Kinetics of potassium transport across single distal tubules of rat kidney

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J. Physiol. (1973), 232, pp. 47-70 With 5 text-figures Printed in Great Britain

47

KINETICS OF POTASSIUM TRANSPORT ACROSS SINGLE DISTAL TUBULES OF RAT KIDNEY

BY MARGARIDA DE MELLO-AIRES, GERHARD GIEBISCH AND GERHARD MALNIC From the Department of Physiology, Yale University School of Medicine, New Haven, Connecticut, U.S.A., and Department of Physiology, Sao Paulo University School of Medicine, Sao Paulo, Brazil

(Received 29 August 1972) WITH AN APPENDIX BY PETER F. CURRAN

From the Department of Physiology, Yale University School of Medicine, New Haven, Connecticut, U.S.A. SUMMARY

1. The transport of potassium across the distal tubular epithelium was studied in vivo in rats on a normal potassium intake and in rats in which distal tubular potassium secretion was either stimulated by potassium loading or the I.V. administration of a 5% sodium bicarbonate solution or in which potassium secretion was suppressed by dietary deprivation of potassium or sodium. 2. 42K was used to measure unidirectional fluxes across the luminal and peritubular cell membranes and to assess the magnitude of cellular potassium partaking in the transport process. This was accomplished by the simultaneous perfusion of the peritubular capillary network with 42KRinger and of the distal tubular lumen with initially tracer-free solution. From the steady-state flux and the time course of tracer washout into the lumen after discontinuing the peritubular perfusion, unidirectional fluxes, rate coefficients of ion transfer and cellular transport pools could be measured. 3. Transepithelial movement of potassium involves mixing with a variable cellular potassium transport pool. The latter is significantly elevated in conditions of enhanced distal tubular potassium secretion; cellular potassium labelling is reduced in conditions in which potassium secretion has been suppressed by potassium deprivation. 4. Evidence is presented that changes in the peritubular transport pattern are primarily responsible for modifications of potassium translocation. Thus, stimulation of potassium secretion is associated with inDownloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

48 MARGARIDA DE MELLO-AIRES AND OTHERS creased peritubular potassium uptake; a reduced potassium uptake across the peritubular cell membrane accounts for the fall in potassium secretion in potassium-depleted animals. Whereas passive entry of potassium across the peritubular membrane is augmented in potassium-loaded animals, the induction of metabolic alkalosis by the administration of 5 % sodium bicarbonate stimulates active potassium uptake across the peritubular cell membrane. Sodium deprivation stimulates active reabsorptive transfer of potassium from the tubular lumen. INTRODUCTION

Previous experiments have shown that in the rat kidney the distal tubular epithelium is the main site of potassium secretion into the urine (Malnic, Klose & Giebisch, 1964; Giebisch, 1969, 1971). Based on measurements of the net transepithelial movement of potassium ions and of electrochemical potential differences across the luminal and peritubular cell membranes of distal tubule cells, a transport model with the following properties was developed (Malnic et al. 1964; Giebisch, 1969, 1971; Malnic, Klose & Giebisch, 1966b). Distal tubule cells maintain a high potassium concentration by the exchange of sodium ions for potassium ions across a potassium selective peritubular cell membrane. Evidence in support of an active component of peritubular potassium uptake into the cell interior is available (Sullivan, 1968; Whittembury, 1965). Net movement of potassium from cell to lumen is determined by two opposing transfer mechanisms. Potassium ions move passively into the lumen across the partly depolarized luminal cell membrane at a rate determined by the electrochemical potential difference. Opposed to this movement from cell to lumen is an active reabsorptive potassium pump which effects movement from lumen to the cell interior against an electrochemical potential difference. Normally, the secretary flux component exceeds reabsorptive potassium transport. Obvious control sites of tubular potassium secretion could be passive or active transport components residing within either the luminal or peritubular cell membrane. The flux studies to be described indicate that variations in the rate of active potassium uptake into the cell across the peritubular cell membrane are fundamental to modulating net potassium transport in conditions in which variations in potassium or acid-base metabolism lead to significant changes in distal tubular potassium secretion. Part of this work has been published in abstract form (Proc. int. Union Physiol. Sci. 9, 203, 1971).

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DISTAL TUBULAR POTASSIUM TRANSPORT

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METHODS Male albino rats weighing 250-350 g were used. They were anaesthetized by i.P. injection of pentobarbitone (40 mg/kg body wt.). Control rats received a standard laboratory rat diet enriched with 10 % casein and had free access to tap water until the time of the experiment. During the experiment they received an infusion of 3% mannitol in saline at a rate of 0-1 ml./min. Dietary potassium deficiency was induced by maintaining rats for at least 4 weeks on a potassium-free diet consisting of 64-2 % corn starch, 30% vitamin-free casein (Nutritional Biochemicals Corp., Cleveland, Ohio, U.S.A.), 3-5% butter fat and a supplement of vitamins and minerals. They were given distilled water to drink and received, during the experiment, an infusion similar to that of control rats. Urinary potassium excretion was stimulated in another group of rats by the addition of 4 % potassium chloride to the control diet and the substitution of potassium chloride 75 m-equiv/l., for drinking water. Animals were kept on this diet for at least 2 weeks and received, during the experiment, an infusion of 3 % mannitol in 0-15 M potassium chloride at a rate of 0-1 ml./min. A fourth group of rats was made acutely alkalotic by the intravenous administration of a 5% solution of sodium bicarbonate at a rate of 0-1 ml./min. for at least 1 hr before the start of the experiment. A fifth group was sodium depleted by maintenance for at least 3 weeks on a low sodium diet. They received an infusion of 5 % mannitol at a rate of 0-1 ml./min. during the experiment. The general methods of preparation, micropuncture and localization of tubular puncture sites have been described in previous publications (Malnic et al. 1964; Malnic, de Mello-Aires & Giebisch, 1971; Malnic, Klose & Giebisch, 1966a, b). Clearance experiments were carried out in the various groups of animals to assess urinary potassium excretion. For this purpose, the ureter of the left kidney was cannulated for timed collection of urine samples. Arterial blood was sampled from the left carotid artery. For the measurement of inulin clearance, all animals received a priming infusion of 50 mg inulin, followed by the delivery of inulin, 100 mg/hr, in the various sustaining infusions. Inulin was analysed in plasma and urine by a modification of the method of Fuehr, Kaczmarczy & Kruettgen (1955). Sodium and potassium concentrations were measured in plasma and urine samples by standard flame photometry. Blood pH was determined anaerobically in a Metrolhm Model EA 520 capillary pH electrode by means of a Model E 322 compensator. Total plasma CO2 levels were measured by means of a Natelson microgasometer. The movement of 42K across the distal tubular epithelium was measured by observing the rate of appearance of tracer in the lumen of single distal tubules under conditions approaching zero solute and fluid movement. The theoretical considerations relevant to the cell model used for analysis and the derivation of the equations for estimating rate coefficients, unidirectional fluxes across individual tubular cell membranes and transport pools are given in the Appendix. The epithelium was analysed as a three compartment system consisting of the tubular lumen, the tubule cells and the infinite peritubular fluid pool. In the present series of experiments both the lumen and the peritubular capillary network were perfused continuously by methods described (Giebisch, Malnic, Klose & Windhager, 1966; Malnic & Giebisch, 1972; Spitzer & Windhager, 1970). In a first step, the tubular lumen is continuously perfused with an equilibrium (steady state) solution containing nonradioactive potassium and sodium at concentrations which have been shown previously to abolish net solute movement (Malnic et al. 1966b). An appropriate peritubular capillary irrigating the distal tubular loop is continuously perfused by means of a micropipette made of 1-5 mm o.d. Pyrex glass tubing having a tip diameter of about 5 #sm. Pressure, regulated by a reduction valve and a three-way Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

