/HCO-3) is the predominant or sole mechanism of apical membrane NaCl entry in this epithelium.     

Highly selective blockade of the frog skin sodium channels by monovalent copper cations.

.5 mM Cu+ added to the mucosal side of frog skin caused rapid reversible inhibition of short-circuit current while no effect of Cu+ could be observed at the serosal side. In both cases Cu2+ was reduced to Cu+ by adding 10 mM ascorbic acid. Cu+ being similar to Na+ both in charge and crystal radius (0.096 and 0.095 nm, respectively) appears to block Na+ channels in the apical membrane. Cu2+ being of a smaller size (crystal radius 0.072 nm) was ineffective at the mucosal side causing only a rather slow irreversible inhibition of Na+ transport when added to the serosal bathing solution.     

Sodium-dependent copper uptake across epithelia: a review of rationale with experimental evidence from gill and intestine

The paper reviews the evidence for apparent sodium-dependent copper (Cu) uptake across epithelia such as frog skin, fish gills and vertebrate intestine. Potential interactions between Na(+) and Cu during transfer through epithelial cells is rationalized into the major steps of solute transfer: (i) adsorption on to the apical/mucosal membrane, (ii) import in to the cell (iii) intracellular trafficking, and (iv) export from the cell to the blood. Interactions between Na(+) and Cu transport are most likely during steps (i) and (ii). These ions have similar mobilities (lambda) in solution (lambda, Na(+), 50.1; Cu(2+), 53.6 cm(2) Int. ohms(-1) equiv(-1)); consequently, Cu(2+) may compete equally with Na(+) for diffusion to membrane surfaces. We present new data on the Na(+) binding characteristics of the gill surface (gill microenvironment) of rainbow trout. The binding characteristics of Na(+) and Cu(2+) to the external surface of trout gills are similar with saturation of ligands at nanomolar concentrations of solutes. At the mucosal/apical membrane of several epithelia (fish gills, frog skin, vertebrate intestine), there is evidence for both a Cu-specific channel (CTR1 homologues) and Cu leak through epithelial Na(+) channels (ENaC). Cu(2+) slows the amiloride-sensitive short circuit current (I(sc)) in frog skin, suggesting Cu(2+) binding to the amiloride-binding site of ENaC. We present examples of data from the isolated perfused catfish intestine showing that Cu uptake across the whole intestine was reduced by 50% in the presence of 2 mM luminal amiloride, with 75% of the overall inhibition attributed to an amiloride-sensitive region in the middle intestine. Removal of luminal Na(+) produced more variable results, but also reduced Cu uptake in catfish intestine. These data together support Cu(2+) modulation of ENaC, but not competitive entry of Cu(2+) through ENaC. However, in situations where external Na(+) is only a few millimoles (fish gills, frogs in freshwater), Cu(2+) leak through ENaC is possible. CTR1 is a likely route of Cu(2+) entry when external Na(+) is higher (e.g. intestinal epithelia). Interactions between Na(+) and Cu ions during intracellular trafficking or export from the cell are unlikely. However, effects of intracellular chloride on the Cu-ATPase or ENaC indicate that Na(+) might indirectly alter Cu flux. Conversely, Cu ions inhibit basolateral Na(+)K(+)-ATPase and may increase [Na(+)](i).     

A simple procedure for NMR measurements of intra- and extracellular sodium in intact tissues

The total Na+ and both the intra- and extracellular Na+ content of excised rat and frog tissues was quantitated by 23Na NMR at 95.51 MHz. An external capillary containing 33 mM Na7[Dy(P3O10)2], resonating about 30 ppm upfield relative to the 0.00 ppm of the intracellular Na+, was inserted into the tissues. The capillary was calibrated against a concentration range of pure NaCl solution, for measurement of intracellular Na+, and against the same concentrations of NaCl solutions containing 4-6 mM K7[Dy(P3O10)2] in 50 mM histidine. Cl and 100 mM choline. Cl, for measurement of extracellular Na+. Spectra were recorded on tissues first in the absence of the shift reagent for determination of the total Na+. After addition of a K7[Dy(P3O10)2] solution to the sample, the 23Na spectra were recorded immediately so that data accumulation was completed within 15 min. Under these conditions, the extracellular Na+ resonated from 10 to 20 ppm upfield relative to the intracellular Na+, and no loss in the intensity of the extracellular Na+ resonance occurred due to the lability of dysprosium(III)tripolyphosphate (cf. Matwiyoff et al., Magn. Reson. Med. 3: 164, 1986). The intra- and extracellular Na+ content of the tissue was calculated from the integrated areas of the respective Na+ resonances and that of the calibrated capillary, from the known weight of the tissue, and from the known volumes of the solutions added.(ABSTRACT TRUNCATED AT 250 WORDS)