41s
CIinical Science (1981) 61,41s-43s
Intracellular cation activities and concentrations in
spontaneously hypertensive and normotensive rats
W. ZIDEK, H. VETTER, H. ZUMKLEY AND H. LOSSE
Medizinische Poliklinik, University of Munster, Munster, F.R. Germany
Summary
1. The intracellular concentrations of Na+, K+
and Ca2+ were measured in the erythrocytes of
spontaneously hypertensive rats and normotensive Wistar rats.
2. The intracellular Na+ concentration in
hypertensive rats was slightly elevated at 3.16 &
0.25 compared with 2.85 It 0.35 mmol/l
(P z 0.05) and intracellular Na+ activity was
markedly increased in hypertensive rats.
3. Intracellular
CaZ+
activity
was
7519 & 28 990 nmol/l of free water in hypertensive rats compared with 123 ? 98 in controls
(P < 0.01).
4. The cytoplasm of hypertensive animals did
not buffer CaZ+as effectively as that of normal
animals.
5. It is concluded that a decreased binding
capacity of intracellular macromolecules for Na+
and CaZ+ may explain the disturbances of
intracellular electrolyte composition in spontaneously hypertensive rats.
Key words: calcium ions, cations, erythrocytes,
potassium, sodium.
Introduction
Since the early work of our group [ l l and of
Tobian & Binion 121 it has been suggested that
alterations in intracellular Na+ are a major factor
in arterial hypertension. However, the mechanism by which intracellular Na+ causes hypertension has not been clarified. Possible mechanisms are a swelling of arteriolar smooth muscle
cells with a consequent narrowing of the arteriolar lumen [cf. 31, increased sensitivity to pressor
Correspondence: Dr W. Zidek, Medizinische
Polikhik, Domagkstr. 3, D-4400 Munster, F.R.
Germany.
substances [41 and an increase of CaZ+inward
transport t51.
In this study in spontaneously hypertensive
rats, which may be regarded as a model of human
essential hypertension, intracellular total and free
Na+ and K+ and free CaZf were determined in
order to shed light on the ionic disturbances
accompanying the alterations in Na+ metabolism.
Methods
Determinations of intracellular total and free ion
concentrations were performed in erythrocytes of
18 normotensive Wistar rats and 17 spontaneously hypertensive rats of the Munster strain.
Total intracellular Na+ and K+ concentrations
were measured by flame photometry. The erythrocytes were prepared by three-fold washing in
isotonic MgCl, solution. This was followed by a
200-fold dilution of the cell suspension with a
LiCl solution (3 mmol/l). Free intracellular ion
concentrations were determined as ion activities
by ion-selective electrodes. In most experiments
ion-selective microelectrodes were used [61,
which contained neutral carriers selective for Na+
and CaZ+ [7], and for K+ measurements the
Corning exchanger no. 477317 181. In some
experiments ion-selective macroelectrodes were
used instead. These were based on CaZ+selective
membranes containing a neutral ligand and on
Na+ or K+ selective glass [cf. 71. Ca2+ buffer
capacity was measured by adding 5 pl of CaC1,
solutions with increasing Ca2+ concentrations to
50 pl samples of erythrocyte intracellular fluid.
Results
The intracellular total K+ concentration in
normal rats was 99.42 f 4 - 6 0 mmol/l and in
spontaneously hypertensive rats 101.82 If: 3.35
mmol/l; this difference is not significant ( P >
W. Zidek et al.
42s
2-
,00
0,'
m
U
a"
'-
0
5
50
500
Ca'+ added (nmol/55 pi of intracellular fluid)
FIG. 1. Ca2+ buffer capacity of intracellular fluid from erythrocytes of spontaneously
hypertensive rats (0)and normotensive rats (0).Abscissa: amount of Ca2+added to a 5 5 p1
sample. Ordinate: changes of pCa (ApCa).
0.05).The intracellular total Na+ concentrations
showed slight, but not significant, differences. In
hypertensive animals intracellular Na+ concentration was 3-16 f 0.25 mmol/l compared with
2.85 0.35 mmol/l in the controls (Pz 0.05).
