Clinical Science and Molecular Medicine (1974)46,613-617.
S O D I U M A N D POTASSIUM TRANSPORT RATES I N
N O R M A L H U M A N LEUCOCYTES I N HYPO-OSMOLAL
EXTRACELLULAR F L U I D
P. J. H I L T O N
AND
J. PATRICK
Renal Laboratory, St Thomas’ Hospital, London
(Received 11 October 1973)
SUMMARY
1. Sodium and potassium transport rates were studied in normal human leucocytes
exposed to iso-osmolal and hypo-osmolal extracellular fluid.
2. Hypo-osmolality of the extracellular fluid led to an increase in sodium influx
and a decrease in potassium influx expressed as mmol h-’ kg-’ cell dry weight. The
fall in potassium influx was smaller than the rise in sodium influx and was confined
to the ouabain-insensitive portion of the flux.
3. The rate constants for sodium and potassium efflux did not differ significantly
between the iso-osmolal and hypo-osmolal media.
Key words : leucocytes, sodium and potassium transport, hypo-osmolal media.
In a previous publication (Patrick & Hilton, 1973), the effect of altering the osmolality of the
extracellular fluid on the water and electrolyte composition of human leucocytes was reported.
In a hypo-osmolal medium, the leucocyte was shown to gain sodium and lose potassium with
an overall increase in measured intracellular cation expressed as mmol kg - dry tissue.
Associated with this increase in cation content was an increase in cell water greater than that
predicted from the van’t Hoff-Marriotte equation for a perfect osmometer. In order to study
the mechanisms underlying these changes in cell cation content, we have performed experiments on human leucocytes in iso-osmolal and hypo-osmolal media in which the flux rates of
sodium and potassium were measured with the use of radioactive isotopes.
MATERIALS A N D M E T H O D S
Sodium chloride-free tissue culture fluid (T.C. 199) was obtained from Burroughs, Wellcome
and Co., choline chloride from British Drug Houses Ltd and 22NaC1and 42KCl from The
Radiochemical Centre, Amersham.
Correspondence: Dr P. J. Hilton, Renal Laboratory, St Thomas’ Hospital, London, S.E.1.
61 3
614
P . J. Hilton and J. Patrick
All experiments to be described were performed on leucocytes derived from whole blood
taken from healthy medical students and members of the laboratory staff. The leucocytes were
separated from the blood by the differential dextran sedimentation method of Baron & Ahmed
(1969). After the leucocyte sample had been obtained, it was divided into two approximately
equal portions, which were resuspended in the appropriate tissue culture fluid for the particular experiment.
In every experiment cation flux in leucocytes suspended in 'normal' T.C. 199 (osmolality
285 mosmol kg-' and sodium concentration 134 mmol 1-I) was compared with that in
leucocytes suspended in an 'abnormal' medium. Usually the 'abnormal' medium was one of
low sodium concentration and low osmolality (103 mmol 1-' and 215 mosmol kg-' respectively) but, in four experiments on sodium influx, a medium of low sodium concentration and
normal osmolality was used (103 mmol 1-' and 285 mosmol 1-1 respectively). The osmolality
of this medium was made up with choline chloride. The tissue culture media used for incubation
of the cells were prepared by addition of the appropriate amount of sodium chloride or choline
chloride to the original sodium chloride-free tissue culture fluid. The p H of the media was in
the range 7-35-7.45 and was identical in the two media of each study.
The techniques for measurements of sodium and potassium transport rates have been fully
described elsewhere (Hilton & Patrick, 1973). "Na and 42K were counted in a well-type
scintillation counter and scaler. Specimens were timed for a minimum of lo4 counts. Osmoldity was measured with an Advanced Osmometer.
Sodium eflux
The iso-osmolal and hypo-osmolal cell suspensions were incubated at 37°C for 20 rnin in the
presence of 5 pCi of "NaCI, washed once in 10 ml of the appropriate tissue culture fluid and
resuspended in that fluid. The cell suspensions were returned to the water bath at 37°C and a
period of 5 min was allowed for temperature equilibration. Over a period of 20 min, aliquots
were removed simultaneously from iso-osmolal and hypo-osmolal suspensions, rapidly cooled
in an ice bath and centrifuged at 0°C and 160 g for 2 min. The extracellular fluid was removed as
completely as possible and the cell specimen counted as described previously. Specimen dry
weight was determined by heating at 100°C to constant weight. Depending on the size of the
original leucocyte sample either three or four observations were made over a period of up to 20
rnin in each experiment. The effect of ouabain at a concentration of 1.4 pmol 1- (100 mg 1- ') was
studied in iso-osmolal and hypo-osmolal cells in separate experiments.