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MARGARIDA DE MELLO-AIRES AND OTHERS

stopcock, is applied to the system from a gas tank of compressed oxygen connected via polyethylene tubing to the perfusion assembly. Sequential collection of the tubular perfusate is performed by means of a concentric glass pipette, shown on the right-hand side of Fig. 1. It was fabricated by first preparing a regular collection pipette into which a constant bore glass capillary of 50 #rm i.d. and 100 ,um o.d. (Drummond Scientific Company, Broomall, Pa.) was fused with the aid of a De Fonbrune microforge. The incorporation of the constant-bore capillary tubing into the collecting pipette reduced its effective volume thus permitting the isolation of aspirated fluid samples by the interposition of oil blocks during the perfusion period.

*;T-l *I~l~o

Continuous perfusion of tubule with cold steady-state solution. Measurement of 42K influx into lumen

-V

/9t/ Peritubular perfusion with 42K Ringer

Second step

/

Stationary perfusion with cold I -1 I FL .1

steady-state solution until equilibrium with respect to 42K is reached

.

,,e/ Peritubular perfusion with 4K Ringer Third step/

/hird se Continuous perfusion with cold

-m\ . are- I I

/

steady-state solution. Measurement of washout of 42K from distal tubule cells into the tubular lumen

Peritubular perfusion with 42K Ringer switched off

Fig. 1. Schematic summary of luminal and peritubular perfusion methods (see Methods for details).

The oil used for the separation was coloured castor oil injected by the doublebarrelled perfusion pipette and aspirated from the tubular lumen. In some instances several single-barrel collection pipettes were used and oil aspirated from either the lumen or the kidney surface which was covered with a thick layer of mineral oil. By carrying out timed collections of tubular perfusate during the perfusion of the peritubular capillaries with an artificial peritubular perfusion solution containing 42K, it was possible to measure the steady-state influx of potassium from

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DISTAL TUBULAR POTASSIUM TRANSPORT

51

capillary to lumen (see Fig. 1, top). Care was taken to begin the series of timed collections not earlier than 2 min after the start of both luminal and peritubular perfusions. As will be discussed later, this time period is of adequate length to assure attainment of steady-state transepithelial flux conditions. A second step of the perfusion experiment consisted in establishing stationary perfusion conditions during continued peritubular perfusion with 42K. The purpose of this phase of the experiment was to establish steady-state conditions between the cellular and extracellular compartments before beginning the third step of the experiments. To assure tracer equilibration, a column of perfusate was separated from the rest of the tubular contents by appropriate oil blocks for at least 2 min. This is schematically shown in the middle panel of Fig. 1. A third phase of the experiment consisted in following the washout of tracer from tubule cells into the lumen. As shown in the lower part of Fig. 1, this was accomplished by abrupt discontinuation of the peritubular perfusion with tracer and the subsequent timed collection of the perfusate from the lumen. As pointed out in the Appendix, the measurement of steady-state influx of 42K from peritubular to luminal compartment, followed by observing the time course of washout of the radioactivity from tubule cells to the lumen provides the information necessary to evaluate the kinetics of transepithelial potassium movement across the distal tubular epithelium. However, to check on the validity of the cell model, an additional type of experiment was carried out in which the time course of tracer appearance in the lumen was followed by timed collections of perfusate after abruptly starting the peritubular perfusion with 42K containing perfusion fluid. As shown in the Appendix, the variation in the rate of 42K entrance into the tubular lumen with time is given by the relationship P1 =

Pilz [1- e(k_,+kS3)

where P1 = dPl/dt, the rate of appearance of radioactivity in compartment 1 (the lumen) at time t, and Pll. is the rate of appearance after reaching the steady-state conditions. k2, and k23 are the rate coefficients of 42K transfer from compartment 2 (the cell) to the lumen (k2l) or the peritubular fluid compartment (k23), respectively. From the mean value of (k2l + k23) of 1-15 min' in control rats (see Table 2) it can be calculated that after 2 min the influx of 42K into the lumen (compartment 1) has reached 90% of the steady-state value. For this reason, the first phase of the experiment, i.e. the measurement of steady-state transepithelial 42K fluxes, was always started at least 2 min after beginning the peritubular perfusion with tracer. The measurement of the time course of tracer appearance in the lumen before the attainment of steady-state conditions also allowed validation of the parameters of the cell model calculated from steady-state transepithelial 42K-fluxes and cellular washout-curves. The samples of collected perfusate were quantitatively transferred, after their volume had been measured in a constant bore tubing, to counting vials filled with 10 ml. Bray solution (1960), and counted in a Beckman Model LS 100 Liquid Scintillation Spectrometer. High specific activity 42K (100-1204uCi/mg at morning of day of delivery) was obtained from the Instituto de Energia Atomica, Sio Paulo. The samples were counted several times and appropriate corrections made for isotopic decay. Transmembrane fluxes and transport pool size are expressed per mm length of distal tubule. This length was measured with an ocular micrometer at the time of the experiment, or by measuring tubular length after latex injection and subsequent microdissection. The latter procedure was chosen if perfusion was carried out over a long distal tubular segment, part of which extended below the surface. In the perfusion experiments in which either steady-state transepithelial, or cell

