Ouabain-Insensitive Salt and
Water Movements in Duck Red Cells
I. Kinetics of Cation Transport
under Hypertonic Conditions
WILLIAM
F. S C H M I D T
I I I and T H O M A S
J. McMANUS
From the Department of Physiology and Pharmacology, Duke University Medical Center,
Durham, North Carolina 27710. Dr. Schmidt's present address is The Children's Hospital of
Philadelphia, Philadelphia, Pennsylvania 19104.
A B S T R A C T Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake
of salt and water is self limiting and demands a specific ionic composition of the
external solution. Although ouabain (10 -4 M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the
reaction, or the total a m o u n t of salt or water taken up. To study the response
without the complications of active Na-K transport, ouabain was added to most
incubations. All water accumulated by the cells can be accounted for by net salt
uptake. Specific external cation requirements for reswelling include: sufficient
sodium (>23 mM), and elevated potassium (>7 mM). I n the absence of external
potassium cells lose potassium without gaining sodium and continue to shrink
instead of reswelling. Adding r u b i d i u m to the potassium-free solution promotes an
even greater loss of cell potassium, yet causes swelling due to a net uptake of
sodium and r u b i d i u m followed by chloride. T h e diuretic furosemide (10 -a M)
inhibits net sodium uptake which depends on potassium (or rubidium), as well as
net potassium (or rubidium) uptake which depends on sodium. As a result, cell
volume is stabilized in the presence of this drug by inhibition of shrinkage, at low,
and of swelling at high external potassium. T h e response has a high apparent
energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling,
or c/s-interaction, between sodium and potassium so that the uphill movement of
one is driven by the downhill movement of the other in the same direction.
INTRODUCTION
T w o e x p e r i m e n t a l c o n d i t i o n s c a u s e a v i a n r e d cells t r a n s i e n t l y to a c c u m u l a t e salt 1
a n d water. T h e first involves i n c u b a t i o n i n a h y p e r t o n i c m e d i u m , as o r i g i n a l l y
r e p o r t e d for p i g e o n r e d cells by O r s k o v (1954). A f t e r a n initial r a p i d o s m o t i c
s h r i n k a g e , t h e cells reswell t o w a r d t h e i r o r i g i n a l v o l u m e e v e n t h o u g h t h e y
1 When the term "salt" is used in this series of papers with respect to uptake or loss by the cells, it
refers to net movements of NaCI and/or KCI across the membrane.
T H E J O U R N A L OF GENERAL P H Y S I O L O G Y " V O L U M E 7 0 ,
1977 •
pages
59-79
59
60
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
" VOLUME
70
"
1977
remain in the hypertonic solution. Alternatively, addition of catecholamine to
pigeon cells suspended in an appropriate isotonic solution will also initiate net
uptake of salt and water, as first reported by Orskov in 1956. These effects have
also been studied in red cells from the Muscovy duck (Cairina raoschata) by
Riddick and by Kregenow. Although propranolol effectively prevents the swelling response to catecholamine (Riddick et al., 1971), this /3-adrenergic blocker
has no effect on reswelling in hypertonic solutions (Kregenow, 1971).
The two systems demonstrate important similarities. In both, the volume
responses are self limiting and demand a specific ionic composition of the
extracellular solution. Sodium is required and external potassium must also be
elevated above the 2.5 mM normally found in duck plasma. Ouabain shifts the
net cation uptake from mainly potassium to mainly sodium. Nevertheless, the
sum of the uptakes of these two cations remains constant in both the hypertonic
(Kregenow, 1971) and isotonic plus catecholamine (Kregenow, 1973) conditions.
Water uptake is also unaffected by ouabain in both conditions, suggesting that
control of the responses under these circumstances does not directly involve the
Na-K exchange pump.
Such similarities in the swelling response of Muscovy duck red cells to hypertonicity and catecholamines led Kregenow (1973) to postulate a common transport pathway responsible for net salt uptake in both instances. A rigorous test of
this hypothesis is not straightforward since the experimental conditions are
really quite different. For example, external ion manipulation, or addition of
proposed transport inhibitors, might not affect the pathway of net salt movement, but rather the interaction of catecholamine with its membrane receptor.
For this reason, we decided to investigate first salt and water uptakes induced by
hypertonicity with red cells from the Pekin duck (Anas platyrhynchos). Insights
gained from these studies were then used to interpret the more complex system
of cells incubated in isotonic solutions containing catecholamine. These results
are presented in the next paper (Schmidt and McManus, 1977a).
It is clear from Kregenow that the volume response induced by hypertonicity
is unaffected by ouabain poisoning of the pump. Therefore, that agent was
added to the incubation medium in most of the experiments reported in these
papers to permit elucidation of the kinetics of ion movements via this ouabainindependent pathway. Ouabain-independent cation movements in mammalian
cells have often been thought to be "leaks" in the sense of simple electrodiffusion
through conducting channels in the membrane, but much evidence points to a
more complex situation (Blum and Hoffman, 1971; Beaug6 and Adragna, 1971;
Sachs, 1971; Wiley and Cooper, 1974; Beaug6, 1975).
The results of this investigation suggest that changes in the simple diffusion of
sodium and potassium cannot account for increased salt and water movements
in the duck red cell. We propose a model in which a cotransport pathway
mediates movement of sodium plus potassium into or out of the cell. This
pathway is stimulated by hypertonicity or by catecholamines and is inhibited by
several drugs but not by ouabain.
The third paper in this series (Schmidt and McManus, 1977b) demonstrates
the effect on the two responses of varying external chloride, changing the pH,
and manipulating the membrane potential. From these results a general concept
SCHMIDT AND McMANus Salt and WaterMovementsin Duck Red Cstls. I
61
is d e v e l o p e d w h i c h p r e d i c t s t h a t t h e cells swell o r s h r i n k in r e s p o n s e t o D o n n a n
f o r c e s o p e r a t i n g to m o v e salt i n t o o r o u t o f t h e cells via t h e c o t r a n s p o r t p a t h w a y .
A p r e l i m i n a r y r e p o r t o f s o m e o f t h e s e r e s u l t s was p r e s e n t e d to t h e B i o p h y s i c a l
S o c i e t y in J u n e , 1974 ( S c h m i d t a n d M c M a n u s , 1974).
MATERIALS
AND
METHODS
Preparation of Cells
Adult white Pekin ducks were used in this investigation. Blood was drawn into heparinized syringes after cardiac puncture. A sample was set aside for ion and water determinations. T h e cells were washed four times in 5 vol o f ice-cold 170 mM NaCI with removal o f
the buffy coat. With complete exsanguination, 30-40 ml o f packed red cells could be
obtained from each duck.
Washed packed cells were preincubated at 41°C (normal body t e m p e r a t u r e for birds
(Irving and Krog, 1954; McNab, 1966)) for at least 90 rain in a standard buffer isotonic
with duck plasma (323 mosmol). This p r o c e d u r e was employed because Riddick et al.
(1971) r e p o r t e d that a period o f 90 rain is required for duck r e d cells to attain a steady
state with respect to ions and water in artificial incubation solutions devoid o f catecholamines. T h e preincubation solution contained (raM): glucose, 10; phosphate, 1.0; K, 2.5;
Mg, 1.0, and was buffered to a final p H o f 7.4 at 41°C with Na-TES (see below). Sufficient
NaCI was a d d e d to adjust the osmolality to 323 mosmol (determined by freezing-point
depression).
Preparation of Reagents
Stock buffer solutions o f sodium and potassium TES (N-Tris(hydroxy methyl)methyl-2aminoethane sulfonic acid) (Sigma Chemical Co., St. Louis, Mo.) were p r e p a r e d by
titrating 200 mM TES to the desired p H with either 1.0 N N a O H or 1.0 N K O H . At p H
7.42 (41°C) final concentrations of Na or K were approximately 56 mM. Mg-TES was
p r e p a r e d by dissolving Mg (OH)2 crystals in 200 mM TES to give an alkaline p H , then
back-titrating to p H 7.4 (41°C) witli additional 200 mM TES. Buffer concentration in the
final incubation solutions was 10-30 raM.
Choline chloride (Eastman Kodak Co., Rochester, N.Y.) was routinely recrystallized
from ligroine according to G a r r a h a n and Glynn (1967). T h e dry salt was stored in a sealed
container at -20°C to prevent water absorption.