Sodium injlux
These experiments were performed on cell suspensions which had been incubated for 20 rnin
in the appropriate tissue culture fluid. At the end of this preincubation period, the extracellular
fluid was replaced with fresh medium and, after allowing a further 5 rnin for temperature
equilibration, sodium influx was observed over a period of 8 rnin after the addition of 5 pCi of
"NaC1 to the cell suspension.
In the main series of experiments, sodium influx was compared in cell suspensions derived
from one donor which had been incubated in iso-osmolal T.C. 199 (285 mosmol kg-l and
134 mmol 1-l sodium) or hypo-osmolal T.C. 199 (215 mosmol kg-' and 103 mmol 1-'
sodium).
Four observations of sodium influx were made in a medium which was iso-osmolal (285
Transport rates in hypo-osmolal leucocytes
615
mosmol kg-') but of low sodium concentration (103 mmol 1-I), the osmolality being made up
with choline chloride. Sodium idlux was compared with that in iso-osmolal, normal sodium
medium.
Potassium e@ux
Iso-osmolal and hypo-osmolal cell suspensions were loaded with radioactive potassium by
with
C 1 an activity of 10 pCi 1-'. After a
incubation for 30 min with T.C. 199 ~ o n t a i n i n g ~ ~ K
single wash and resuspension as described for sodium efflux, potassium efflux was observed
at intervals up to 30 min.
Potassium influx
Equal portions of the original cell suspension were incubated at 37°C for 20 min in isoosmolal and hypo-osmolal T.C. 199. Potassium influx was studied after the addition of T.C.
199 containing 42KClat an activity of 1OpCi1-I. Aliquots were taken from the cell suspension
at periods up to 30 min. The effect of ouabain at a concentration of 1-4pmol 1- (1OOmg 1- ')
on potassium influx at both osmolalities was studied simultaneously.
'
Calculation of results
Sodium and potassium efflux rate constants were calculated from the graph of log (residual
radioactivity) per unit cell dry weight against time, as previously described (Hilton & Patrick,
1973). In these experiments limitations of sample size precluded measurement of intracellular
sodium and potassium content and it was therefore not possible to calculate absolute efflux
rates. Values for sodium and potassium contents of leucocytesin hypo-osmolalmedia have been
reported by Patrick & Hilton (1973). Sodium and potassium influx were calculated as described
by Hilton & Patrick (1973).
Results were expressed as the mean ( ~ S E M )of a group of experiments. The significance of
differences between groups was determined by Student's t-test since the distribution of individual results was approximately normal.
RESULTS
The results of the main series of experiments in which cation fluxes were compared in hypoosmolal and iso-osmolal media are given in Table 1. It can be seen that hypo-osmolality of the
medium leads to a highly significant increase in sodium influx but it has no demonstrable
effect on the rate constant for sodium efflux. In addition, four experiments were performed in
which the influx of 22Nawas studied in cells exposed to iso-osmolal media of different sodium
concentration, the osmolality being adjusted with choline chloride. The osmolality of both
media was 285 mosmol kg-l but the sodium concentrations were 134 and 103 mmol 1-'
respectively. The mean value for sodium influx in cells in the high sodium medium was 373 f 39
mmol h-I kg-l cell dry weight and in the low sodium medium was 271 +31 mmol h-' kg-'
cell dry weight. The difference between the two groups was significant (P<0-05).
In a limited number of experiments on potassium efflux, no change in the rate constant could
be shown between cells incubated in iso-osmolal and hypo-osmolal media. Total potassium
influx showed a small but significant fall in celIs in a hypo-osmolal medium and the difference
D
616
P . J. Hilton and J . Patrick
TABLE
1. Sodium and potassium influx and eflux rates
Results are expressed as means f SEM; P is the probability that the difference in the values for iso-osmolal and
hypo-osmolal media was a chance effect; n is the number of paired measurements. N.S., not significant.
Sodium efflux rate constant (h-l)
Ouabain-insensitivesodium efflux rate constant (h-l)
Sodium influx ( m o l h-' kg-' cell dry weight)
Potassium efflux rate constant (h-l)
Potassium influx (total) (mmol h-' kg-' cell dry weight)
Potassium influx (ouabain-insensitive)(mmol h- kg- '
cell dry weight)
Potassium influx (ouabain-sensitive) (mmol h- kg- l
cell dry weight)
P
n
N.S.
7
7
15
3
9
Iso-osmolal
Hypo-osmolal
3.7 2 0.2
0.91 20.12
312k 16
0.85 & 0.08
367232
3.850.2
1.17k0.18
439 _+ 25
0.82 k 0.06
320k21
N.S.
<O-OOOl
N.S.
< 0.05
227k35
170k25
< 0.05
140+21
151+22
N.S.