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MARGARIDA DE MELLO-AIRES AND OTHERS

to lumen fluxes were measured, an attempt was made to keep the luminal concentra tion of 42K low in order to minimize tracer back-flux from lumen to cell. This was accomplished by choosing a relatively fast perfusion rate to assure tracer dilution into a large volume. The rate of perfusion was measured by timed collection of perfusate and a mean value of 208 + 27 nl./min. obtained in thirty-three determinations. This value exceeds the normal flow rate within distal tubules by more than an order of magnitude. The mean ratio of radioactivity of collected perfusate/ peritubular perfusion fluid was 0-037 + 0-0065 (n = 24) indicating that the magnitude of the back-flux component must have been small.

TABLE 1. Composition of luminal perfusion fluid (m-equiv/l.)* C1 Na+ 30 40 Control 33 Low K 30 50 70 High K 70 20 5% NaHCO4 30 Low Na 35 * Isotonicity of solutions was achieved by addition of of raffinose.

HCO-

K+ 10 3 20 20 5

70

the appropriate amount

TABLE 2. Inulin, potassium and sodium ratios of collectedlinjected perfusion solutions

Control Low K High K

Inulin 0-97 ± 0-021 (12) 1-01+ 0-014 (11) 0-98 + 0-019 (21)

K 0-98 0-020 (9) 0-99 0-017 (13) 0-99 ± 0-016 (12)

Na 1-05 + 0-025 (9) 1-01+ 0-015 (12) 1-06 ± 0-027 (6)

Means ± s.E. (no. of observations).

Another condition inherent in the compartmental analysis used is the existence of steady-state conditions and absence of net potassium transport throughout the experiment. Accordingly, solutions chosen for the luminal perfusion had ionic concentrations which had been shown previously to minimize transepithelial net movement of fluid or ions (Malnic et al. 1966 b). Their composition is summarized in Table 1. The solutions were coloured with Lissamine Green (0-05 %). The effectiveness of these solutions in abolishing significant fluid and electrolyte transport was established by comparing inulin, sodium and potassium concentrations in the perfusion fluid with that in the collected perfusate. [3H]inulin (New England Nuclear, Inc.) was added to the perfusion fluid and measured by liquid scintillation spectrometry. Sodium and potassium concentrations were measured by ultramicroflame photometry (Malnic et al. 1964; Malnic et al. 1971). As shown in Table 2, there was no significant net fluid or electrolyte movement during the perfusion experiments. The composition of the peritubular perfusion solutions is given in Table 3. They correspond to the ionic concentrations observed previously in the metabolic conditions described (Malnic et al. 1964; Malnic et al. 1971; Malnic et al. 1966b). All solutions were equilibrated with 5 % CO2 in air.

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DISTAL TUBULAR POTASSIUM TRANSPORT TABLE 3. Composition of peritubular perfusion fluid

Control Low K High K 5 % NaHCO4 Low Na

Na+ 145 145 140 145 125

Cl127-5 125-0 127-5 101-5 107-5

K+ 50 2-5 10.0 40 4-5

53

(m-equivpl.) HCO25 25 25 50 25

Ca2+ 2-5 2-5 2-5 2*5 2-5

RESULTS

Table 4 summarizes data on plasma levels of potassium and on fractional potassium excretion rates in the different experimental conditions. In agreement with previous observations (Malnic et al. 1964), the plasma potassium level is decreased in animals on a low-potassium diet or subjected to acute metabolic alkalosis (Malnic et al. 1971). In the latter group TABLE 4. Summary of potassium and sodium plasma levels and fractional excretion rates in different experimental conditions

Experiment Control High K Low K 5 % NaHCO3 Low Na

Control Low Na

CKICI,

[K]p 4417 + 0-062 7-35 + 0X553 2.69 + 0187 3.85 + 0.146 4.53 + 0227

(21) (13) (10) (13) (9)

0-26 ± 0X016 0-71 ± 0-099 0065± 0020 0*39 0*032 0.10± 0.021

(29) (13) (10) (15) (14)

[Na]p

C.N.CIn

137.7 +050 (35) 1204 +3-36 (9)

0 028 + 0-0043 (29) 0.0022 + 0.0003 (16)

of animals, mean blood pH was elevated to 7-64 + 0-028 (n = 12) and the mean blood bicarbonate was 47-8 + 3 09 m-equiv/l. (n = 12). Fractional excretion rates of potassium were reduced in the low-potassium and low-sodium animals and elevated in animals receiving 5 % sodium bicarbonate i.v. On the other hand, the combination of acute and chronic potassium loading was effective in elevating the plasma potassium concentration in addition to stimulating the urinary excretion rate of potassium. Both free-flow micropuncture studies (Malnic et al. 1964; Giebisch, 1969, 1971; Malnic et al. 1971; Malnic et al. 1966a) and stationary microperfusion experiments (Malnic et al. 1966 b) have demonstrated that the distal tubule is the nephron site where the most important changes in potassium transport occur in response to potassium deprivation (Malnic et at. 1964), potassium loading (Malnic et al. 1964, 1966a) or during induction of metabolic alkalosis (Malnic et at. 1971). In sodium deprivation, Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

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55 DISTAL TUBULAR POTASSIUM TRANSPORT distal potassium secretion is reduced. In addition, avid potassium reabsorption along the collecting ducts contributes to the decreased potassium excretion (Malnic et al. 1966a). A summary of the mean values of the most important kinetic parameters in control and various experimental conditions is given in Table 5. These data were obtained from steady-state transepithelial flux measurements and cellular washout experiments (steps 1-3, Fig. 1). 100

S=2 3 log 90/14 9=1 79 P,, =322 c.p.m./min k23=1 79 x 39.2/90=0.64 min' k2,,=179-0 64=1 15 min' 0b32=90x 0-64/11 5 x 2-72 x 109=1 84x10-8 m-equiv/min

so -HH

(Spec. act.=2-72x109 c.p.m./m-equiv K) Length of perfumed segment=0 21 mm Therefore: 032=8 75 x 10-8 m-equiv/min. mm S2=8-75x10-8/0 64=13 6x 10-8 m-equiv/min SI = 0 314 x 10-8 m-eq uiv/m m (Calc. from perfusion soln) 1x 5=157 x 10-8 1=08 36 x x 25,-I m-equiv/min mm k,2=15 7x10-810 314x10-8=50 0 min'

20 C

E E 10 ci.