Furosemide (Hoechst-Roussel Pharmaceuticals, Inc., Somerville, N .J .) was dissolved in
water and stock Na-TES (or K-TES). After adjustment of the p H to 7.4 (410C) with N a O H
or K O H , water was a d d e d to yield a final concentration o f 50 mM furosemide and 20 mM
TES buffer. Furosemide was generally used at a final concentration of 1.0 mM and the
a p p r o p r i a t e stock was selected d e p e n d i n g o n whether sodium or potassium was omitted
from the incubation. Ethacrynic acid (Merck, Sharp & Dohme, Montreal, Que., Canada),
trifiocin (Lederle Laboratories, Pearl River, N.Y.) a n d phloretin (Nutritional Biochemicals Corp., Cleveland, Ohio) were p r e p a r e d in the same m a n n e r as furosemide. Triflocin
was a generous gift from Dr. Michael Dunn (University o f Vermont School o f Medicine,
Burlington, Vt.). Ouabain, 10 mM in water, was a d d e d to incubation solutions to give a
final concentration o f 0.1 raM.
Incubation Solutions
Specific concentrations of [Na]o, [K]o, [Rb]o, and [Clio are given in the legend to each
figure. In addition, incubation solutions always contained (raM): glucose, 10; inorganic
phosphate, 2.0-5.0; and magnesium, 1.0. Osmolality was adjusted by suitable addition o f
choline chloride.
62
THE JOURNAL OF GENERALPHYSIOLOGY" VOLUME 70 • 1977
EXPERIMENTAL PROCEDURES
General Experimental Protocol
A f t e r p r e i n c u b a t i o n , cells were washed once with ice-cold MgCl2 ($23 m o s m o l ) , t h e n
routinely test i n c u b a t e d at a 2% h e m a t o c r i t in isotonic ( - 3 2 3 mosmol) or h y p e r t o n i c
( - 4 0 0 mosmol) solutions. Constituents o f these solutions for individual e x p e r i m e n t s are
given in the l e g e n d o f each figure and table. Substitution o f choline c h l o r i d e (323
mosmol) for MgCI2 as wash solution did not affect the results.
U p o n contact with the h y p e r t o n i c solution, the ceils instantly shrink. This water loss is
associated with a loss o f chloride and a gain o f potassium as shown in T a b l e I, which
c o m p a r e s initial values o f cell water a n d ion contents in the isotonic and h y p e r t o n i c test
TABLE
I
C E L L W A T E R A N D I O N C O N T E N T S IN I S O T O N I C A N D H Y P E R T O N I C
S O L U T I O N S A F T E R T H E 90-min P R E I N C U B A T I O N
W~
kg/kg
Nae
Isotonic (~323 mosmol)
Mean
---SEM
Range
n
1.50
0.01
1.38-1.60
(59)
8.9
0.3
5.3-15.7
(88)
Hypertonic (~400 mosmol)
Mean
-+SEM
Range
n
1.26
0.01
1.21-1.32
(92)
9.9
0.2
5.5-15.9
(92)
P
0.0t
Kc
mraol/kgcellsolid
CIc
rcl
231
2.0
190-265
(88)
153
1.0
137-178
(77)
0.66
0.004
0.58-0.72
(77)
242
1.0
202-264
(92)
143
2.0
101-158
(80)
0.60
0.01
0.51-0.66
(80)
0.001
0.001
0.001
Preincubated cells were washed as described in the text, then added to fresh isotonic or hypertonic
incubation solutions. Samples were taken immediately. [Na]c, [K]e, and [C1], (millimoles/liter cell
water) can be obtained by dividing the values of ion contents (miUimoles/kiiogram cell solids) by the
cell water content, We. Millimoles/kilogram cell solids can be converted to millimoles/kilogram cells
in the isotonic solution by multiplying by 0.40. P is the probability that the differences of the means
found in the two test solutions (isotonic versus hypertonic) can be accounted for by chance, n is the
number of observations.
solutions. N o t e that a l t h o u g h the large sample size permits g o o d statistics, t h e r e is
considerable variation b e t w e e n ducks, as shown by the wide r a n g e o f values. For e x a m ple, in Fig. 1, initial Ke in the h y p e r t o n i c solution is ~210 m m o l / k g cell solids or 15% lower
than m e a n K, o f the h y p e r t o n i c series p r e s e n t e d in T a b l e I.
A f t e r initial equilibration in the test solution, ion and water m o v e m e n t s were followed.
At t i m e d intervals, 25 ml of whole suspension were p o u r e d into chilled, precalibrated
polycarbonate tubes and the cells p r o m p t l y separated in a Sorvall RC-2B r e f r i g e r a t e d
c e n t r i f u g e ( D u P o n t I n s t r u m e n t s , Sorvall O p e r a t i o n s , N e w t o w n , C o n n . ) at 4°C. In this
m a n n e r , eight samples could be collected, cooled, and c e n t r i f u g e d in less t h a n 3 min.
A f t e r c e n t r i f u g a t i o n , excess s u p e r n a t e was aspirated to a p r e d e t e r m i n e d level so that the
packed cell v o l u m e o f the resuspension was a p p r o x i m a t e l y 35%. T h a t resuspension was
quickly d r a w n u p into l o n g - s t e m m e d Pasteur pipettes and t r a n s f e r r e d to p r e c o o l e d nylon
tubes which were c e n t r i f u g e d at 10,000 r p m for 10 rain. T h e s e specially p r e p a r e d tubes
SCHMIDTAND McMANVS Salt and Water Movements in Duck Red Cells. I
63
were first described by F u n d e r and Wieth (1967). T h e y can contain up to 0.7 ml.
Precooling was accomplished by inserting them into holes in an aluminum block set in a
large icebath. For centrifugation, the tubes were s u p p o r t e d by specially machined nylon
holders which fit into the SS-34 rotor o f the RC-2B Sorvall centrifuge.
Occasionally, it was necessary to incubate cells at 30% hematocrit (e.g., 2*Na influx
determinations), These suspensions were introduced directly from the incubation flask
into precooled nylon tubes and centrifuged. This procedure provided for rapid cooling
of the cell suspension to slow the high rate of ion movement observed in these experiments.
Separation o f Packed Cells
For each timed sample at least two nylon tubes were filled with cell suspension. After
centrifugation, the packed cell mass was separated from its supernate by slicing the tube
with a razor blade several millimeters below the top of the red cell column. This method
of cell separation avoids multiple washings which might result in a loss of i m p o r t a n t
solutes from the cells. Supernates were saved for analysis. Cells from duplicate tubes were
then p r e p a r e d for analysis o f (a) cell water and (b) ion contents.
CELL WATER After the tube bottom had been sliced off, the packed cell mass was
expressed with a close-fitting plastic r o d onto an a l u m i n u m foil tare that had been d r i e d
at 150°C and preweighed. After initial weighing, the wet packed cell mass was dried to
constant weight for 15 h at 99°C and reweighed. When a m i n i m u m o f 100 mg of wet
packed cells was treated in this m a n n e r , duplicate cell water fractions always agreed
within 0.3% and usually within 0.15%.
ION CONTENTS Wet packed cells were carefully e x t r u d e d from the duplicate nylon
tube into the bottom o f a tared, 12-ml polycarbonate centrifuge tube and weighed. 2 g o f
0.56 M perchloric acid (PCA) were a d d e d by means o f a reagent dispenser (Glencoe
Scientific, Inc., Houston, Tex.). T h o r o u g h mixing by h a n d with a glass stirring rod
insured complete extraction and precipitation of cell proteins. After centrifugation at
10,000 r p m for 10 min, the clear supernate was saved for analysis of cations and chloride.
Sodium I n f l u x
Carrier-free 2~NaC1 (International Chemical & Nuclear Corp., Irvine, Calif.) was evaporated to dryness, then reconstituted with 5 mM Mg-TES, p H 7.4, to give 0.2 mCi/ml. For
sodium influx determinations, 5 ~,1 (1.0 ~Ci) of buffered tracer were a d d e d to a 3 ml
suspension o f r e d cells (hematocrit 35%).
