85
High Calcium Diet Augments Vascular
Potassium Relaxation in Hypertensive Rats
Ilkka Porsti, Pertti Arvola, Heikki Wuorela, and Heikki Vapaatalo
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
The effects of increased dietary calcium on the development of hypertension and vascular
smooth muscle responses were studied in spontaneously hypertensive rats and normotensive
Wistar-Kyoto rats. Both hypertensive and normotensive animals were divided into two groups;
the calcium content of the normal diet was 1.1% and that of the high calcium diet 3.1%. During
the 12-week study, calcium supplementation significantly attenuated the increase in systolic
blood pressure in the hypertensive rats but did not affect blood pressure in the normotensive
rats. The contractile responses of endothelium-denuded mesenteric arterial rings to potassium
chloride were similar in all study groups. The contractions to norepinephrine were not altered
by the high calcium diet either, but smooth muscle sensitivity to this agonist was lower in the
normotensive than in the hypertensive rats. Potassium relaxation was used to evaluate the
activity of vascular smooth muscle Na+,K+-ATPas«. The maximal rate of potassium relaxation
was fastest in the normotensive groups but was also clearly faster in calcium-treated
hypertensive rats when compared with hypertensive rats on a normal diet Platelets were used
as a cell model for the analysis of intracellular free calcium concentration, which was measured
by the fluorescent indicator quin-2. Intracellular free calcium was significantly reduced in the
hypertensive rats by calcium supplementation and was not affected in the normotensive rats. In
conclusion, a reduction of intracellular free calcium concentration indicating improved calcium
regulation and a concomitant alteration in vascular relaxation probably reflecting increased
activity of smooth muscle Na+,K+-ATPase may contribute to the blood pressure-lowering effect
of a high calcium diet (Hypertension 1992;19:85-92)
pidemiological studies have suggested an inverse correlation between calcium intake and
blood pressure,1'2 and a hypothesis has been
developed whereby dietary calcium deficiency is associated with essential hypertension.3 Both clinical and
experimental studies suggest that blood pressure can be
reduced by oral calcium supplementation.4-6 However,
contradictory results have also been published.7-8
The mechanisms underlying the possible blood
pressure-lowering effect of calcium have not been
fully clarified. Dietary calcium may correct the generalized membrane defect observed in hypertension
and have a direct membrane-stabilizing effect on
vascular smooth muscle, resulting in decreased conductance of ions across the cell membrane and
reduced intracellular free calcium concentration.6-9
E
Calcium supplementation may also affect the overall sodium and calcium balance1011 and lead to
phosphate depletion, thus impairing cardiovascular
regulation.5 A high calcium diet has been found to
reduce aortic smooth muscle reactivity in spontaneously hypertensive rats (SHR).12 We have reported
augmented smooth muscle relaxation with a concomitant reduction in tissue sodium/potassium ratio in
calcium-supplemented SHR.13
In the present study, we tested the hypothesis that
dietary calcium alters functional properties of vascular smooth muscle. We investigated the effects of oral
calcium supplementation on blood pressure and intracellular free calcium concentration in SHR and
Wistar-Kyoto (WKY) rats and examined the associated changes in vascular smooth muscle reactivity.
From the Department of Biomedical Sciences, University of
Tampere, Tampere, Finland.
Supported by grants from the University of Tampere, Finland
(I.P.), the Sigrid Juselius Foundation, Finland (H.W.), and the
Academy of Finland (H.V.).
Address for correspondence: Dkka P5rsti, MD, Department of
Biomedical Sciences, University of Tampere, P.O. Box 607, SF33101 Tampere, Finland.
Received March 22, 1991; accepted in revised form September
16, 1991.
Methods
Animals, Blood Pressure, and Plasma Electrolytes
Thirty-two male SHR of the Okamoto-Aoki strain
and eighteen WKY rats (M0llegaard's Breeding Centre, Ejby, Denmark) were used to study the effect of
dietary calcium on blood pressure (age 8 weeks,
average weight 190 g). At the beginning of the study,
the SHR and the WKY rats were divided in two
86
Hypertension
Vol 19, No 1 January 1992
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
groups (n=16 and n=9, respectively) of equal mean
systolic blood pressures (155-156 and 144-145
mm Hg, respectively). Thus, the study groups were
SHR, WKY rats, calcium-supplemented SHR (CaSHR), and calcium-supplemented WKY rats (CaWKY). An additional 24 SHR and 18 WKY rats on a
normal calcium diet were used to study smooth
muscle responses in vitro.
