Clinical Science (2003) 104, 323–327 (Printed in Great Britain) 323

Sodium/lithium countertransport and

intracellular calcium concentration in
patients with essential hypertension
and coronary heart disease

Sybille GRUSKA*, Ingo JENDRAL*, Rainer RETTIG† and Gu$ nter KRAATZ*
*Department A of Internal Medicine, Ernst Moritz Arndt University Greifswald, Loefflerstrasse 23a, D-17487 Greifswald,
Germany, and †Department of Physiology, Ernst Moritz Arndt University Greifswald, Greifswalder Strass 11C, D-17495
Karlsburg, Germany

A B S T R A C T

The present study was designed to test the hypothesis that enhanced intracellular calcium
signalling and increased sodium/lithium countertransport (Na+/Li+ CT) activity may be
associated with coronary heart disease (CHD) in non-diabetic patients with essential hyper-
tension. Platelet-activating factor (PAF)-evoked rises in the intracellular calcium concen-
tration ([Ca2+]i) were measured in Epstein–Barr-virus-immortalized lymphoblasts from 62
hypertensive patients with CHD and 34 patients without CHD. Na+/Li+ CT activity was assessed
in erythrocytes from 80 hypertensive patients with CHD and 46 patients without CHD. Baseline
values of unstimulated and PAF-stimulated [Ca2+]i were not significantly different between
hypertensive subjects with (baseline, 126p5 nmol/l ; stimulated, 550p43 nmol/l) and without
(baseline, 125p5 nmol/l ; stimulated, 654p105 nmol/l) CHD. Similarly, Na+/Li+ CT activity was
not significantly different between the two groups (patients with CHD, 219p8 µmol : l−1 : h−1 ;
patients without CHD, 234p10 µmol : l−1 : h−1). We conclude that intracellular signal trans-
duction, as indicated by PAF-induced rises in [Ca2+]i and Na+/Li+ CT activity, is not associated
with an increased risk of CHD in non-diabetic patients with essential hypertension.

INTRODUCTION hypertension-induced end-organ damage rather than

Essential hypertension is associated with several abnor-
malities in cellular ion transport systems, including
Na+\Li+ countertransport (Na+\Li+ CT) [1–8], Na+\H+
exchange [3,9–14], Na+–K+ co-transport [6,15], and
intracellular Ca#+ signalling [9,12,15–17]. Several of
these abnormalities have been shown to be particularly
frequent in subsets of subjects with hypertension, giving
rise to the hypothesis that they may be associated with
with hypertension per se [18,19]. Thus a high intracellular
calcium concentration ([Ca#+]i) and high activity of the
Na+\H+ exchanger have been reported to be associated
with a high proliferation rate and growth of vascular
smooth muscle cells [13,20–23], as well as with left
ventricular hypertrophy [24]. Furthermore, increased
Na+\Li+ CT activity has been shown to be associated
with left ventricular hypertrophy [18,19,24–26], cor-
onary heart disease (CHD) [27], hyperlipidaemia [4,7,8]

Key words : calcium signalling, cellular ion transport system, coronary heart disease, hypertension, platelet-activating factor,
sodium\lithium countertransport.
Abbreviations : BMI, body mass index ; [Ca#+] , intracellular calcium concentration ; CHD, coronary heart disease ; Na+\Li+ CT,
i
sodium\lithium countertransport ; PAF, platelet-activating factor.
Correspondence : Dr Sybille Gruska (e-mail gruska!uni-greifswald.de).