5

2

I

1

I

2

3

min

Fig. 2. Results of a perfusion experiment including plot of 42K washout from cells into lumen (see Appendix for details relating to the calculations of kinetic parameters).

A representative example of such an experiment is given in Fig. 2 which depicts the slope of disappearance of 42K from the tubule cells. The quantities necessary to evaluate rate constants and pool size are Y, the slope of tracer washout, A, the intercept, Pico, the steady-state influx of 42K into the lumen, the specific activity p* of the peritubular perfusion fluid and the potassium concentrations in the tubule and peritubular fluid. Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

56 MARGARIDA DE MELLO-AIRES AND OTHERS As described in the Appendix, this information is adequate to determine the several quantities of interest in the system. The following are the most important conclusions: (1) there were in general, no major differences in the rate of 42K washout from cells to lumen since the value of .Y9 was quite similar in the different experimental conditions. (2) The rate of unidirectional potassium flux from peritubular space to cell, qS32 was significantly lowered in potassium deprived, and elevated in potassium-loaded and alkalotic animals. Similarly, 021 (the flux from cell to lumen) is decreased in the potassium-deficient state, whereas elevated values were found under conditions of potassium loading and alkalosis, experimental situations which are characterized by increased potassium secretion across the distal tubular epithelium. On the other hand, in sodium-depleted rats, 021, 532 and the potassium transport pool are all increased despite a reduction in distal tubular potassium secretion. (3) The cellular transport pool of potassium, S2, was significantly reduced TABLE 6. Summary of 42K appearance in the distal tubular lumen during peritubular perfusions with 42K

Experiment Control KF 12-D KF 13-B KF 13-D KF 13-E KF 13-F KBF 6 5 % NaHCO3 KBF 7-C Mean

Slope (min-) 1-38

0-82 1-27 1-44 0-84 1-69 1-84

1-15 + 0- 15 (s.E. of mean)

in distal tubules from potassium-deficient animals and increased in potassium-loaded and alkalotic animals. In a number of experiments, the rate at which 42K influx into the tubular lumen approaches steady-state values after beginning perfusion of the peritubular capillaries with 42K-containing solutions was also evaluated. Relevant data are summarized in Table 6. A representative example of the time course of tracer appearance is given in Fig. 3 where the data have been plotted according to eqn. (11) of the Appendix. The magnitude of the observed slopes is quite similar to that of Y°, determined from the rate of cellular tracer washout in the different experimental conditions (Table 5). This observation provides an independent method of checking the kinetic behaviour of the system and supports the view that the series three compartment system shown in Figs. 1, 4 and 5 is a reasonable representation of the distal tubular potassium transport mechanism.

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DISTAL TUBULAR POTASSIUM TRANSPORT

57

1-0

Ln 5[1-] =-(k2,+k23)t -S=1 27 min'

0-2

0.1

005

002

Expt. KF 13 _

.I

I

_

I

I

2 3 4 min Fig. 3. Time course of 42K flux into the lumen after perfusion of peritubular capillaries with 42K-containing Ringer solution. 1

DISCUSSION

The distal tubule of the rat nephron has been the subject of a large number of micropuncture and microperfusion studies that have firmly established that this nephron segment is the main control site of urinary potassium excretion (Malnic et al. 1964; Giebisch, 1969, 1971; Malnic et al. 1971; Malnic et al. 1966a). Under conditions of normal potassium balance, a large fraction of potassium appearing in the urine can be shown to be derived from distal tubular secretion. During dietary potassium deprivation and urinary potassium conservation, distal tubular potassium secretion is diminished or abolished (Malnic et at. 1964), whereas during exogenous potassium loading (Malnic et al. 1964, 1966a) and during metabolic alkalosis (Malnic et al. 1971) distal tubular potassium secretion and urinary potassium excretion are stimulated. Potassium secretion is diminished along the distal nephron in low-sodium states (Giebisch, 1971; Malnic et al. Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

58 MARGARIDA DE MELLO-AIRES AND OTHERS 1966 a). A recent study of potassium transport across an amphibian nephron has shown that the distal tubule of Amphiuma shares many properties with the corresponding mammalian nephron segment (Wiederholt, Sullivan, Giebisch, Curran & Solomon, 1971). On the basis of free-flow micropuncture and microperfusion studies and measurements of electrochemical potential differences, it was proposed (Giebisch, 1969, 1971; Malnic et at. 1966a, b) that the most important functional properties of the mammalian distal tubule cell included: (1) A high intracellular potassium concentration which is maintained by a peritubular sodium-potassium exchange pump. Little is known at present about the exact nature of the coupling of sodium to potassium. (2) Asymmetrical electrical polarization maintaining a lower potential difference across the luminal than across the peritubular cell membrane. (3) A reabsorptive potassium pump at the luminal cell membrane. Evidence for the presence of such a pump is threefold: (a) presence of transepithelial net potassium reabsorption in some experimental conditions (low potassium-diet) from the electrically negative distal tubular lumen into the cell against a steep concentration gradient; (b) the observation that the transtubular or transmembrane concentration difference of potassium ions is always less than that to be expected from either the transepithelial or luminal transmembrane electrical potential difference (Giebisch, 1969, 1971; Malnic et al. 1966a, b); (c) the finding that cardiac glycosides, known to inhibit cellular potassium uptake (Glynn, 1964), increase the distal luminal potassium concentration (Duarte, Chomety & Giebisch, 1971; Wiederholt et al. 1971). This last observation is consistent with the view that the luminal membrane is a site of action of cardiac glycosides and that the latter blocks an active reabsorptive transport mechanism of potassium (Wiederholt et al. 1971; Duarte et al. 1971). Net movement from peritubular fluid into the lumen is envisaged to occur by the following mechanism: a high potassium concentration in distal tubule cells is maintained by the peritubular potassium pump. Potassium movement from cell to lumen is determined by two opposing mechanisms: a passive secretary leak which allows potassium ions to move down an electrochemical potential gradient across the partially depolarized luminal cell membrane, and an opposing luminal reabsorptive pumping mechanism. The present series of tracer flux experiments provides additional information on the behaviour of distal tubular potassium transport. In particular, the results of the present estimates of the kinetic parameters of potassium transfer allow some insight as to the localization of the mechanism by which net potassium transport is altered at the distal tubular level. Fig. 4 illustrates schematically the main points. Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