The rats were housed two animals to a cage and
had free access to drinking fluid (tap water) and food
pellets (Ewos, Sodertalje, Sweden) that in the SHR
and WKY groups contained 1.1% calcium and in the
Ca-SHR and Ca-WKY groups, 3.1% calcium
(weight/weight, the extra calcium was supplied as
carbonate salt). The consumption of food and drinking fluid was measured by weighing the chow and
bottles, respectively.
The systolic blood pressures and heart rates of the
unanesthetized animals were measured by the tailcuff method at +28°C (model 129 Blood Pressure
Meter, IITC Inc., Woodland Hills, Calif.).
After 12 weeks of study, the rats were weighed,
decapitated, and exsanguinated. The hearts were
excised and weighed whole. Blood samples were
drawn into chilled tubes on ice with heparin (100
units/ml, Sigma Chemical Co., St. Louis, Mo.) as
anticoagulant.
For plasma electrolyte determinations, 2 ml blood
was centrifuged (15 minutes, 1.000& +4°C), and the
samples were stored at —70°C until assayed. Sodium,
potassium, and calcium concentrations were analyzed with an atomic absorption spectrophotometer
(AAS) (Spectr AA-30, Varian, Techtron Ltd., Victoria, Australia). In the calcium samples, lanthanum
chloride (final concentration 5 mM) was used as
ionization suppressant. This was prepared by dissolving La2O3 (AAS grade, Aldrich Chemical Co., Milwaukee, Wis.) in concentrated hydrochloric acid and
diluting it in deionized water.
Platelet Isolation and Measurement of Intracellular
Free Calcium Concentration
Whole blood was diluted 1:2 with RPMI-1640
medium (Flow Laboratories, Irvine, Scotland), and 4
ml diluted sample was layered on 3 ml Ficoll-Paque
solution (Pharmacia, LKB Biotechnology Inc., Piscataway, NJ.). After centrifugation (25 minutes,
120g +20°C), the supernatant was removed, and
platelets and lymphocytes were harvested from the
plasma-Ficoll interface. The cells were resuspended
in 4 ml RPMI-1640 medium, and 0.4 ml ACD solution (8% citric acid, 2.2% trisodium citrate, 2.45%
dextrose, pH 6.5) was added to the suspension.
Lymphocytes were separated by centrifugation (10
minutes, lOOg, +20°C), and platelets were spun down
from the supernatant (10 minutes, 500& +20°C). The
platelets were washed twice with HEPES medium (in
mM, NaCl 145, MgSO, 5, glucose 10, HEPES/NaOH
10, pH 7.4) by aspirating the cell pellet into a
polypropylene Pasteur pipette and finally were resuspended in this medium.
The cell suspension (l-2xlO 9 platelets/ml) was
loaded with 50 ^.M (final concentration) acetoxymethyl ester quin-2 (quin-2 AM, Aldrich) for 30
minutes at +37°C without shaking. Thereafter, the
cell suspension was washed with 2 ml HEPES medium to remove extracellular quin-2. Finally, the
platelets were resuspended in prewarmed (+37°C)
Hanks' balanced salt solution (2-2.5 x 10s platelets/
ml). After a 5-minute equilibration period, fluorescence was measured with a Shimadzu RF-5000 spectrofiuorometer (Shimadzu Corp., Kyoto, Japan) in
quartz cuvettes with continuous stirring at +37°C.
The excitation and emission wavelengths were set at
339 and 492 nm, respectively.
The calibration of intracellular fluorescence as a
function of intracellular free calcium concentration
([Ca2+]|) was performed essentially according to the
method described by Tsien et al.14 The maximal
fluorescence (F,^) was measured after the addition
of 2 fiM ionomycin (Sigma) and 5 mM calcium
chloride (final concentrations), which saturated the
binding capacity of quin-2. To measure the minimal
fluorescence (F,^), 25 mM Tris-EGTA (final concentration), pH 8.6, and 0.1% (vol/vol) Triton X100
(Aldrich) were added to the suspension. The intracellular free calcium concentration was calculated by
the equation
[Ca 2+ ] i =115(F-F min )/(F mM -F)
where 115 represents the dissociation constant in
nanomoles and F is the fluorescence of the intact
cell suspension.