When influx studies were p e r f o r m e d in hypertonic solutions, cells were a d d e d directly
to solutions at 41°C already containing 22Na. An initial sample was taken immediately
after mixing, and additional samples removed at 15-min intervals. An aliquot o f cell
suspension was removed from the incubation flask and immediately t r a n s f e r r e d into
precooled nylon tubes. Two tubes were filled for each timed sample: one for cell water
determination, the other for radioactivity and ion measurements. After centrifugation,
cells and supernate were separated by slicing tubes in the usual m a n n e r . Supernates from
both tubes were saved and diluted to a final volume o f 2.2 ml for counting. T h e packed
cell mass from one tube was weighed, extracted with 2 g PCA, centrifuged, and saved for
counting. 2~Na was d e t e r m i n e d in the Packard Auto-gamma System (Packard I n s t r u m e n t
Co., Inc., Downers Grove, Ill.). T h e entire PCA extract, including precipitated proteins,
was counted. 25 #i o f the extracellular fluid were a d d e d to 2.2 ml PCA to make the
volume counted comparable to that o f the PCA-extracted cells. At least 10,000 counts
were collected for each sample. Sodium influx was calculated according to Tosteson and
Robertson (1956).
64
THE JOURNAL
OF GENERAL
PHYSIOLOGY
" VOLUME
70 • 1977
Trapping Corrections
Fluid trapped between the packed cells after centrifugation in the nylon tube was
estimated with lstI-albumin. Osmolality of test solutions was varied from 200 to 435
mosmol by addition of NaCI, producing cell volumes more than adequate to reproduce
the range observed when duck cells accumulate water in response to hypertonicity.
T r a p p e d fluid was found to vary between 1.8% in the most hypotonic and 2.17% in the
most hypertonic solution. Therefore, a trapping correction of 2.0% was routinely included in all final calculations.
Osmolality, pH, and Ion Measurements
Osmotic concentration of incubation solutions was determined by freezing point depression with an Osmette Precision Osmometer (Precision Systems, Framingham, Mass.).
TABLE II
D E F I N I T I O N OF SYMBOLS
Bc,
we,
[B1~,
[Blo,
rcl,
pHc,
pHo,
tMBnet,
tMB,
°Mn,
Es,
millimoles B per kilogram cell solid where B represents Na, K, Rb, or CI.
kilograms cell water per kilogram cell solids.
millimoles B per liter cell water (raM); [B]e = BdWc.
millimoles B per liter medium (raM).
chloride distribution ratio = [Gl]d[Cl]o.
cell pH.
incubation solution pH.
net influx of Be (millimoles B per kilogram cell solid per time interval).
unidirectional influx of Be,
unidirectional efflux of Be, calculated from net and unidirectional influxes (°Mn -tMB - IMsnet)
T l n [~lo
Nernst potential of B; EB = a~-~ (miUivolts).Where R, T, z, and F have their
usual meanings.
Commercial standards were employed whenever u n k n o w n s were analyzed, pH was
measured with a Radiometer pH meter (type PHM-22r, Radiometer Co., Copenhagen,
Denmark) equipped with a scale e x p a n d e r (type PHA 630T) and a thermostated microelectrode unit (type E5021). When necessary, the volume of packed cells in the centrifuged
nylon tubes was measured with a very accurate (+--1%)optical cathetometer designed and
developed by T. J. McManus. Chloride was analyzed electrometrically in duplicate
samples on a Cotlove Chloridometer (Buchler Instruments, Inc., Fort Lee, N.J.). A
Perkin-Elmer model 303 Atomic Absorption Spectrophotometer (Perkin-Elmer Corp.,
Norwalk, Conn.) was used for cation determinations. Duplicate dilutions were assayed
and the results averaged. Duplicate determinations never varied by too.re than 2%.
Recovery was complete for serially diluted samples over the range of 5-125 /~M for
sodium and potassium, and 25-150/~M for rubidium.
Unless stated otherwise, all data shown are representative of several other experiments
of similar design. A definition of symbols used throughout this series of papers appears in
Table II.
RESULTS
Effect of Ouabain
T a b l e I I I illustrates t h e effect o f o u a b a i n o n cell w a t e r a n d i o n c o n t e n t s d u r i n g a
3-h i n c u b a t i o n i n isotonic s o l u t i o n s . I n the a b s e n c e o f t h e d r u g , t h e s e p r e i n c u -
SCHMIDT AND MCMANUS Salt and Water Movements in Duck Red Cells. I
65
bated cells usually continue to lose 2% of their water over the next 3 h. This is in
contrast to the apparent steady state achieved by Muscovy duck red cells as
reported by Riddick et al. (1971). Kregenow (1973), however, has observed a
similar 2% shrinkage of Muscovy duck cells under these circumstances. With
ouabain present, they exchange potassium for sodium at a rapid rate without
affecting this gradual loss of water. This ouabain-induced cation exchange rate
( - 2 0 mmol/kg cell solids × h) is somewhat greater than that reported for
Muscovy duck red cells (Kregenow, 1971). Net movements o f both cations are
down their electrochemical gradients as would be expected from inhibition of an
Na-K coupled pump.
Fig. 1 illustrates water and ion shifts occurring in the presence and absence
of ouabain in solutions made hypertonic by addition of choline chloride. Initial
samples were taken immediately after washed packed cells were introduced into
TABLE
III
EFFECT OF OUABAIN (10 -4 M) ON DUCK RED CELLS IN ISOTONIC
SOLUTIONS
Ouabain
Hours
We
Nae
Ke
Cle
mmo~ ceUso~Is
~/kg
0
0
0
0
0
1
2
3
1.46
1.45
1.43
1.43
7.4
8.7
8.7
8.7
234
229
229
227
151
143
144
144
+
+
+
+
0
1
2
3
1.46
1.45
1.44
1.43
10.2
28.4
58.1
75.2
230
210
195
171
144
144
146
151
C o m p o s i t i o n o f the i n c u b a t i o n soluti on (raM): [Na]o = 160; [K]o = 15; [Cl]o = 160; N a -TES = 10. pHo
= 7.4; 41°C; 323 m o s m o l .
400 mosmol solutions. T h e cells had already shrunk to their hypertonic volume.
Concentrations of both [K]o and [Na]o are similar to those used in the isotonic
solutions of Table III, yet it is apparent that salt and water adjustments over 3 h
under hypertonic conditions differ markedly from the isotonic case. First, cell
volume and total salt content are not constant, but progressively increase.
Second, ouabain is without significant effect on this volume increase, although
the glycoside does completely reverse the pattern of cation accumulation. In the
absence of ouabain, cells take up predominantly Ke, CI~, and water with little
change in Na~. With ouabain, they take up the same amount of water by
accumulating Nae and CI~, with little change in Ke. T h e large Ke loss shown in
Table III is no longer evident with ouabain in hypertonic solution. This reversal
of net potassium loss b y hypertonicity is considered in greater detail below.
Kregenow (1971) showed that net water accumulation, in the absence of
ouabain (Fig. 1) can be prevented by lowering [K]o to 2.5 mM, or by replacing
[Na]o with choline. T o investigate these ion movements in detail without the
complicating effects of active transport, we varied both [K]o and [Na]o in the
presence o f ouabain.
THE JOURNAL O F
66
GENERAL
PHYSIOLOGY
" VOLUME
70 • 1977
Effect of External Potassium
Fig. 2 illustrates changes in water a n d cation contents o f cells in h y p e r t o n i c
solutions containing o u a b a i n , high [Na]o, a n d various [K]o. In the absence o f
[K]o, the cells rapidly shrink. This water loss can be attributed to the large net
efflux o f K~. In this case, t h e r e is little c h a n g e in [Na]~ d u r i n g the incubation.
I n t e r p o l a t i o n o f the 15-min w a t e r data shows that a p p r o x i m a t e l y 7 m M [K]o is
1.40
-- CELL
,CONTROL
WATER/~
" 1.35
O
(/3
.J
-J
/
260250-
"+OUABAIN
L3o
240
~¢:
o 1.25
~u) 230
JJ 220
e
','
°
I
I
1801-- CELL
/ CHLORIDE
~70t--
I
O
d ~6o
~
#
",~.CONTROL
(
1.20
Q
C
POE L T A L s s ~ , D
I
(.9
o,
I
1
I
I
I
60[--- CELL
SODIUM
50~
4o~
I
I
I
I
I
2
3
I
2
3
HOURS
HOURS
FIGURE l. Effect of ouabain (10.4 M) on the hypertonic response. All incubation
solutions contain (mM): [Na]o = 155; [K]o = 15; [Cl]o = 196; Na-TES = 10. pHo =
7.4, 41°C, 440 mosmol. At zero time, cells contain (millimoles/liter cell water): [Na]c
-- 7.2; [Klc= 175; [Cl]~ -- 110.
necessary to maintain a stable cell v o l u m e u n d e r these conditions. As [K]o is
elevated, less K~ is lost. Significant net K~ u p t a k e is seen at 15 min w h e n [K]o is 22
mM. This a c c u m u l a t i o n occurs even t h o u g h the potassium electrochemical
g r a d i e n t would be e x p e c t e d to p r o m o t e a loss o f Ke. S o d i u m a c c u m u l a t i o n is also
m a r k e d l y d e p e n d e n t on [K]o. Na~ r e m a i n s stable at zero [K]o, but increases as
[K]o increases. It is a p p a r e n t f r o m these data that the role o f [K]o is c o m p l e x ,
since sodium as well as potassium net m o v e m e n t s are altered by varying [K]o in
the presence o f ouabain.