59 DISTAL TUBULAR POTASSIUM TRANSPORT An important finding is the observation that the transport pool of distal tubular potassium (S2) decreases in dietary potassium deprivation at a time of greatly diminished or abolished net potassium secretion. In contrast, the transport pool is elevated under those conditions in which potassium secretion is stimulated by chronic and acute potassium loading. The values of the cellular potassium pool calculated in the control experiments can be compared to the amount of cell potassium in the distal tubules. Assuming that potassium distributes evenly in cell water, that intracellular potassium concentration is 150 m-equiv/l. (Burg, Grantham, Abramon & Orloff, 1966) and that potassium is distributed in a hollow cell cylinder of 37,tm external and 24,um internal diameter (Thurau & Deetjen, 1961; and own observations), the expected potassium content Control

k21=-66 mink23=0-59 _15=_0I 12 6 k,2=29.1 _x10-@ 032=676 1v/m ) 2 =9 15 x1 0- m-equiv/min. mm Low K

k2,,=0 4202=2 41

High K

k2l=064

k23=0-82 $ hS, 032=1k63273

k2l=063

k18

5% NaHCO3

k23=0 57

52= 3

k2l=0 75 [21=1876

Fig. 4. Summary of main kinetic parameters of distal tubular potassium transfer in control, potassium-loaded, alkalctic and potassium-deprived animals (data from Table 5).

per mm tubular length can be calculated to be 20-9 x 108 m-equiv. This value is somewhat larger than that measured by 42K in control rats (Table 5). The values indicate that normally a large fraction of cellular potassium is involved in the translocation of potassium from peritubular to luminal fluid. With respect to the participation of cell potassium in potassium transport in cortical collecting tubules of the rabbit it has been shown that the total cell content of potassium participates in transport (Stoner & Orloff, 1972). In contrast, a significant difference between cell potassium content and transport pool has been observed in the distal tubule of the amphibian kidney of Amphiuma (Wiederholt et al. 1971), in the colonic mucosa of rats (Edmonds, 1969) and in the midgut of Cecropia (Harvey & Zerahn, 1969). It should be noted that the volume in which the exchangeable potassium distributes is unknown and this introduces uncertainties with respect to the effective potassium concentration in the tubular cell compartment. In spite of such uncertainties, an attempt was made to approximate intracellular potassium concentrations Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

MARGARIDA DE MELLO-AIRES AND OTHERS 60 in the different experimental conditions. Using the above discussed geometry and assuming that cellular volume remains unchanged, the following 'effective' concentrations of exchangeable potassium are obtained: controls 91 m-equiv/l., low K: 47 m-equiv/l., high K: 159 mequiv/l., low Na: 155 m-equiv/l. and 5 % NaHCO3: 176 m-equiv/l. It should be emphasized that these data have comparative rather than absolute significance due to the uncertainties concerning possible variations in cell volume (see below), cell distribution and cell compartmentalization. Also, the calculated cellular potassium concentrations are highly sensitive to small differences in tubular geometry. A comparison of the magnitude of the distal tubular transport pool in states of low- and high-potassium secretion shows significant changes in cellular potassium labelling. A considerable fall was observed in low potassium animals, indicating that the amount of potassium partaking in the transport process is reduced. We believe that this fall in cell potassium is a major factor in the reduction of net potassium secretion under freeflow conditions. It is apparent from inspection of Table 5 and Fig. 4 that in low-potassium rats the unidirectional potassium flux from cell to lumen (021) is considerably reduced at a time when the rate coefficient k21 is not significantly changed. This supports the view that decreased potassium secretion in low potassium rats is caused by reduced intracellular potassium content rather than reduced potassium permeability of the luminal cell membrane. The view that cellular potassium concentration is reduced after dietary potassium restriction is also supported by the recent observation of Wright (1971) that the peritubular transmembrane potential of distal tubule cells falls significantly after several weeks of potassium deprivation and the effective curtailment of urinary potassium excretion. The peritubular membrane of distal tubule cells in Amphiuma has been shown to have a relatively high potassium permeability and the magnitude of its transmembrane potential is largely determined by the concentration difference of potassium ions between cellular and peritubular fluid compartment (Sullivan, 1968). A similar sensitivity of the peritubular potential difference of distal tubule cells has been observed in the rat in vivo, although the precise quantitative relationship between cell polarization and transmembrane potassium concentration has been less extensively explored (Giebisch et al. 1966; Giebisch, 1971; Wright, 1971: Malnic & Giebisch, 1972). In vitro studies on cells of mammalian kidney slices have also shown that potassium ions carry most of the current across the peritubular cell membrane and are the main ion species contributing to the generation of a diffusion potential (Whittembury, 1965). Accordingly, the observed reduction of the electrical potential difference at this site in Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