Smooth Muscle Responses In Vitro
The mesenteric artery was excised and cleaned of
connective tissue. A 3-mm-long standard section
taken approximately 10 mm from the proximal part of
the artery was cut, and the endothelium was removed
by gently rubbing the preparation with a scuffled
injection needle. The vascular ring was placed between two stainless steel hooks and was mounted in an
organ bath chamber in physiological salt solution
(PSS) (pH 7.4) of the following composition (mM):
Nad 118.0, NaHCO3 25.0, glucose 11.1, CaOz 2.5,
KC1 4.7, KH2PO41.2, MgSO41.2. The ring was equilibrated for 1 hour at +37°C with a resting tension of
1.5 g and was aerated with 95% O2 and 5% CO2. The
force of contraction was measured with an isometric
force-displacement transducer (model FT03, Grass
Instrument Co., Quincy, Mass.) and registered on a
Grass polygraph (model 7 E Polygraph).
The removal of the endothelium was confirmed by
adding 1 fiM acetylcholine (Sigma) to a norepinephrine (1 fiM) (Fluka Chemie AG, Buchs, Switzerland)
precontracted vascular ring. If any relaxation was
observed, the endothelium was further rubbed.
After a 30-minute stabilization period, dose-response curves for potassium chloride and norepinephrine were determined cumulatively for all preparations. In solutions containing high concentrations
Porsti et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
of potassium, N a d was substituted with KG on an
equimolar basis.
After another 30 minutes of stabilization, potassium was omitted from the PSS. The K+-free solution
was prepared by substituting KH2PO4 and KC1 with
NaH2PO4 and NaO, respectively, on an equimolar
basis (pH 7.4). The K+-free buffer solution resulted
in a gradual contraction in all vascular preparations.
After the contraction had reached a plateau, normal
PSS (containing 5.9 mM potassium) was reintroduced to the preparations and the subsequent relaxation registered. The K+-free contraction and K+relaxation scheme was also performed in some
endothelium-intact preparations and in some endothelium-denuded preparations in the presence of 1
mM ouabain (Sigma) or after chemical sympathectomy; these preparations were obtained from SHR
and WKY rats given a normal calcium diet. The
vascular rings were denervated in vitro by exposing
them to a buffer-free solution containing 1.2 mM
6-OH-dopamine (Sigma), and by vigorously gassing
them with N2 for 15 minutes, followed by a 2-hour
recovery period in standard PSS.15
After registration of responses, all preparations
were dried overnight at +100°C and then were
weighed. The concentration of agonist at which 50%
of maximal force was developed (EQo) for norepinephrine and KC1 in each preparation was calculated
from plots of the concentration of agonist versus the
percentage of maximal tension response. Contractile
responses in K+-free solution were normalized by
comparing them with the maximal response to KG
(124 mM KG). After potassium repletion, the greatest reduction in smooth muscle contractile force
during a 1-minute period was considered as maximal
relaxation rate, which was normalized by comparing
it with the maximal 124 mM KG-induced contraction
or by relating it to tissue dry weight. Only one
vascular preparation from each animal was used in
the study.
The experimental design was approved by the
Animal Experimentation Committee of the University of Tampere.
Statistics
Statistical analysis was carried out using one-way
analysis of variance (ANOVA), supported by Bonferroni confidence intervals in the case of pairwise
comparisons between the test groups. When the data
consisted of repeated observations at successive time
points, ANOVA for repeated measurements was
applied to study differences between groups. Comparisons were made between all groups or between
two treatment groups as appropriate. The results are
expressed as mean±SEM. The data were analyzed
with BMDP Statistical Software, Los Angeles, Calif.