Gain or loss o f cell water with d i f f e r e n t [K]o can be a c c o u n t e d for by the net
salt gained or lost by the cells. Fig. 3 depicts cell water content plotted as a
function o f the s u m o f the m a j o r inorganic ions in the cells. T h e s e data are
derived f r o m the e x p e r i m e n t shown in Fig. 2. A correlation coefficient o f 0.983
s u p p o r t s the i n t e r p r e t a t i o n that, once cells have e x p e r i e n c e d initial s h r i n k a g e in
67
SCHMIDT AND McMANUS S ~ t and Water Movements in Duck Red Cells. I
- (A) CELL
:
WATER
O
m.l~les
(~
22
1.3C
(/}
1.25
~
21C L201
2.8
~20C'~B) CELL
6
2.8 33oi--
uo~9c -
ON I.15
,~ 1.10
D~
Q
O
_1
~
"1
7TASS'UM
"~t..O
nL
m Moles I
v ] ( C ) CELL
(K)o
401-- SODIUMj........- ..'''4 22 I
~
_.111------"
"
I
1.05
-0
1
15
I
MINUTES
MINUTES
60
FIGURE 9. Effect o f [K]o o n cell w a t e r , p o t a s s i u m , a n d s o d i u m in h y p e r t o n i c
s o l u t i o n s . All i n c u b a t i o n s o l u t i o n s c o n t a i n ( m M ) : o u a b a i n = 0.1; [Na]o = 155; [C1]o
= 200; N a - T E S = 10. pHo = 7.42, 41°C, 440 m o s m o l . C h o l i n e was u s e d as a
r e p l a c e m e n t c a t i o n f o r [K]o. A t z e r o t i m e , cells c o n t a i n ( m i l l i m o l e s / l i t e r cell water):
[Na]e = 10.4 + 0.4 (SEM); [K]e = 196 + 1; [Cl]e = 126 + 1.
1,300
•
0
~-I 1,250
•
•
•
I
I
1,200
"~
1,150
•
•
I,I00
1,050
I
280
I
I
I
I
I
I
320
360
400
440
MILLIMOLES (Nac + Kc + CIc )
KILOGRAM CELL SOLID
I
FIGURE 3. R e l a t i o n s h i p b e t w e e n cell w a t e r a n d t h e s u m o f Nae + Ke + Cle.
Results w e r e o b t a i n e d at 0, 15, a n d 60 m i n for cells i n c u b a t e d in h y p e r t o n i c
s o l u t i o n s c o n t a i n i n g d i f f e r e n t [K]o. All d a t a s h o w n in Fig. 2 w e r e u s e d , with t h e
a d d i t i o n o f values f o r cell c h l o r i d e . T h e a r r o w i n d i c a t e s m e a n values o f cell w a t e r
a n d t h e m a j o r ions at z e r o t i m e in Fig. 2. T h e r e g r e s s i o n e q u a t i o n d e s c r i b i n g all
d a t a is i n d i c a t e d at t h e l o w e r r i g h t . T h e r e g r e s s i o n c o e f f i c i e n t is 0.983.
68
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E
70
• 1977
hypertonic solutions, any subsequent volume changes can be explained by net
movements of salt.
T he arrow in Fig. 3 indicates the mean values of both variables at zero time:
1,203 ± 6 (SEM) g cell water/kg cell solids, and 400 ± 3 (SEM) mmol [Nac+ Ke +
Cl~]/kg cell solids. A separate analysis of the shrinking phase to the left of the
arrow ([K]o ~ 6 mM), and the swelling phase to the right of the arrow ([K]o ~ 11
mM), yields regressions which do not differ significantly. This justifies the use of
a single equation to describe the curve under both of these conditions.
If one assumes that all water taken up or lost by cells is available to ions, it is
possible to estimate the apparent osmolality of the fluid taken up from the
reciprocal of the slope in Fig. 3 and the osmolality of the incubation solutions.
This calculation reveals that each gram of water entering or leaving the cell
contains 0.57 mmol [Na + K + C1]. This is equivalent to 534 mosmol. T h e
measured osmolality of the medium is 440 mosmol, which is equivalent to 0.48
mmol [Na + K + Cl]/g medium. Thus, the fluid taken up by the cells appears to
be slightly hypertonic to the medium.
Effect of External Sodium
[Na]o is also important in regulating ouabain-insensitive net salt and water
movement in cells incubated in hypertonic solutions. Fig. 4 depicts water and
cation changes in cells incubated with 23 mM [K]o and various [Na]o.
Interpolation of the water data indicates that the cells will neither swell nor
shrink at approximately 27 mM [Na]o. Lowering [Na]o results in diminished rate
of increase of Na~. Note that 22 mM [Na]o allows steady-state Nae levels and that
cells actually lose sodium at 12 mM [Na]o. This occurs even though the electrochemical gradient still favors net sodium gain. Reduction of [Na]o also accelerates loss of Ke. These results agree qualitatively with those describing effects of
[K]o (Fig. 2). Both [Na]o and [K]o promote Na~ gain and reduce K¢ loss. Such
reciprocal effects of [K]o on net sodium movements and o f [Na]o on net potassium movements are difficult to explain by assuming independent diffusion
pathways for these two ions through the cell membrane.
Several alternative hypotheses were considered. Because the system appears
more sensitive to [K]o, these are presented in terms of altered [K]o. Equally valid
predictions could be made by considering [Na]o as the agent variable.
In principle, [K]o could stimulate net sodium gain either by increasing sodium
influx or decreasing efflux. Similarly, [K]o could decrease net potassium loss
either by increasing potassium influx or decreasing efflux. These statements
offer four possible explanations for the observed effect of [K]o on both sodium
and potassium net movements. All require interaction of one ion with transport
of the other.
Sodium Fluxes
T he effect of [K]o on the sodium fluxes is illustrated in Fig. 5. Increasing [K]o
causes marked stimulation of influx which approaches saturation at 20 mM [K]o.
Efflux is not significantly changed. This implies that the considerable net
sodium uptake promoted by [K]o (Fig. 2) is primarily a direct result of increased
influx rather than of decreased efflux.
SCHMIDT AND McM^Nus Salt and Water Movements in Duck Red Cells. I
CELL
CELL
WATE R
mMolel ]
POTASSIUM
m Moles
(No) o
1.40
(N°)o I
2201
164
i
69
210~
1.35
|
~1, 22
:2oo-
64
I
/
lal
~,, t.30
601- CEL'
SODIUM
50-
,.25
=<
~
0
.~
30
22
12
mMoles
164
,
2
1.15
E
7
22
I0,I~
I
I
I
2
4,
II
HOURS
12
I
2
HOURS
FIGURE 4. Effect o f [Na]o on cell water, potassium, and sodium. All incubation
solutions contain (mM): o u a b a i n = 0.1; [K]o = 23; [CI] o = 190; M g - T E S = 20. pHo =
7.45, 41°C, 400 m o s m o l . C h o l i n e was used as a r e p l a c e m e n t cation for [Na]o. At zero
time, cells contain (millimoles/liter cell water): [Na]c = 11.4 -+ 0.7 (SEM); [K]c = 174
-+ 2; [Clio = 103 + 1.
8-
~7-
.2.
d4
0MNo
(
o
E
o
o
I
5
I
I
i0
15
(K)o mM
I
20
FIGURE 5. Effect o f [K]o on u n i d i r e c t i o n a l s o d i u m fluxes. All incubation solutions
contain (mM): ouabain = 0.1; [Na]o = 100; [CI]o = 195; N a - T E S = I0. pHo = 7.39,
41°C, 400 m o s m o l . C h o l i n e c h l o r i d e was used as a r e p l a c e m e n t cation for [K]o. IMNa
r e p r e s e n t s s o d i u m influx in the first 15 min o f incubation in h y p e r t o n i c solutions.