61 DISTAL TUBULAR POTASSIUM TRANSPORT low potassium animals is in principal agreement with the conclusion that the effective cellular potassium concentration in tubule cells is diminished. From the observation that the distal tubular potassium transport pool increases after potassium loading and after induction of metabolic alkalosis it appears safe to conclude that some increase in intracellular potassium concentration has occurred. Similar arguments to those advanced in the case of the low-potassium group hold. In both potassium and bicarbonateloaded animals, the unidirectional potassium fluxes from cell to lumen are increased in the absence of consonant changes in the rate coefficient k2l. This finding stresses the fact that the effective potassium concentration in distal tubule cells must have increased. It is also relevant, and in support of an elevated cell potassium concentration, that the cell potential measured across the peritubular membrane of distal tubule cells of potassium-loaded animals is significantly increased (Wright, 1971). Indirect evidence also supports the view that potassium uptake into body cells is stimulated by metabolic alkalosis (Brown & Goot, 1963; Giebisch, Berger & Pitts, 1955; Swan, Axelrod, Seip & Pitts, 1955; Toussaint & Vereerenstraeten, 1962). The present series of experiments provides direct and strong evidence that distal tubule cells share in this process. Similar to the situation in potassium-loaded animals, the transition to a state of enhanced distal tubular potassium secretion appears to be associated with an increase in the cellular potassium concentration since the enhancement of 021 is not accompanied by significant changes in the rate constant k21. Cellular changes in tubular potassium concentrations are thus an important factor in the alterations of potassium secretion occurring both in response to variations in potassium intake and during alkalinization of the body fluids by the infusion of sodium bicarbonate. Although the arguments cited support in general the view that the distal tubular concentration of cell potassium is elevated in potassiumloaded and alkalotic animals, the absolute magnitude of the concentration change is probably less than is to be expected from the increase in the size of the cellular potassium pool S2. Cell swelling is known to occur in conditions of enhanced potassium uptake in a variety of organs including renal tissue (Conway, Fitzgerald & MacDougald, 1946); Whittembury, 1965; Conway, 1957), and it is reasonable to assume that such osmotically induced fluid shifts would not obviate but reduce the concentration increase of potassium in distal tubule cells. In low-sodium rats, on the other hand, an increased cellular potassium pool was found in spite of reduced over-all net potassium secretion. Likewise, increased cell-lumen unidirectional potassium fluxes were found. These findings at first appear to be paradoxical. However, the resulting Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

62 MARCARIDA DE MELLO-AIRES AND OTHERS reduced net flux into the lumen characteristic of this experimental situation appears to be maintained by an increased back-flux from lumen to cell, due to the significantly increased rate coefficient k12. Several previous observations have shown that there exists an active reabsorption of potassium from cell to lumen against an electrochemical potential gradient (Giebisch, 1969, 1971; Malnic et al. 1966a, b). The increase in k12 suggests that in sodium-depleted rats this reabsorptive mechanism is significantly enhanced, and responsible for reduced over-all potassium excretion. The same appears to occur along the collecting duct, since extensive net reabsorption of potassium along this segment was shown to occur under similar experimental conditions. The finding of an increased cellular potassium pool in sodium-depleted rats deserves further comment. McCance (1938) has found an increased potassium concentration in saliva and in sweat in sodium-depleted humans, indicating that the cellular potassium concentration in these glands might be enhanced under these conditions. It is known that aldosterone levels are enhanced in sodium-depleted subjects (Crabbe, Ross & Thorn, 1958), which might lead to an increased sodium-potassium exchange at the peritubular cell membrane, and to lower sodium as well as higher potassium concentrations in epithelial cells. The data of Edmonds (1969) obtained in sodium-depleted rats are likewise of interest. This author found an increased blood-lumen flow of potassium in colonic mucosa, and an increased uptake of radioactive potassium from the luminal side, indicating a greater permeability of the luminal membrane for potassium in sodium-depleted rats. These findings might indicate that an increased potassium content is a general characteristic of epithelial cells in sodiumdepleted animals; in the distal tubule, however, this situation which would otherwise lead to increased potassium secretion, is compensated for by a markedly enhanced reabsorptive mechanism. The present series of experiments also allows some insight into the mechanism underlying the changes in the cellular transport pool of potassium in the different experimental situations. In principle, these changes could be the consequence of either or both of the following events. The potassium permeability of the peritubular membrane could change and allow passive entry of potassium ions to be modulated by the electrical potential difference and the transmembrane concentration difference. Alternatively, alterations in the active flux component from peritubular fluid into the cells could be responsible for the observed changes in intracellular pool size of potassium. The changes in the cellular pool size are, in general, paralleled by commensurate alterations in the fluxes of potassium from the peritubular fluid into the cells (see Table 5). Thus, compared to control observations, Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

DISTAL TUBULAR POTASSIUM TRANSPORT 63 032 values are elevated in high-potassium and alkalotic animals and depressed in potassium-deprived animals. Since the egress of potassium ions across the peritubular cell boundary is due to diffusion, the rate constant k32 provides an estimate of its potassium permeability. Since the electrical potential difference increases across the peritubular membrane of high-potassium animals (mean p.d.: - 93-8 + 3-6 mV, compared to a control value of -70+ 3-2 mV (Wright, F., personal communication), the passive efflux should decrease. We have observed no significant change in k23 between control and high-potassium animals, indicating some increase in peritubular potassium permeability. This could contribute to the increased influx of potassium across the peritubular cell wall. A consideration of the relevant data in low-potassium animals indicates that the fall in q32 is associated with no significant alteration in the rate constant k2,. Since the peritubular potential difference is lowered in potassium-depleted rats (mean p.d.: - 55-6 + 4-2 mV, compared to a control value of - 70-1 + 3-2 mV (Wright, F., personal communication), the passive efflux of potassium should be increased if the permeability of the peritubular cell membrane had remained unchanged. From the fact that k23 is not increased, some fall in the potassium permeability of the peritubular cell membrane may be assumed. This could contribute to the observed fall in 5O32 in the low-potassium state. Another factor to be considered is the change in the plasma potassium level. The latter is sharply increased in potassium-loaded animals and reduced in both low-potassium and alkalotic conditions (see Table 3). These concentration changes also contribute to the enhancement of 032 in high-potassium, and the reduction of 032 in low-potassium rats. Since the absolute magnitude of the peritubular potassium pool cannot be established, direct evaluation of k32 is not possible (k32 = 032/S3). However, the following relationship can be considered: 032

=32

]3,

where the unidrectional potassium flux is proportional to the potassium concentration [K]3 in the peritubular compartment. The latter is considered to be of infinite magnitude and thus having a constant potassium concentration. The proportionality constant, k32, has the characteristics of a permeability coefficient, its units being k' cm3 cm.10-8 32- m-equiv.10-8 min. cm2 m-equiv min A summary of the relative changes of the fluxes, 032 and of the permeability coefficients k32 is given in Table 7. It is apparent that the changes in concentration in the peritubular fluid play an important role in altering the 3