Whereas the measurements of total intracellular
Na+ and K+ did not reveal major differences
between both groups, intracellular Na+ and Ca2+
activities, which represent free ion concentrations, showed significant differences. Intracellular Na+ activity in spontaneously hypertensive animals was 4.10 f 0.99 mmoI/l, clearly
exceeding the Na+ activity in control animals,
which amounted to 2.53 5 0.38 (P < 0.001 with
the Wilcoxon test). It was also noteworthy that
the standard deviations of Na+ activity indicated
much wider fluctuations in spontaneously hypertensive than in normal rats. In contrast, the range
of the intracellular Na+ concentration was similar in both groups. Measurements of intracellular
K+ activity did not reveal clear differences. K+
activity was 113.72 f 7.88 mmol/l in normotensive rats and 112.99 L-6.02 mmol/l in hypertensive animals. The most prominent changes
were noted in intracellular Ca2+ activity. The
value of 7519 f 28 990 nmol/l in hypertensive
animals differed significantly from the control
value of 123 f 98 nmol/l (P < 0.01). Wide
fluctuations of intracellular CaZ+activity occurred in hypertensive rats compared to the controls. This corresponded to the wider range of
intracellular Na+ activity in spontaneously hypertensive rats.
To determine the cause of these wide fluctuations and to test whether this could be
attributed to defective buffering properties of the
cytoplasm the CaZ+ buffer capacity of the
cytoplasm of the erythrocytes was determined.
When the total CaZ+content of the intracellular
fluid was increased stepwise by adding CaCI,, the
intracellular CaZ+ activity rose more steeply in
spontaneously hypertensive rats (Fig. 1). The
Ca2+ buffering capacity of the cytoplasm of
normotensive rats was about 8.8 x
mol/l,
whereas the cytoplasm of hypertensive animals
had a buffering capacity of about 2-0 x
moM.
Discussion
Since an elevated intracellular Na+ concentration was described in essential hypertension
[ 11, several studies have been concerned with the
underlying mechanisms. Among these, Canessa
et al. [91 detected an enhanced Na+-Li+ exchange
in the erythrocyte membrane of essential hypertensive patients. It seems difficult to account for
an increased intracellular Na+ on the basis of
such an exchange mechanism unless it is supposed that normally an exchange of Na+ takes
place against another monovalent cation. In view
of the intracellular ionic composition this could
only mean that increased amounts of K+ would
be extruded from the cells. Such an explanation,
however, would be in contrast with the findings of
Losse et al. [lo], who showed that the intra-
Intracellular cations in hypertension
cellular K+ concentration tends to bc elevated in
essential hypertensive patients. Furthermore our
results indicate that in spontaneously hypertensive rats neither intracellular K+ concentration
nor activity is lowered. A second mechanism
which may elevate intracellular Na+ was proposed by Garay et al. [ l l ] , who claim that an
inwardly directed Na+-K+ cotransport is more
active in essential hypertension.
In contrast, there is evidence that ouabainsensitive Na+-K+ countertransport is depressed
in hypertensive patients [12]. At present these
conflicting results cannot be reconciled easily.
Furthermore changes in intracellular K+, which
should accompany those of Na+, have not been
impressive in essential hypertensive patients and
in animal experiments.
A third possibility was proposed by Blaustein
[51: according to this hypothesis a Na+-Ca2+
exchange operates with an Na+ outward transport and a Ca2+ inward transport. When this
exchange mechanism is stimulated by an increase in intracellular Na+, intracellular Ca2+ is
increased. Thereby the contractility of the contractile filaments in the arteriolar smooth muscle
cells may be enhanced. The cause of the elevated
intracellular Na+ remains open, but might be the
result of an unidentified natriuretic factor. The
increased intracellular Ca2+ in spontaneously
hypertensive rats, which was demonstrated
above, is consistent with this theory. However,
there is no explanation for the increased fluctuations of intracellular Ca2+in hypertensive rats.
Furthermore there was no clear correlation
between intracellular free Na+ and Ca2+, which
would be expected if both ion activities depended
on a transmembranous exchange mechanism
with a fixed coupling rate. On the other hand, the
decreased buffering capacity for Ca2+ in erythrocytes of spontaneously hypertensive rats
provides a good explanation for the observed
alterations. Both the lack of correlation between
intracellular Na+ and Ca2+ and the wide fluctuations of CaZ+can be explained by a defective
Ca2+ buffering by intracellular macromolecules.
Similar findings. have been presented by Postnov
& Orlov [131, who showed that cellular membranes from spontaneously hypertensive rats did
not accumulate Cat+ as effectively as normal
membranes. The part of the erythrocytes which is
primarily responsible for the decreased Ca2+
binding cannot be inferred from our experiments.
Either the cytoplasm or the cell membranes might
play a role. When the results are regarded in the
light of those of Postnov & Orlov [131 it seems
likely that defective Ca2+ binding by macromolecules occurs in essential hypertension. Such
43s
a concept, which is based on intracellular ion
adsorption [141 is controversial [9, 11, 121.
Nevertheless altered intracellular ion binding
might be one important factor in the pathogenesis of primary hypertension.
Acknowledgment
We thank Professor W. Simon, Lab. f. Organ.
Chemie, ETH Zurich, Switzerland, who supplied
us with Ca2+selective membranes.
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