°MNa was calculated as indicated in T a b l e II.
7O
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E 7 0 "
1977
T h e r e are two m e c h a n i s m s by which [K]o m i g h t increase sodium influx: (a) by
an allosteric m e c h a n i s m without [K]o itself e n t e r i n g the cell; (b) by [K]o serving as
an obligate p a s s e n g e r on a carrier molecule. A c c o r d i n g to the first m e c h a n i s m ,
[K]o could r e d u c e net potassium loss only by r e d u c i n g unidirectional efflux,
either by lowering potassium permeability or by r e d u c i n g the g r a d i e n t driving
the ions across the m e m b r a n e . T h e fact that cells actually gain K¢ in the p r e s e n c e
o f ouabain at 22 mM [K]o (Fig. 2) makes this hypothesis unlikely. T h e second
m e c h a n i s m predicts that [K]o stimulates both potassium a n d sodium influx. It
f u r t h e r implies that the system should have c a r r i e r - t r a n s p o r t p r o p e r t i e s , such as
analog substitution, saturability, c o u n t e r t r a n s p o r t effects, a n d d r u g sensitivity.
R u b i d i u m is k n o w n to substitute for [K]o in m a m m a l i a n red cell ion t r a n s p o r t
systems (Solomon, 1952; Beaug$ a n d Ortiz, 1970, 1971). It t h e r e f o r e m i g h t be
e x p e c t e d to b e h a v e like [K]o in its effect on sodium a n d chloride a d j u s t m e n t s in
duck r e d cells.
TABLE
IV
COMPARISON OF NET CATION MOVEMENTS IN ISOTONIC AND
HYPERTONIC SOLUTIONS C O N T A I N I N G 21 mM [Rb]o
Min
We
Nac
kg[kg
Isotonic (323 mosmol)
Hypertonic (400 mosmol)
Ke
Rbe
Kc +
Rbc
mmol[kg cell solids
0
30
60
0
30
1.49
1.46
1.45
1.27
1.30
8.5
12.1
17.9
9.4
21.6
250
232
217
253
218
0.9
9.6
15.3
1.0
39.7
251
242
232
254
258
60
1.31
28.5
199
58.9
258
Both incubation solutions contain (mM): ouabain = 0.1; [Na]o = 92; [Rb]~ = 21; Na-TES = 10. pHo =
7.45, 41°C. Conditions for isotonic incubation (raM): [CI]o = 152; [Clio = 103; [Na]e = 5.7; [K]~ = 170.
Conditions for hypertonic incubation: lC1]o = 191; [Cl]e = 127; [Na]~ --- 7.3; [K]c = 200.
Effect of External Rubidium
T a b l e IV gives a c o m p a r i s o n o f water a n d cation changes in cells i n c u b a t e d in
isotonic a n d h y p e r t o n i c media, respectively, in the p r e s e n c e o f 21 m M [Rb]o a n d
ouabain. Fig. 6 shows an e x p e r i m e n t c o m p a r i n g the effect o f [K]o or [Rb]o in
h y p e r t o n i c solutions. Several points are o f interest. First, [Rb]o is as effective as
[K]o in p r o m o t i n g cell swelling a n d s o d i u m u p t a k e (Fig. 6). Second, if [Rb]o can
i n d e e d substitute for [K]o, t h e n these results o f f e r an e x p l a n a t i o n o f the relative
constancy o f I~ in h y p e r t o n i c incubation with o u a b a i n c o m p a r e d with its decrease u n d e r similar isotonic conditions (Table I I I ) . Potassium loss shown in
T a b l e IV is similar in both isotonic a n d hypertonic conditions. H o w e v e r , rubidi u m u p t a k e is increased almost f o u r f o l d in the h y p e r t o n i c case. T h u s , the s u m o f
K~ a n d Rbc a p p e a r s relatively constant. T h e actual concentration o f r u b i d i u m in
these cells, [Rb]c, has risen to 45 mmol/liter cell water a f t e r 1 h, which is 13 m M
g r e a t e r t h a n the equilibrium p r e d i c t e d f r o m the chloride distribution ratio (rct -0.66). T h e r e f o r e , it a p p e a r s that the m e c h a n i s m o f decreased net p o t a s s i u m loss
in hypertonic solutions involves an increased u p t a k e o f [K]o (or, in this case,
[Rb]o) against its electrochemical g r a d i e n t , r a t h e r than a decrease in efflux.
SCHM1DT AND M c M A N u S
71
Salt and Water Movements in Duck Red Cells. I
W h e n [Rb]o is substituted for [K]o, an identical v o l u m e r e s p o n s e a n d net
sodium u p t a k e occurs (Fig. 6). Cell chloride (not shown) also increased by the
same a m o u n t (22 m m o l / k g cell solids after 60 rain) with [Rb]o as with [K]o. Cell
potassium results indicate that [Rb]o p r o m o t e s a m a r k e d stimulation o f Kc loss,
even g r e a t e r t h a n that usually o b s e r v e d in the absence o f either [K]o or [Rb]o.
After 1 h, the r u b i d i u m content o f these cells had increased to 64 m m o l / k g cell
solids. Again, r u b i d i u m a c c u m u l a t i o n in the cells was g r e a t e r t h a n e x p e c t e d
f r o m equilibrium considerations ( c o m p a r e T a b l e IV).
I~(A)~LLF~
g
-~
F
2o.~
, . 2 s E /
,20
co
_("~,,,C~,~, 2~..-----'--'°-------~(,~,.20..
~
~ 22ol- \
.
E
200-
~ .
(K)o -0
J
40
30
60
MINUTES
\
(C) CELL
50
60
MINUTES
FIGURE 6. Effects of substituting [Rb]o for [K]o. All incubation solutions contain
(mM): ouabain = 0.1; [Na]o = 110; [Cl]o -- 191. pH o = 7.45, 41°C, 400 mosmol. Cell
chloride content increased from 135 to 157 mmol/kg cell solids in 1 h with [Rb]o and
[K]o. In the rubidium incubation, Rb e increased to 64 mmol/kg cell solids at the end
of 1 h. Although it is not obvious from the scale of the plot of cell sodium, cells
incubated without [K]o or [Rb]o actually lose Na c (from 9.5 to 5.5 mmol/kg cell
solids) over the 60-min incubation.
Effect of Inhibitors
Various drugs, most o f which are diuretics, have b e e n shown to inhibit ouabaininsensitive cation m o v e m e n t s in h u m a n r e d cells ( H o f f m a n a n d K r e g e n o w , 1966;
D u n n , 1970, 1972, 1973; Sachs, 1971; Wiley a n d C o o p e r , 1974). Since the net salt
a n d water m o v e m e n t s p r o d u c e d by h y p e r t o n i c solutions in the duck r e d cell are
not inhibited by o u a b a i n (Fig. 1), it was o f interest to see if the overall h y p e r t o n i c
r e s p o n s e was also sensitive to these agents. T h e effect o f these c o m p o u n d s is
illustrated in Fig. 7. Triflocin ( D u n n , 1972) inhibits Na~ a n d water gain, but is
less effective t h a n f u r o s e m i d e . Ethacrynic acid, on the o t h e r h a n d , causes
m a r k e d loss o f K~ a n d water. T h i s m a y be related to the increased potassium leak
p r o d u c e d in h u m a n red cells by this a g e n t (Dunn, 1970). Like f u r o s e m i d e ,
phloretin inhibits a c o m p o n e n t o f net s o d i u m influx. H o w e v e r , this a g e n t also
increases net p o t a s s i u m efflux. T h e result is a loss o f water f r o m the cells.
Similar increases o f net potassium loss in h u m a n r e d cells e x p o s e d to p h l o r e t i n
72
THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 70 " 1977
or phloridzin (the glycoside of phloretin) have b e e n previously noted, but only in
cells p r e t r e a t e d with valinomycin (Wieth et al., 1973; K a p l a n a n d Passow, 1974).
C o m p o u n d s (10 -a M) which failed to affect salt a n d water a c c u m u l a t i o n in
hypertonic solutions include: amiloride, tetrodotoxin, d i p y r i d a m o l e , acetazolamide, E D T A , a n d E G T A . Addition o f 10 m M calcium or omission o f glucose
was also without effect.