PH Y 232

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64 MARGARIDA DE MELLO-AIRES AND OTHERS unidirectional fluxes, 032' in potassium-depleted and potassium-loaded animals since k32 changes considerably less than the corresponding potassium fluxes. During metabolic alkalosis (infusion of sodium 5 % bicarbonate) the increase in peritubular potassium influx, 032 seems to be due primarily to the stimulation of active uptake of potassium. It was pointed out that the rate constant k23 is an estimate of the potassium permeability provided that there are no changes in the electrical potential difference across the peritubular membrane. We have found that the latter is the case, since the mean values of the transmembrane potential difference across the peritubular cell membrane in 5 % bicarbonate-loaded animals was 72-7 + 1*29 mV, a potential difference not different from that observed in our control group, 71f1 + 1*38 mV (Malnic & Giebisch, 1972). The fact that the potassium permeability is not changed suggests that the observed TABLE 7. Permeability of peritubular cell membrane to potassium

632

Control Low K High K 5% NaHCO3 Low Na

632032 6-76 2-41 16.7 13-4 12-7

[K]3

k'

50

1-35 0-96 1-67 3.35 2.83

2.5 10 0 4-0 4.5

~~~~~~~~32

exp contr 1.00 0-36 2-47 1.98 1-87

32

exp contr

1.00 0.71

1*23 2*48 2.09

032, K flow in m-equiv/min. mm x 10-8; [K]3, K concentration in compartment 3 in m-equiv/l. k'2, permeability coefficient of peritubular cell membrane, cm. 10-8.

increase in the intracellular potassium transport pool is caused by enhanced active cellular potassium uptake across the peritubular cell membrane of distal tubule cells. The cellular mechanism underlying the stimulation of peritubular active potassium uptake in alkalosis is unknown but the following possibility should be considered. If part of peritubular potassium uptake is coupled to active sodium extrusion, it is possible that intracellular sodium and hydrogen ions compete for carrier sites. Evidence obtained in muscle (Keynes, 1965) demonstrating pH sensitivity of sodium extrusion has been interpreted as some sort of competition between hydrogen and sodium ions for carrier-mediated transport. Provided that such peritubular sodium-potassium exchange is less efficient with a larger fraction of sites occupied by hydrogen instead of sodium ions, cellular uptake of potassium would be expected to decrease with cell acidosis and increase with alkalosis. Clearly, more experiments are necessary to clarify this point. Similarly to the previous experimental situation, in low sodium rats Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

DISTAL TUBULAR POTASSIUM TRANSPORT 65 it appears that most of the increase in O32 is due to changes in the rate coefficient k32, indicating that this increase may be due to either changes in permeability or of active uptake of potassium at the peritubular membrane. It is of interest that the changes in the transport properties of the distal tubular epithelium, i.e. stimulation of potassium secretion in potassiumloaded and alkalotic animals and suppression of potassium secretion in low-potassium animals, are not associated with major changes of the kinetic parameters characterizing the luminal cell membrane. Also, no significant alterations in the electrical potential difference across the luminal cell membrane have been observed in high-potassium, lowpotassium (Wright, F., unpublished observation) or alkalotic animals (Malnic & Giebisch, 1972). This makes it unlikely that changes in the electrical driving force are of importance in the modification of potassium transport at this cell site. In conclusion the present experiments indicate that the distal tubular epithelium of the rat responds to stimuli known to modify the rate of potassium secretion by significant changes in cellular potassium labelling. An increase in this transport pool is observed with stimulation of potassium secretion (potassium-loading, metabolic alkalosis) whereas a decline is associated with suppression of potassium secretion (potassium deprivation). Under these conditions, changes in potassium translocation across the luminal cell membrane are thought to be secondary to corresponding changes in cell potassium concentration, and evidence is provided to indicate that alterations of the rate at which potassium ions are transferred across the peritubular membrane are of primary importance in modulating distal tubular potassium secretion. On the other hand, luminal potassium reabsorption was significantly stimulated in sodium-deprived animals and appears responsible for reduced net secretion of potassium ions. Work done by the authors was supported by grants from the National Institutes of Health, the American Heart Association and the Fund de Amparo a Pesquisa do Est. S. Paulo and Conselho de Pesquisas (Brazil). APPENDIX

By PETER F. CURRAN Analysis of tracer behaviour The data presented above can be used to obtain rate constants and pool sizes describing potassium transport by the tubular cells. As shown in Fig. 5, we assume that the system can be described in terms of three compartments, tubular lumen (1), cytoplasm (2), and peritubular space (3) 3-2

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66 MARGARIDA DE MELLO-AIRES AND OTHERS and that cellular potassium is in a single exchangeable compartment. With tracer initially present in compartment 3 only, the rate of change in amount of tracer in the other two compartments is given by

d-P P2 = dPi

=

=

k21P2-k12P1,

(1)

=

-(k2l + 23)P2 +k32P3+ k12PI

(2)

in which P1 is total tracer in compartment i and klj is the rate coefficient for movement of tracer from compartment i to compartment j; kxj = 0i1/Si where Oij is unidirectional flux from compartment i to compartment j and Si is the pool of potassium in compartment i.

Capillary

oltmt3 k23

Tubular cell

3

k32

Compartment 2

S2

k2,

k12

Lumen

Compartment I

S.

Fig. 5. Schematic illustration of three-compartment system made up of the tubular lumen (compartment 1), the cell compartment (compartment 2) and the peritubular compartment (compartment 3). S1 and S2 represent the amount of solute in compartment 1 and 2. The peritubular compartment is considered infinite. k12, k23, k24 and k23 are rate constants. It is assumed that the system is in the steady state and that net transport of potassium is zero.