# 2003 The Biochemical Society and the Medical Research Society

324 S. Gruska and others

and urinary albumin excretion [1,18,28]. A relationship Table 2 Baseline characteristics of patients in the NaT/LiT
between Na+\Li+ CT activity and diabetic nephropathy CT study
is less evident [4,29,30]. HDL, high-density lipoprotein ; LDL, low-density lipoprotein. Variations in n are
In the present study we tested the hypothesis that indicated in parentheses. *P 0.05 compared with group without CHD.
increased Na+\Li+ CT activity and changes in intra-
Parameter Without CHD With CHD
cellular Ca#+ signalling may be associated with CHD in
non-diabetic patients with essential hypertension. To test Number 46 80
this hypothesis, we compared erythrocyte Na+\Li+ CT Sex (M/F) 20/26 62/18*
activity and platelet-activating factor (PAF)-evoked rises Age (years) 63.5p1.2 65.4p1.0
in [Ca#+]i in lymphoblasts from hypertensive patients BMI (kg/m2) 29.0p0.6 27.0p0.4*
with or without CHD. Creatinine (µmol/l) 95.9p2.8 100.3p3.4
Cholesterol (mmol/l) 5.9p0.2 5.8p0.1
HDL-cholesterol (mmol/l) 1.3p0.1 1.2p0.1
LDL-cholesterol (mmol/l) 3.7p0.1 3.6p0.1
METHODS Triacylglycerols (µmol/l) 2.2p0.2 2.3p0.2
Uric acid (µmol/l) 353p16 (n l 39) 369p12 (n l 71)
Patients Lipoprotein (a) (mg/l) 239p39 (n l 38) 442p69 (n l 55)
The study population consisted of 126 non-diabetic
patients with essential hypertension who attended the
University Hospital of Internal Medicine of the Univer-
clinical and biological parameters used to determine
sity of Greifswald between December 1997 and Febru-
associated cardiovascular risk factors were estimated
ary 2001 for symptoms of CHD and who were subjected
during the stay in hospital. PAF-evoked Ca#+ signals
to coronary angiography. The study was approved by
were determined in immortalized B lymphocytes isolated
the institutional review committee of the University of
from 10 ml of blood. Na+\Li+ CT activity was quantified
Greifswald. Prior to inclusion in the study, all patients
in isolated erythrocytes from an additional sample of
gave informed consent.
10 ml of blood.
Characteristics of the patients are presented in Tables 1
and 2. Intracellular Ca#+ signalling was determined in
immortalized lymphoblasts from 96 patients (59 males Cell culture
and 37 females), 62 of whom suffered from CHD. Lymphocytes were isolated on a Ficoll density gradient
Na+\Li+ CT activity was determined in erythrocytes (Lymphocyte Separation Medium, density 1.077 g\ml ;
from 126 patients (82 males and 44 females), 80 of whom Roche Diagnostics, Mannheim, Germany). B-lympho-
suffered from CHD. All patients were under anti- cytes were immortalized by incubation with Epstein–
hypertensive treatment with calcium channel blockers Barr-virus-containing supernatant from the B 95-8 cell
(27 %), β-blockers (77 %), angiotensin-converting en- line (Deutsche Sammlung von Mikroorganismen und
zyme inhibitors (75 %), AT receptor antagonists (7 %) Zellkulturen GmbH, Braunschweig, Germany). The
" preparation and immortalization has been described in
or α-blockers (8 %), given alone or in combination. All
detail elsewhere [13]. Cells were routinely maintained
in culture with RPMI 1640 medium (Gibco, Eggenstein,
Table 1 Baseline characteristics of patients in the [Ca2T]i Germany) supplemented with 2 mmol\l L-glutamine,
study 100 units\ml streptomycin, 100 units\ml penicillin and
HDL, high-density lipoprotein ; LDL, low-density lipoprotein. Variations in n are 10 % (v\v) fetal bovine serum (Gibco, Eggenstein,
indicated in parentheses. *P 0.05 compared with group without CHD. Germany). Passages were performed twice a week. After
immortalization, lymphoblasts were grown for 16 weeks,
Parameter Without CHD With CHD
then stock cultures were frozen and cells were used for
Number 34 62 measurements.
Sex (M/F) 17/17 42/20*
Age (years) 62.4p1.6 63.9p1.2 Measurement of [Ca2T]i
BMI (kg/m2) 28.7p0.7 26.6p0.5* [Ca#+]i was measured by the fura-2 fluorescence tech-
Creatinine (µmol/l) 91.8p2.9 96.8p2.8 nique, as described previously [31].
Cholesterol (mmol/l) 6.0p0.2 5.8p0.2
HDL-cholesterol (mmol/l) 1.4p0.1 1.2p0.4
LDL-cholesterol (mmol/l) 3.8p0.2 3.6p0.1
Preparation of erythrocyte suspension
EDTA-treated blood was centrifuged at 1500 g. Erythro-
Triacylglycerols (µmol/l) 2.2p0.2 2.2p0.2
cytes were washed four times with iso-osmotic washing
Uric acid (µmol/l) 352p21 (n l 30) 367p15 (n l 57)
buffer (75 mmol\l MgCl , 80 mmol\l sucrose, 5 mmol\l
Lipoprotein (a) (mg/l) 210p41 (n l 25) 372p61 (n l 45) #
glucose, 10 mmol\l Tris\Mops, pH 7.55) at room tem-