CELL
_ ~
2~ol
. ~ T R O L
CELL
~ O N T p0TASSIUM
ROL
1.34
r
o 1.30
~
2,
o---- ~ F
2101
i
1.26'
w
d
U
JgO I
I
~ 1.22
170
O
...i
~1.18
o
,2
%
T'
m 1.14
60-
rr
(.9
O 1.10
v
1.06
1.02
\
CELL
SODIUM
•
20
I
2
HOURS
1,
I
3
I
I
2
HOURS
I
3
FIGURE 7. Inhibitory effects of several diuretics and phloretin on the hypertonic
response. Triflocin (T), furosemide (F), phloretin (P), and ethacrynic acid (E) are
all present in final concentrations of 10-s M. All incubation solutions contain (raM):
ouabain = 0.1; [Na]0 = 110; [K]o = 19; [Cl]o = 193; Na-TES = 10. pHo = 7.45, 41°C,
400 mosmol.
Since f u r o s e m i d e caused neither gain n o r loss of water, it a p p e a r s to r e t u r n
the system to a situation c o m p a r a b l e to that f o u n d in isotonic solutions containing ouabain (Table I I I ) . It was t h e r e f o r e selected for f u r t h e r study. This choice
is admittedly s o m e w h a t arbitrary, as will be discussed below.
In the e x p e r i m e n t shown in Fig. 8, two conditions were investigated: cell
swelling with 15 m M [K]o, a n d cell s h r i n k a g e with 2.2 m M [K]o. With [K]o
elevated, f u r o s e m i d e blocks the usual net u p t a k e o f salt a n d water. Net Na¢ gain
is r e d u c e d , while loss o f K¢ is increased by the d r u g . T h u s , cell water content
remains stable because net s o d i u m u p t a k e balances net potassium loss.
With low [K] o, cells c o n t i n u e d to lose salt a n d water into h y p e r t o n i c solutions
(Fig. 2). U n d e r these conditions, f u r o s e m i d e p r o d u c e d an u n e x p e c t e d result. It
decreased potassium loss a n d increased s o d i u m gain. T h e s e alterations are
SCHMII)T AND M c M A N u s
73
Salt and Water Movements m Duck Red Cells. I
opposite to those caused by the diuretic in high [K]o solutions. Clearly, f u r o s e m ide also blocks a pathway responsible f o r net cation loss in low [K]o h y p e r t o n i c
media. In the p r e s e n c e o f this d r u g , net s o d i u m a n d potassium fluxes are
a p p r o x i m a t e l y balanced in b o t h high [K]o a n d low [K]o solutions a n d t h e r e f o r e
little c h a n g e occurs in cell v o l u m e .
In Fig. 9, changes in cell water (W~) a n d Nae at 1 h with a n d without
f u r o s e m i d e are plotted as a function o f [K]o. F u r o s e m i d e a p p e a r s to stabilize cell
v o l u m e by p r e v e n t i n g s h r i n k a g e at [K]o < 5 m M a n d p r e v e n t i n g swelling at [K] o
> 5 m M . Similarly, in the p r e s e n c e o f this d r u g net s o d i u m u p t a k e b e c o m e s
3
L(A) CELL
~[
WATER
-
5m.
.
o
i
l.202
\
---I
•
1.3o~
21o I-
o~
\
J
FUROSEMIDE
I
"~
115
(K)0-2.2 mM "~o
I
I
I
2
HOURS
I
5
I
2
HOURS
3
FIGURE 8. Effects of furosemide (10-3 M) on cell water, potassium, and sodium in
high and low [K]o hypertonic solutions. All incubation solutions contain (mM):
"ouabain = 0.1; [Na]o = 160; [CI]o = 200; Na-TES = 10. pH 0 = 7.40, 41°C, 400
mosmol. Choline was used as a replacement cation for [K]o. At zero time, cells
contain (millimoles/liter cell water): [ N a ] c = 6.7 -+ 0.3 ( S E M ) ; [K]c = 184 - 1; [C1]~ =
117 -+ 1.
i n d e p e n d e n t o f [K]o. T h u s , furosemide-insensitive net sodium influx r e m a i n s
a p p r o x i m a t e l y 10 m m o l / k g cell solids x h, regardless o f [K]o. Curves identical to
those o f Fig. 9 were o b t a i n e d w h e n [Rb]o substituted for [K]o.
T a b l e V illustrates the effect o f f u r o s e m i d e on unidirectional fluxes o f rubidium a n d sodium. Again, the e x t e r n a l solutions contain ouabain as well as high
a n d low [Na]o a n d [Rb]o. I n c r e a s i n g [Na]o f r o m 2 m M to 91 m M stimulates b o t h
sodium a n d r u b i d i u m influx (7.9 a n d 9.2 m m o l / k g cell solid x 15 min, respecti'vely). In the p r e s e n c e o f f u r o s e m i d e , r u b i d i u m - d e p e n d e n t s o d i u m influx is
practically abolished, but a s o d i u m - d e p e n d e n t s o d i u m influx persists. F u r o s e m ide also inhibits s o d i u m . d e p e n d e n t r u b i d i u m influx as well as a c o m p o n e n t that
is i n d e p e n d e n t o f [Na]o. In the p r e s e n c e o f f u r o s e m i d e , r u b i d i u m influx is little
affected by either [Na]o or [Rb]o.
A d o s e - r e s p o n s e c u r v e o f the inhibition by f u r o s e m i d e o f cell swelling in
o u a b a i n - c o n t a i n i n g solutions is p r e s e n t e d in Fig. 10. Inhibition o f net u p t a k e o f
74
T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME
|
NET CHANGE IN
+0.08~-- CELL WATER
70
• 1977
•
•
!
I/
-°'°o| if'+"
I
I
5
I0
I
.+ I
I
20
25
15
(K) o mM
NET CHANGE IN
CELL SODIUM
-I-
30
~
20
"
iO L___..~ ~
•
z
++
m
II"
oo
o
~
,,+,,,,M+,°
I
I
I
os
I
5
I0
15
(K) o mM
20
I
25
FIGURE 9. Effects o f [K]o a n d f u r o s e m i d e (10 -s M) o n l - h n e t c h a n g e s in cell w a t e r
a n d s o d i u m . All i n c u b a t i o n solutions c o n t a i n 0.1 m M o u a b a i n . Osmolality was
a d j u s t e d to ~ 4 0 0 m o s m o l with c h o l i n e c h l o r i d e . Data w e r e calculated by s u b t r a c t i n g
values o b s e r v e d at 1 h f r o m initial values at zero time f o r e a c h g r o u p . Initial m e a n
(---SEM) cell w a t e r a n d s o d i u m c o n t e n t s : We = 1.25 - 0.01 kg H 2 0 / k g cell solids,
Nae = 11.4 _ 0.5 m m o l / k g cell solids. Data w e r e o b t a i n e d f r o m f o u r s e p a r a t e
experiments.
TABLE
V
EFFECT OF F U R O S E M I D E O N C A T I O N FLUXES IN H Y P E R T O N I C
SOLUTIONS CONTAINING OUABAIN
|Na]o
[Rb]o
Furosemide
IMa~
91
91
2
91
91
2
20.0
2.5
20.5
20.0
2.5
20.0
0
0
0
+
+
+
14.7
8.1
5.5
1.2
0.9
1.4
ARb
,XNa
6.6
9.2
0.3
-0.2
IMs.
9.2
2.9
1.3
3.0
2.6
0.2
ARb
ANa
6.3
7.9
0.4
2.8
Incubations solutions contain (mM): ouabain = 0.1; [Clio = 191; Mg-TES = 10. pH0 = 7.41, 41°C, 400
mosmoi. IM,b represents net uptake of rubidium in the first 15 min. IMab was linear with respect to
time over this initial period. 'MN, was estimated with ==Na as described in Materials and Methods.
ARb represents the effect on tM~ or IMNa of increasing [Rb]o from 2.5 mM to 20 mM. Similarly, ANa
shows the effect of raising [Na]o from 2 mM to 91 raM.
75
Salt and Water Movements in Duck Red Cells. I
SCHMIDT A N D M c M A N u S
sodium and rubidium is also shown. Both cell water and Rbc uptakes were
completely inhibited by 10-3 M furosemidc, with 5 0 % inhibitionoccurring at 5-6
× I0-~ M. Net sodium uptake is not completely blocked by 10-8 M furosemide
(Fig. 9). However, m a x i m u m inhibitionof Nac uptake is achieved above 5 × 10-4
M. If the assumption is m a d e that the furosemide-sensitivc component of net
sodium uptake is fullyinhibited at 10-3 M, then the half-maximum value of this
component is also 5 × I0-s M.