In the initial experiment (first step, Fig. 1), the steady-state rate of tracer appearance in compartment 1 is determined when compartment 3 is perfused with tracer at constant specific activity. Under these conditions, we assume that the term k12P, can be neglected in eqns. (1) and (2) Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

DISTAL TUBULAR POTASSIUM TRANSPORT 67 (because P1 is relatively quite small as described in the Methods section). Under steady-state conditions (second step, Fig. 2), P3= constant, dP2/dt = 0, and the rate of tracer appearance in compartment 1, P1j,, is given by (3) i= 1211k32P3 = k21 0y32P3 -

co

21

k21+ k23

in which q32 is flux from (3) to (2) (/,t-equiv/min) and p4 is specific activity (c.p.m./u-equiv) in compartment 3. The next step (third step, Fig. 1) in the experiment is to stop perfusion of compartment 1, leaving fluid in the lumen and allow sufficient time for the system to come to tracer equilibrium. This procedure is then followed by perfusion of compartments 1 and 3 with unlabeled solution and determination of washout of tracer into compartment 1. To describe this procedure, we must first evaluate the final amount of tracer in compartment 2 at tracer equilibrium. (Note that this quantity then becomes the initial condition for the washout study.) At tracer equilibrium dP,/dt = 0 = dP2/dt and the amount of tracer in compartment 2, P2-0 is given by P

32

= 20

P3 _32P3

(4)

kw km During the washout phase of the study, P1 = P3 O 0 SO that eqn. (2) becomes P2 = -(k2l + k23)P2 which, upon integration with the initial condition P2 = P2, at t = 0, yields P2

=

P2. e(k2l+k23)t.

Under these conditions, eqn. (1) becomes

P1= k21P2

or

P1 = k21P2 e-(k2l+k23)t.

Thus,

In Pi =

(k2l +k23)t +ln k21 P2,0 (5) and from a plot of In P1 vs. time we can determine (k2l + k1c) and In k2l P2- .

This information suffices to determine the several quantities of interest for the system. We denote the slope of the line In P1 vs. time as A, Y= k2l+k23 (6) and the quantity k21P211. as A. From eqn. (4),

A =

k21P20

=

1`21032PT/123-

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(7)

MARGARIDA DE MELLO-AIRES AND OTHERS 68 From eqns. (3), (6) and (7), we find that

P1co

or

k23

=

=

k23 A/I°,

P100 YIA.

(8)

Since all quantities on the right hand side of eqn. (8) are measured experimentally, k23 can be calculated. Then from eqn. (6), (9) k2l = eY-k232 and from eqn. (7), 032 =

k23A

(10)

Since the experiments were carried out under conditions of no net potassium flux (see Table 2), P32 = 023 = k23S2 from which S2 can be calculated since 032 and k23 are known. If S2 is known, 521 can be calculated since 021 = k2lS2 and finally, the condition of no net flux also requires

012 = k12S1 from which k12 can be calculated since S, is known. There is a second method for evaluating the quantity 5° (eqn. (6)). This approach involves determination of the rate at which isotope influx into compartment 1 approaches a steady-state value after initiating perfusion of compartment 3. The behaviour of the system is described by eqns. (1) and (2) assuming that the term k1c2P. is negligible. Since k32P3 is constant, integration of eqn. (2) yields 521 =

P2 - P'w [1 -e-(k21+k23)t in which P' is the steady-state amount of tracer in compartment 2. (Note that PI00 is not identical with P2co of eqn. (4)). Since the rate of tracer appearance in compartment 1, P1, is given under these conditions by k21P2, P1

=

k21P2', [1-ee(k2a+k23)t]

=

P1,, [1 -e-(k2+k23)t].

Thus

[nI

P,

-(k2

+

(11)

and a plot of the left-hand side of this expression against time should be a straight line with a slope of k2l + k23 ( =Y). The data in Table 6 show that this approach yields a mean value of 1*15 + 0-15 for (k21 + k23). This value Downloaded from J Physiol (jp.physoc.org) by guest on July 11, 2011

69 DISTAL TUBULAR POTASSIUM TRANSPORT does not differ from those obtained from the usual method (eqns. (5) and (6)) suggesting that the three compartment system provides a reasonable description of tubular potassium transport.

REFERENCES

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MALNIC, G., KLOSE, R. M. & GIEBISCH, G. (1964). Micropuncture study of renal potassium excretion in the rat. Am. J. Physiol. 206, 674-686. MALNIC, G., KLOSE, R. M. & GIEBIScH, G. (1966a). Micropuncture study of distal tubular potassium and sodium transport in rat nephron. Am. J. Physiol. 211, 529-547. MALNIC, G., KLOSE, R. M. & GIEBISCH, G. (1966b). Microperfusion study of distal tubular potassium and sodium transfer in rat kidney. Am. J. Physiol. 211, 548559. SPITZER, A. & WINDHAGER, E. E. (1970). Effect of peritubular oncotic pressure changes in proximal tubular f uic reabsorption. Am. J. Physiol. 218, 1188-1193. STONER, L. & ORLOFF, J. (1972). K transport pool in cortical collecting tubules. Fedn Proc. 31, 343, Abst. SULLIVAN, W. J. (1968). Electrical potential differences across distal tubules of Amphiuma. Am. J. Physiol. 214, 1096-1103. SwAN, R. C., AXELROD, D. R., SEIP, M. & PITTS, R. F. (1955). Distribution of sodium bicarbonate infused into nephrectomized dogs. J. cdin. Invest. 34, 1795-1801. THimRAU, K. & DEETJEN, P. (1961). Kinematographische Untersuchungen am Warmblueternephron. Nachr. Akad. Wiss. Goettingen 2, 27-37. ToussAINT, C. & VEREERENSTRAETEN, P. (1962). Effect of blood pH changes on potassium excretion in the dog. Am. J. Physiol. 202, 768-772. USSING, H. H. & WINDHAGER, E. E. (1964). Nature of shunt path and active transport path through frog skin epithelium. Acta physiol. scand. 61, 484-504. WHITTEMBURY, G. (1965). Sodium extrusion and potassium uptake in guinea pig kidney cortex slices. J. gen. Physiol. 48, 699-717. WIEDERHOLT, M., SULLIVAN, W. J., GIEBISCH, G., CURRAN, P. F. & SOLOMON, A. K. (1971). Potassium and sodium transport across single distal tubules of Amphiuma. J. gen. Physiol. 57, 495-525. WRIGHT, F. S. (1971). Alterations in electrical potential and ionic conductance of renal distal tubule cells in potassium adaptation. Proc. int. Union Physiol. Sci. 9, 609.

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