# 2003 The Biochemical Society and the Medical Research Society


perature. The erythrocyte suspension was kept at room
temperature and used within 1 h.
Loading with lithium bicarbonate
A sample of 4 ml of washed erythrocytes was incubated
for 15 min at 37 mC with shaking in 20 ml of lithium
bicarbonate medium (150 mmol\l LiHCO , 10 mmol\l
$
glucose, 10 mmol\l Tris\Mops, gassed with CO until
#
the pH value had adjusted to 7.55) [32]. To remove
extracellular lithium, cells were washed five times with
washing buffer.
Measurement of lithium efflux
Lithium efflux was measured by incubating 1.8 ml of cell
)
suspension (haematocrit 0.05–0.08) in a sodium-free
medium (150 mmol\l KCl, 1 mmol\l MgCl , 10 mmol\l
#
glucose, 0.1 mmol\l ouabain, 10 mmol\l Tris\Mops,
pH 7.55) or in a sodium-containing medium (KCl in the
efflux medium was replaced by 150 mmol\l NaCl). After
60 min, incubation in either medium was stopped by
cooling the tubes on ice for 2 min and subsequent
centrifugation at 1500 g for 4 min at 4 mC. Then 1 ml of
the supernatant was mixed with 10 µl of 20 % (w\v)
CsCl solution, and lithium concentrations were
#
measured by atomic absorption spectrophotometry
(Unicam Model Solaar 939).
Measurement of NaT/LiT CT activity
Na+\Li+ CT activity (µmol : l−" : h−") was determined as
the difference between sodium-stimulated lithium efflux
and passive lithium efflux in the sodium-free medium
over time.
Statistics
Data were analysed using the statistical package
Statgrafics Plus (STSC Inc., Rockville, MD, U.S.A.). Data
in the text are presented as meanspS.E.M. Differences
between the two groups were compared by a non-
parametric two-sample test (Mann–Whitney U test).
Differences in gender distribution between the groups
were analysed using chi-squared analysis. Stepwise mul-
tiple linear regression analyses were carried out to
identify independent determinants of Na+\Li+ CT ac-
tivity and PAF-stimulated [Ca#+]i. Values of P 0.05
were considered statistically significant.
RESULTS
Baseline clinical and laboratory data of patients are shown
in Tables 1 and 2. The proportion of male compared with
female patients was significantly higher in the group of
hypertensive patients with CHD than in the group
without CHD. Hypertensives without CHD had a
significantly higher body mass index (BMI) than patients
Signal transduction in patients with hypertension 325
Figure 1 [Ca2T]i at baseline and after stimulation with
0.1 µM PAF in patients without CHD (n U 34 ; ) or with CHD
(n U 62 ;
There were no statistically significant differences between the two groups.
with CHD. All other clinical and biological parameters
were not significantly different between both groups.
Data on PAF-evoked rises in [Ca#+]i are shown in
Figure 1. Baseline values of [Ca#+]i were not significantly
different between patients with (126p5 nmol\l) and
without (125p5 nmol\l) CHD. PAF stimulation led to
significant increases in [Ca#+]i in both groups. PAF-
stimulated [Ca#+]i values were not significantly different
between hypertensives with CHD (550p43 nmol\l) and
those without CHD (654p105 nmol\l). Similarly, there
were no significant differences in Na+\Li+ CT activity
between patients with (219p8 µmol : l−" : h−") and with-
out (234p10 µmol : l−" : h−") CHD.
In a multiple regression analysis with Na+\Li+ CT
activity as the dependent variable and gender, age,
BMI, plasma triacylglycerol concentration and plasma
cholesterol concentration as independent variables, only
age (P 0.05) and plasma triacylglycerol concentra-
tion (P 0.01) emerged as significant and independent
determinants of Na+\Li+ CT activity. In this model,
age and plasma triacylglycerol concentration explained
17.7 % of the variation in Na+\Li+ CT activity. In a
further multiple regression analysis using the same
independent variables and PAF-stimulated [Ca#+]i as the
dependent variable, none of the independent variables
had a statistically significant effect.
DISCUSSION
PAF-evoked increases in both [Ca#+]i and Na+\Li+ CT
activity have been reported to be enhanced in patients
with essential hypertension compared with normo-
tensives [3,5,16,17,33–35]. One study [31] showed that
only a subgroup of hypertensive patients was affected by