Effect of Temperature
Cells were incubated at various t e m p e r a t u r e s f r o m 41°C to 0°C in h y p e r t o n i c
solutions with ouabain, 140 mM [Na]o, and 16 mM [Rb]o. C o m p l e t e inhibition o f
n,"
22,
0"1"
8o
uJ
z
0
,~ 60
/
0rv
z
0
40
L2
LL
0
20
I 5 o : 5X 10-4M
•
I
I
I
i0-6
10-5
10-4
'~_
.t
1
I
1(5-3
10-6
i0-~
FUROSEMIDE
10-4
10-3
(M)
FIGURE 10. Concentration dependence of furosemide inhibition in hypertonic
solutions. Results are expressed as percent of increase in cell water, Na~, and Rb¢
observed at 1 h in the absence of furosemide. All incubation solutions contain
(raM): ouabain = 0.1; [Na]o = 140; [Rb]o = 15; [Clio = 196; Na-TES = 10. pHo =
7.44, 41°C, 400 mosmol. In the absence of furosemide, cell water increased 59 g/kg
cell solid in 1 h; Na~ and Rbc increased 23.2 and 40.6 mmol/kg cell solids, respectively, in 1 h.
net water a n d ion m o v e m e n t s o c c u r r e d at 0°C. I n the latter incubation, s o d i u m
a n d r u b i d i u m influxes were, respectively: 0.8 and 0.6 m m o l / k g cell solids x 15
min. At 41°C, influxes o f 13.5 and 12.0 m m o l / k g cell solids x 15 min for s o d i u m
and potassium were observed. A p p a r e n t activation energies calculated f r o m an
A r r h e n i u s plot o f the data were obtained for net sodium and r u b i d i u m movements, as well as unidirectional sodium influx. T h e y all fell in the r a n g e o f 16-20
kcal/mol.
DISCUSSION
T h e substantial salt a n d water uptakes which occur in d u c k red cells incubated in
h y p e r t o n i c solutions have been cited by K r e g e n o w (1971) as evidence for a
unique volume-controlling m e c h a n i s m not included in the steady-state " p u m p leak" t h e o r y (Tosteson and H o f f m a n , 1960). His observation that ouabain does
76
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E 7 0 " 1 9 7 7
not alter the rate or extent o f water uptake was c o n f i r m e d in this study (Fig. 1).
This suggests that the mechanism mediating net salt uptake is i n d e e d i n d e p e n d ent o f the ouabain-sensitive Na-K p u m p . Water m o v e m e n t s in the presence o f
ouabain are linearly related to net movements Of salt (Fig. 3), although the
calculated osmolality o f the solution crossing the m e m b r a n e is hypertonic compared to the bathing solution (534 vs. 440 mosmol). Such an uptake o f hypertonic
fluid was also f o u n d by Kregenow (1971) in the absence o f ouabain. It could
reflect a change in hemoglobin charge characteristics in the s h r u n k e n cells
(Gary-Bobo and Solomon, 1968). Whatever the explanation, it can at least be
concluded that sufficient salt (NaCI plus KCI) is taken u p by the cells in these
experiments to account for observed volume changes. O t h e r solutes, such as
amino acids, need not be considered as making a significant contribution to
water m o v e m e n t .
It appears f r o m the data p r e s e n t e d h e r e that net salt uptake by cells in
hypertonic solutions containing ouabain results f r o m neither a decrease in the
simple diffusion o r "leak" o f potassium from the cells, n o r an increased simple
diffusion o f sodium in the inward direction. T h e unique properties o f this
transport system may be s u m m a r i z e d as follows:
(a) a d e p e n d e n c e o f sodium uptake on [K]o and o f potassium uptake on
[Na]o(Figs. 2, 4, 5, and 9; Table V);
(b) [Rb]o behaves in a m a n n e r analogous to [K]o in terms o f net water, sodium,
and chloride movements (Fig. 6);
(c) A net potassium uptake when its electrochemical gradient favors a net loss
(Fig. 2), and a net sodium loss when its electrochemical gradient favors a net gain
(Figs. 4, 6, and 9);
(d) saturation characteristics o f ouabain-insensitive, unidirectional sodium
influx as a function o f [K]o (Fig. 5);
(e) inhibitory effects o f f u r o s e m i d e and o t h e r drugs (Figs. 7-10; Table V);
(f) a relatively high activation e n e r g y for sodium and r u b i d i u m uptakes.
All o f these characteristics are typical o f a facilitated diffusion mechanism
(Stein, 1967). T h e y also s u p p o r t the hypothesis that a direct coupling exists
between the t r a n s p o r t o f sodium and potassium in the ouabain-poisoned duck
red cell. Such coupling implies a cis-interaction o f both cations with some
m e m b r a n e c o m p o n e n t (carrier) b e f o r e transport can occur. T h a t is, each species
facilitates m o v e m e n t o f its co-ion in the same direction. Both [K]o and [Rb]o
enhance sodium influx (Fig. 5, Table V). T h e r u b i d i u m data o f Table IV and
Fig. 10 d e m o n s t r a t e that this ion does indeed e n t e r the cell with sodium.
F u r t h e r m o r e , calculations (Table IV) show that m o r e r u b i d i u m enters the cell
than can be predicted f r o m the chloride distribution. When early points are
taken (Fig. 2) a transient accumulation o f potassium against its electrochemical
gradient can also readily be d e m o n s t r a t e d . Net potassium m o v e m e n t s also
d e p e n d on [Na]o. Reduction o f [Na]o, without change in the potassium electrochemical gradient, causes net potassium loss (Fig. 4). This net loss with low [Na]o
can be attributed to reduction in the unidirectional potassium influx (estimated
with r u b i d i u m in Table V).
Cosubstrate d e p e n d e n c y for t r a n s p o r t is not unique to duck red cells. As early
as 1902 Reid suggested that glucose absorption in the small intestine is stimulated
SCHMIDT AND McMANus
Salt and Water Movements in Duck Red Cells. I
77
by sodium. More recently, sodium has been shown to enhance the transport o f
many solutes in a wide variety of cells. Particular emphasis has been placed on
the linkage of sugar and amino acid transport with sodium movements. In
general, sodium-sugar and sodium-amino acid transport mechanisms are
thought to derive their energy from the sodium electrochemical gradient and by
this means produce net uphill movements of the cosubstrate of the transport
system (Schultz and Curran, 1070).
Co-ion interaction between sodium and potassium influx was suggested by
Sachs's (1071) observation that [K]o increases ouabain-insensitive sodium influx
in human red cells. Beaug~ and Adragna (1071) also described in detail a ouabain-insensitive, [Na]o-dependent potassium influx in human red cells. Wiley
and Cooper (1074) confirmed the presence of a [K]o-dependent sodium influx,
as well as a [Na]o-dependent potassium influx, in human red cells incubated
with ouahain. Significantly, both of these co-ion-dependent fluxes were inhibited by furosemide. Compared to the duck cells activated by hypertonicity, the
ability of this system to move salt into the human red cell is, at best, minimal.
Diuretics such as furosemide, ethacrynic acid, and triflocin have often been
employed in the study of ouabain-insensitive cation fluxes in human red cells
(Hoffman and Kregenow, 1066; Dunn, 1070, 1072, 1073; Sachs, 1071; Wiley and
Cooper, 1074). Although they have similar effects on sodium efflux in those
cells, they differ greatly in the way they affect net cation and water movements in
ouabain-treated duck red cells in hypertonic media. Ethacrynic acid appears to
make cells leaky to both sodium and potassium, while triflocin has only about
one-half the inhibitory effect of furosemide (Fig. 7).
It is interesting to compare the effect of furosemide on the net movements of
sodium and potassium shown in Figs. 8 and 9 with its effect on influxes of
sodium and rubidium given in Table V. At high [K]o or [Rb]o, the d r u g inhibits
both influxes which should lead to the expected decrease in the rate of net
sodium uptake and an increase in net potassium loss. On the other hand, at low
[K]o or [Rb]o net sodium uptake is increased by furosemide with little effect on
influx. Fur t her m or e , net potassium loss is decreased in spite of a marked
inhibition of influx. We conclude that this drug inhibits efflux as well as influx.
It is attractive to speculate that its primary effect is on the cotransport system
which is responsible for net influx at high [K]o and net efflux at low [K]o.