Results
During the 12-week study period, blood pressure
increased in both SHR groups, whereas no significant
change was observed in the WKY rat groups. The
Calcium Diet and Potassium Relaxation
87
240
190
140 L
4
8
TlME(<mkB)
12
FIGURE 1. Line graph shows effect of high calcium (Ca) diet
on systolic blood pressure (BP) in the spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) during the
12-week study. Development of hypertension was attenuated in
the Ca-SHRgroup (p<0.0001) (n=16forSHRand
Ca-SHR,
andu=9for WKY and Ca-WKYgroups).
final mean values for the four groups were: SHR
233±3, Ca-SHR 213±3, WKY 155±5, and Ca-WKY
153±4 mm Hg. When compared with SHR, the rise
was significantly attenuated in the Ca-SHR group
(/xO.0001, ANOVA for repeated measurements)
(Figure 1).
Calcium supplementation did not affect heart
weight or heart rate in the SHR and WKY rats or
growth in the SHR, but the Ca-WKY group gained
slightly less weight than the WKY group. Heart rate
and weight were significantly lower in the WKY
groups, and the final body weight was higher when
compared with the SHR groups. Based on food
consumption, the mean intake of calcium was increased about fourfold in the Ca-SHR group when
compared with the SHR group, and about threefold
in the Ca-WKY when compared with the WKY
group (Table 1).
Plasma sodium concentrations were not affected
by the high calcium diet but were slightly higher in
both WKY groups than in the SHR groups. Plasma
potassium was somewhat elevated in the Ca-SHR
group. Plasma total calcium concentration was
slightly higher in both WKY groups when compared
with the SHR groups (Table 1).
The dose-response curves of contractions induced
by potassium chloride and norepinephrine were not
affected by the high calcium diet. For potassium
chloride, both the EQo concentrations and the maximal contractile force related to tissue dry weight
were quite comparable in all study groups (Figure 2,
Table 2). However, the time required for total relaxation after 124 mM KG (i.e., the washout time) was
markedly shorter in the WKY and Ca-WKY groups
than in the SHR and Ca-SHR groups (Table 2). For
norepinephrine, the WKY groups showed higher
ECM concentrations (i.e., lower sensitivity), and the
maximal contractile forces did not differ from those
of the SHR groups (Figure 2, Table 2).
Hypertension
88
Vol 19, No 1 January 1992
TABLE 1. Body Weight and Heart Rate During Weeks 1 and 12, Mean Calcium Intake During the Study, and Heart
Weight and Plasma Electrolytes at the Close of the 12-Week Study
Variable
SHR
Body weight (g)
Weekl
Week 12
Heart rate (bpm)
Week 1
Week 12
Heart weight/body weight
(g/kg)
Mean calcium intake
(mg/kg/day)
Plasma sodium (mM)
Plasma potassium (mM)
Plasma calcium (mM)
Ca-SHR
WKY
Ca-WKY
191+4
368±5
194±6
361 ±5
186±3
417±6*
397±6't
190±5
404+7
388+9
397±7
386±7
360±7*
317±6*
364±8*
325 ± 8 '
3.85 ±0.06
3.95+0.07
2.56±0.06*
2.64±0.09*
554+40
127.9±4.7
4.22±0.12
2.33+0.03
2^14±84*
130.9±2J
4.79±0.16§
2J8±0.02
502±62
143.9±3J§
4.10±0.19
2.56±0.07§
l,454±60*:f:
142.7±4.1§
3.98±0.12
2.69±0.06*
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Values are mean±SEM. n = 16 for SHR and Ca-SHR, n=9 for WKY and Ca-WKY groups. SHR, spontaneously
hypertensive rats; WKY, Wistar-Kyoto rats; Ca, calcium-supplemented animals.
5p<0.05, *p<0.001 compared with SHR.
tp<0.05, 1p<0.00l compared with WKY rats using one-way analysis of variance.
Typical responses of endothelium-denuded mesenteric arterial rings in K+-free solution are shown in
Figure 3, and plots of study groups in Figure 4. The
maximal contractile force generated in K+-free solution was similar in all study groups, but the plateau
phase was reached earlier in the WKY, the Ca-WKY,
and the Ca-SHR groups than in the SHR group.