increased Na+\Li+ CT activity and enhanced PAF-
evoked increases in [Ca#+]i. Since a high [Ca#+]i and
enhanced Na+\Li+ CT activity have been shown to
be associated with hypertension-induced end-organ
damage, such as left ventricular hypertrophy [18,24,25]
# 2003 The Biochemical Society and the Medical Research Society
326 S. Gruska and others
and CHD [27], we hypothesized that enhanced PAF-
evoked increases in [Ca#+]i and increased Na+\Li+ CT
activity may be useful markers to distinguish between
non-diabetic hypertensive patients with and without
CHD. To test this hypothesis, measurements of PAF-
evoked cytosolic calcium transients and Na+\Li+ CT
activity were performed in immortalized lymphoblasts
and erythrocytes respectively obtained from patients
with essential hypertension. Patients were grouped ac-
cording to the presence or absence of CHD, as diag-
nosed by coronary angiography. PAF-evoked cytosolic
calcium transients and Na+\Li+ CT activity were not dif-
ferent between hypertensive patients with and without
CHD. Thus our results argue against an association
between these membrane ion transport abnormalities
and CHD in non-diabetic patients with essential hyper-
tension.
Our results may have been influenced by a number of
factors that have been reported to affect membrane ion
transport systems, including gender [6,36], BMI [6,7,36],
plasma cholesterol concentration [7,8,37] and plasma
triacylglycerol concentration [35,38,39]. To test for
potentially confounding effects of these factors, we
performed multiple regression analyses with Na+\Li+
CT activity and PAF-stimulated [Ca#+]i as dependent
variables. Na+\Li+ CT activity was significantly affected
by age and plasma triacylglycerol concentration, but
not by gender, BMI or plasma cholesterol concentration.
PAF-stimulated [Ca#+]i was not affected by any of the
above-mentioned potential confounders.
Our failure to demonstrate a significant difference in
Na+\Li+ CT activity between hypertensive patients with
and without CHD cannot be explained by the effects of
age and plasma triacylglycerol concentrations, since our
groups were well matched with respect to these factors.
Other factors, such as gender distribution and BMI,
which showed significant differences between the groups
with and without CHD, did not have independent effects
on Na+\Li+ CT activity in a multiple regression analysis,
and can therefore be excluded as confounders in the
present study.
The finding that none of the factors under consider-
ation had a significant and independent effect on PAF-
stimulated [Ca#+]i may be due in part to the fact that
measurements of [Ca#+]i were performed in Epstein–
Barr-virus-transformed lymphoblasts after prolonged
cell culture. Under these conditions, many potentially
confounding effects of variables related to the homoeo-
static environment of the circulating blood, such as
neurohumoral factors or plasma lipid concentrations, can
be excluded. In the present study it is particularly
important that none of the factors that differed between
the groups with and without CHD, i.e. gender and BMI,
showed an independent effect on PAF-stimulated [Ca#+]i.
In summary, we have failed to demonstrate an as-
sociation between PAF-evoked increases in [Ca#+]i in
# 2003 The Biochemical Society and the Medical Research Society
Epstein–Barr-virus-transformed lymphoblasts or Na+\
Li+ CT activity in freshly obtained erythrocytes and
CHD in non-diabetic patients with essential hyper-
tension. Our results do not support the hypothesis that
these membrane ion transport systems contribute to
CHD in essential hypertension.
ACKNOWLEDGMENTS
This work is part of the Community Medicine Research
(CMR) net of the University of Greifswald, Germany,
which is funded by the Federal Ministry of Education
and Research, the Ministry of Cultural Affairs and the
Social Ministry of the Federal State of Mecklenburg-
West Pomerania. The CMR gathers together several
projects that comprise the population-based Study of
Health in Pomerania (SHIP ; http:\\www.medizin.uni-
greifswald.de\cm).
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# 2003 The Biochemical Society and the Medical Research Society