T h e insensitivity of sodium influx to furosemide at low [K]o (Table V) is
somewhat puzzling in view o f the fact that norepinephrine-induced increase in
sodium influx in isotonic solutions remains sensitive to the drug u n d e r these
conditions (Schmidt and McManus, 1977a). This may reflect a nonspecific
increase in the permeability o f the cells to sodium in hypertonic solutions which
obscures the dr ug effect. Such an increase in sodium permeability has been
demonstrated in dog red cells by Parker and Hoffman (1076) and human red
cells by Poznansky and Solomon (1072).
Nevertheless, furosemide appears to be the "drug of choice" in the study of
this system. However, it should not be concluded that we are suggesting it as a
specific agent in the sense that ouabain has been used in this and other red cell
studies. It is useful in this work to observe drug inhibition in various experimental situations, but only limited conclusions can be drawn from these observations.
78
THE JOURNAL
OF GENERAL
PHYSIOLOGY
• VOLUME
70 • 1977
A m u c h b r o a d e r s t u d y o f f u r o s e m i d e effects will h a v e to be m a d e b e f o r e we c a n
say w h a t it is b i n d i n g to a n d w h a t it is i n h i b i t i n g .
We are grateful for the expert technical assistance of Ms. Deloris Rogers.
This investigation was supported by National Institutes of Health grant HL-12157 and a grant to
William F. Schmidt III from the North Carolina Heart Association (1974-75-A-57). Dr. Schmidt was
also the recipient of a predoctoral fellowship from National Institutes of Health Training Grant 5T01-G700929. A portion of this work was presented by William F. Schmidt to the Department of
Physiology and Pharmacology, Duke University, Durham, N.C., in partial fulfillment of the requirements for the Ph.D. degree.
Received for publication 5 October 1976.
REFERENCES
BEAUG~, L. A. 1975. N o n - p u m p e d sodium fluxes in h u m a n red cells: evidence for facilitated diffusion. Biochim. Biophys. Acta. 401:95-108.
BEAUC~, L. A., and N. ADRAGNA. 1971. T h e kinetics of ouabain inhibition and the partition of rubidium influx in h u m a n red cells. J. Gen. Physiol. 57:576-592.
BEAUG~, L. A., and O. ORTIZ. 1970. Rubidium, sodium and ouabain interactions on the
influx of rubidium in rat red blood cells. J. Physiol. (Lond.). 210:519-532.
BEAUG~, L. A., and O. ORTIZ. 1971. Sodium and r u b i d i u m fluxes in rat red blood cells.
J. Physiol. (Lond.). 218:533-549.
BLUM, R. M., and J. F. HOFFMAN. 1971. T h e m e m b r a n e locus of Ca-stimulated K
transport in energy depleted h u m a n red blood cells. J. Membr. Biol. 6:315-328.
DUNN, M.J. 1970. T h e effects of transport inhibitors on sodium outflux and influx in red
blood cells: evidence for exchange diffusion. J. Clin. Invest. 49:1804-1814.
DUNN, M . J . 1972. Ouabain-uninhibited transport in h u m a n erythrocytes: the effects of
triflocin. Biochim. Biophys. Acta. 225:567-571.
DUNN, M. J. 1973. Ouabain-uninhibited sodium transport in h u m a n erythrocytes: evidence against a second p u m p . J . Clin. Invest. 52:658-670.
FUNDER, J., and J. O. WtETH. 1967. Determination of sodium, potassium and water in
h u m a n red blood cells. Scand. J. Clin. Lab. Invest. 18:151-166.
GARRAHAN, P. J., and I. M. GLYNN. 1967. The behaviour of the sodium p u m p in red cells
in the absence of external potassium. J. Physiol. (Lond.).. 192:159-174.
GARY-BoBo, C. M., and A. K. SOLOMON. 1968. Properties of hemoglobin solutions in red
cells. J. Gen. Physiol. 52:825-853.
HOFFMAN, J. F., and F. M. KREGENOW. 1966. The characterization of new energyd e p e n d e n t cation transport processes in red blood cells. Ann. N. Y. Acad. Sci. 157:566576.
IRVtNG, L., and J. KROG. 1954. Body temperature of arctic and subarctic birds and
mammals. J. AppL Physiol. 6:667-680.
KAPLAN, J. H., a n d H. PASSOW. 1974. Effects of phlorizin on net chloride movements
across the valinomycin-treated erythrocyte membrane. J. Membr. Biol. 19:179-194.
KREGENOW, F. M. 1971. The response of duck erythrocytes to hypertonic media: further
evidence for a volume-controlling mechanism. J. Gen. Physiol. 58:396-412.
KREG~NOW, F. M. 1973. The response of duck erythrocytes to n o r e p i n e p h r i n e and an
elevated extracellular potassium: volume regulation in isotonic media. J. Gen. Physiol.
61:509-527.
SCHMIDT AND McMA~uS Salt and Water Movements in Duck Red Cells. I
79
McNAB, B. K. 1966. An analysis o f the body t e m p e r a t u r e o f birds. Condor. 68:47-55.
OaSKOV, S. L. 1954. T h e potassium absorption by pigeon blood cells. Acta Physiol. Scand.
$1-221-229.
ORSKOV, S. L. 1956. Experiments on the influence of adrenaline and noradrenaline on
the potassium absorption o f red blood cells from pigeon and frogs. Acta Physiol. Scand.
37:299-306.
PARKER, J. C., and J. F. HOFrMAN. 1976. Influence of cell volume and adrenalectomy on
cation flux in d o g red blood cells. Biochim. Biophys. Acta. 455:404-408.
POZNANSKV, M., and A. K. SOLOMON. 1972. Regulation of h u m a n red cell volume by
linked cation fluxes.J. Membr. Biol. 10:259-266.
R~ID, E. W. 1902. Intestinal absorption o f solutions.J. Physiol. (Lond.). 28:241-256.
RIDDICK, D. H., F. M. KaEGENOW, and J. ORLOFF. 1971. Effect of n o r e p i n e p h r i n e and
dibutyryl cyclic adenosine m o n o p h o s p h a t e on cation transport in duck erythrocytes.J.
Gen. Physiol. 57:752-766.
SACHS, J. R. 1971. Ouabain-insensitive sodium movements in the h u m a n red blood ceil.J.
Gen. Physiol. 57:259-282.
SCnMmT, W. F., I I I , and T. J. McMANus. 1974. A furosemide-sensitive cotransport of
Na + plus K + into duck red cells activated by hypertonicity or catecholamines. Fed. Proc.
$3:1457.
SCHMmT, W. F., I I I , and T. J. McMAN~S. 1977a. Ouabain-insensitive salt and water
movement in duck red cells: n o r e p i n e p h r i n e stimulation of sodium plus potassium cotransport. J. Gen. Physiol. 70:81-97.
SCRMmT, W. F., I I I , and T. J. McM^Nus. 1977b. Ouabain-insensitive salt and water
movements in duck red cells. III. T h e role o f chloride in the volume r e s p o n s e . J . Gen.
Physiol. 70:99-121.
SHULTZ, S. G., and P. F. CUaRAN. 1970. Coupled transport of sodium a n d organic
solutes. Physiol. Rev. 50:637-718.
SOLOMON, A. K. 1952. T h e permeability o f the h u m a n erythrocyte to sodium and
potassium. J. Gen. Physiol. $6:57-110.
STEIN, W. D. 1967. T h e Movement of Molecules Across Cell Membranes. Chap. 4.
Academic Press, Inc., New York.
TOSTESON, D. C., and J. S. Robertson. 1956. Potassium transport in duck red cells.J. Cell.
Comp. Physiol. 47:147-166.
TOSTESON, D. C., and J. F. HOFFMAm 1960. Regulation of cell volume by active cation
transport in high and low potassium sheep r e d cells.J. Gen. Physiol. 44:169-194.
W~ETH, J. O., M. DALMARK, R. B. GUNN, and D. C , TOSTESON. 1973. T h e transfer o f
monovalent inorganic anions t h r o u g h the red cell membrane. In Erythrocytes, T h r o m bocytes, Leukocytes: Recent Advances in Membrane and Metabolic Research. E.
Gerlach, K. Moser, E. Deutsch, and W. Willmanns, editors. Georg T h i e m e , Stuttgart.
71-79.
WlLrV,J. S., and R. A. COOPER. 1974. A furosemide-sensitive co-transport o f sodium plus
potassium in the h u m a n r e d cell. J. Clin. Invest. 55:745-755.