K+-relaxation time was shortest in the WKY groups,
and that of the Ca-SHR group was also shorter than
in the SHR group (Figure 4, Table 2). The maximal
rate of relaxation was similar and clearly fastest in
the WKY and Ca-WKY rats, and in the Ca-SHR it
was also significantly faster than in the SHR given a
normal diet (Figure 5A). Ouabain (1 mM) effectively
prevented the K+-relaxation (data not shown), indicating that it reflected the activity of smooth muscle
Na+,K+-ATPase.
100
•B
O SHH
• Cfr-SHR
If the endothelium was left intact, the contractile
response in K+-free solution was clearly depressed
when compared with endothelium-denuded preparations in both SHR and WKY rats, but the contraction
remained more pronounced in the SHR (Figure 3,
Table 3). In vitro sympathectomy with 6-OH-dopamine also markedly depressed the K+-free contractions of endothelium-denuded preparations, but the
remaining contractions were clearly greater in the
SHR than in the WKY rats (Figure 3, Table 3). The
rate of potassium relaxation related either to maximal potassium chloride-induced contractile response
or to tissue dry weight was always faster in the WKY
than in the SHR, whether the endothelium or functioning adrenergic nerve endings were present or not
(Table 3). If potassium was returned to the bath
before the plateau was reached, the maximal rate of
relaxation was not noticeably affected (data not
shown).
Platelet intracellular free calcium concentration,
measured by the fluorescent indicator quin-2, was
comparable in the WKY, Ca-WKY, and Ca-SHR
groups and was significantly lower than in the SHR
on the normal calcium diet (Figure 5B).
• Cfr-WKY
Discussion
In the present study, calcium supplementation
lowered blood pressure in SHR, confirming earlier
observations of our group and others.5611-12 Blood
pressure in normotensive WKY rats was not affected
20
30
50
80
124 9
8
7
6
5
4
by the high calcium diet.
POTASSIUM CHLORIDE (mM)
NORBWEPHfTOE (-tog M)
In accordance with previous findings,12-13 dietary
FIGURE 2. Line graphs show dose-response curves of endocalcium had no effect on body or heart weights of the
thelium-denuded mesenteric arterial rings to potassium chlo- SHR. Apparently, the reduction in afterload accomride and norepinephrine. SHR, spontaneously hypertensive panying the lower blood pressure was not sufficient to
rats; WKY, Wistar-Kyoto rats; Co, calcium-supplemented attenuate cardiac hypertrophy. Weight gain was
animals (n=16 for SHR and Ca-SHR, and n=9 for WKY
slightly impaired in the Ca-WKY group during the
and Ca-WKY groups).
study, suggesting a nonbeneficial effect of increased
Pdrsti et a! Calcium Diet and Potassium Relaxation
89
TABLE 2. Parameters of Smooth Musde Responses of EndotheUam-Dennded Mesenteric Arterial Rings
Variable
Norepinephrine EQo (nM)
Potassium chloride ECJO (mM)
Maximal contractile force (g/mg tissue
dry wt)
Norepinephrine
Potassium chloride
Washout time after 124 mM KC3 (min)
Potassium-free contraction
Time to maximum (min)
Maximal force related to 124 mM
Kd(%)
Potassium relaxation time (min)
SHR
Ca-SHR
WKY
Ca-WKY
487±58
46.1 ±2.0
397±44
43.5±2.0
737±68*
43.8±13
765±76*
46.5 ±2.0
5.74±0.44
5.71 ±037
17.2±1.9
6.78±0.70
6.54+0.52
17.6±2.2
6.72±0.49
6.82±0.56
7.0±0.6t
6.88±0.58
6.92+0.58
8.1±0.8t
41.6 ±2.2
35.7±2.0*
25.4±1.8t
26.7±2.0t
66.5 ±5.0
12.0±0.8
68.0±5.5
9.0±0.4f
72.3 ±4.6
67.8±3.6
5.9±0.6t
5.3±0Jt
Values are mean±SEM. ECJO is the concentration of agonist giving 50% of the maximal response; 124 mM KC1
induces the maximal contractile response to high potassium. n = 16 for SHR and Ca-SHR, n=9 for WKY and Ca-WKY
groups. SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; Ca, calcium-supplemented animals.
*p<0.05, t/><0.001 compared with SHR using one-way analysis of variance.