9
9
was more marked if the ouabain-insensitive portion of the flux is considered. Despite this,
the ouabain-sensitive potassium influx was not significantly higher in the hypo-osmolal
cells.
DISCUSSION
In previous studies we showed that when leucocytes were suspended in hypo-osmolal
extracellular fluid there was an increase in intracellular sodium and a fall in intracellular
potassium per unit dry weight (Patrick & Hilton, 1973). It was clear that these results could
be explained on the basis either of a change in the permeability of the cell membrane or as a
consequence of altered activity of the sodium :potassium transport system.
Our present findings demonstrate that the change in the sodium content of the leucocyte in
hypo-osmolal media is a consequence of an increased rate of sodium influx rather than a
change in the rate constant for sodium efflux. This increased influx of sodium occurs in the
face of a lowered external sodium concentration and does not occur when the external sodium
concentration is low but the osmolality maintained by addition of choline chloride. These
observations together suggest that the increase in sodium influx in the hypo-osmolal medium
occurs as a result of the hypo-osmolality of the medium or its immediate consequence--cell
swelling-rather than as a result of the low sodium concentration.
The lack of a demonstrable change in the sodium efflux rate constant in the hypo-osmolal
medium is of interest and calls for some comment. Current theories of sodium efflux assume the
presence of a finite number of carrier sites on the cell membrane through which the major
(ouabain-sensitive) portion of sodium efflux takes place (Glynn, 1967). At these sites carrier
molecules combine with sodium ions on the inner surface of the membrane and the resulting
complex migrates to the outer surface where dissociation takes place and the carrier molecule
returns to the inner surface. The reaction is essentially comparable to that involving an enzyme
and its substrate. Hypo-osmolal swelling would on first consideration be expected to depress
the rate constant for sodium efflux (or at least for its ouabain-sensitive portion) by reducing
Transport rates in hypo-osmolal leucocytes
617
the carrier sitelcell volume ratio. That this does not appear to occur may reflect the simultaneous development of a compensatory change within the sodium pump whereby the existing
carrier molecules become more efficiently deployed. This could be the result of alterations in
carrier-site geometry accompanying cell swelling such as have been postulated to occur in
canine erythrocytes by Romualdez, Sha’afi, Lange & Solomon (1972).
We have shown (Patrick & Hilton, 1973) that the fall in intracellular potassium expressed as
mmol kg-l cell dry weight in leucocytes exposed to hypo-osmolal media is small compared with
the increase in sodium content. Similarly, the changes in potassium flux which could be demonstrated in the present experiments were less than for sodium. There was no detectable alteration
in the rate constant for potassium efflux, but a significant fall occurred in both the total and
ouabain-insensitive potassium influx. The ouabain-sensitive potassium influx tended to be
higher in the hypo-osmolal cells though the difference from the iso-osmolal value was not
significant. The mechanisms responsible for the ouabain-insensitive potassium influx in the
leucocyte are poorly understood, and we have previously discussed the problem of the high
proportion of potassium influx which is ouabain-insensitive in this cell (Hilton & Patrick,
1973). In the face of this uncertainty we prefer not to speculate on the possible reasons for the
fall in this flux in the hypo-osmolal cells.
These experiments elucidate some of the mechanisms which underly the steady-state changes
in leucocyte intracellular electrolytes which are associated with a fall in the osmolality of the
extracellular fluid. The fact that similar changes do not occur in the erythrocyte under equivalent conditions stresses the need for caution in the extrapolation of these results to other body
tissues (Patrick & Hilton, 1973). Nevertheless, the results do show that in one cell system at
least the development of a state of lowered osmolality in the extracellular fluid is accompanied
by significant changes in the fluxes of the major cations between the cells and the extracellular
fluid. Such a finding is of relevance in the understanding of low sodium states in man, in
particular the syndrome of inappropriate secretion of antidiuretic hormone.
REFERENCES
BARON,
D.N. & AHMED,
S.A. (1969) Intracellular concentrations of water and the principal electrolytes determined by the analysis of isolated human leucocytes. Clinical Science, 37,205-219.
GLY”, I.M.(1967) The action of cardiac glycosides on sodium and potassium movements in human red cells.
Journal of Physiology, 136,148-173.
HILTON,
P.J.& PATRICK,
J. (1973) Sodium and potassium flux rates in normal human leucocytes in an artificial
extracellular fluid. Clinical Science, 44,439-445.
PATRICK,3. & HILTON,P.J. (1973) The response of the human leucocyte to alterations in the extracellular
osmolality. Clinical Science, 44,457-465.
ROMUALDEZ,
A., SHA’AFI,
R.I., LANGE,
Y.& SOLOMON,
A.K. (1972) Cation transport in dog red cells. Journal of
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