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
dietary calcium in the WKY rat. SHR have been
reported to have an enhanced appetite for calcium,16
and in our study, the Ca-SHR group consumed about
30% more chow than the other groups, which resulted in an approximately fourfold increase in dietary calcium intake.
ENDOTHELJUM
Extracellular ionized calcium values have been
reported to be decreased both in essential hypertension and in SHR.1718 We did not measure ionized but
total plasma calcium concentration, which was also
lower in SHR than in the normotensive controls and
was not corrected by the high calcium diet. In contrast, plasma potassium concentration was elevated
in the Ca-SHR group. The explanation for this is not
clear from the present results, but in renovascular
hypertension calcium supplementation has been reported to reduce blood pressure by suppressing the
renin-angiotensin system.19 If plasma renin was reduced in calcium-supplemented SHR, this could
explain the slightly higher plasma potassium. Plasma
sodium concentration was somewhat higher in both
WKY groups than in the SHR and was not affected
by the high calcium diet.
In the present study, the high calcium diet did not
affect vascular sensitivity to vasoconstrictors, and
100
1
so
- ENDOTHEUUM + SYMPATHECTOMY
WKY
noK*
SHFt
TME(mki)
FIGURE 3. Representative tracings show typical contractile
responses of endothdium-intact (+endothdium), endotheliumdenuded (—endothdium), and endothdatm-aenuded+sympathectomized (—endothdium+sympathectomy) mesenteric arterial rings from spontaneously hypertensive rats (SHR) and
Wistar-Kyoto (WKY) rats in K+-free buffer solution and the
relaxation after the return of5.9 mMpotassium to the organ bath.
FIGURE 4. Line graph shows contractile responses of endothelium-denuded mesenteric arterial rings in K+-free buffer
solution (where 100% contraction represents the force attained
by 124 mM KCl), and the relaxation response to 5.9 mM
potassium after the contraction had reached the plateau
phase. SHR, spontaneously hypertensive rats; WKY, WistarKyoto rats; Ca, calcium-supplemented animals (n=16 for
SHR and Ca-SHR, and n=9for WKY and Ca-WKY groups).
90
Hypertension
Vol 19, No 1 January 1992
I I SHR
Y7771 Ca-SHH
M WKY
ESi Ca-WKY
E3 SHR
VZ& Ca-SHR
M WKY
ES3 Ca-WKY
|
O
P
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
FIGURE 5. Bar graphs show maximal potassium relaxation
rates related to tissue dry weight (panel A) and platelet
intracelhdar free calcium concentrations (panel B). SHR,
spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; Co,
calcium-supplemented
animals. *p<0.05,
**\><0.01,
"*p<0.001 when compared with SHR; n=16 for SHR and
Ca-SHR, and n=9for WKY and Ca-WKY groups.
maximal contractile responses to both potassium
chloride and norepinephrine were also similar in
SHR and WKY rats. However, smooth muscle sensitivity to norepinephrine but not to potassium chloride was lower in the WKY rats. This indicates
increased sensitivity to a-adrenergic agonists in SHR,
a finding reported in numerous studies (for review,
see Reference 9). We also found a clear difference in
vascular smooth muscle relaxation; the washout time
after maximal potassium chloride-induced contraction was markedly prolonged in both SHR groups
when compared with the WKY groups, which could
result from impaired calcium sequestration, extrusion, or contractile protein dephosphorylation in
SHR smooth muscle.
The removal of extracellular potassium results in
almost total inhibition of Na+,K+-ATPase,20 which
can affect intracellular calcium and promote smooth
muscle contraction in many ways: The inhibition
results in increased intracellular sodium and decreased plasmalemmal sodium gradient, and thus in
decreased extrusion of calcium by the Na+-Ca2+
exchange mechanism.21 Na+,K+-ATPase inhibition
also causes depolarization and increases the influx of
calcium through potential-dependent channels.20
The depolarization of vascular adrenergic nerves
releases norepinephrine from the nerve endings,
which activates a-adrenergic receptors and contributes to smooth muscle contraction.22 In the present
study with endothelium-denuded preparations,
smooth muscle contraction after removal of extracellular potassium was more rapid in the WKY rats than
in the SHR, and calcium supplementation shifted the
response of the SHR in the normotensive direction.
Both intact vascular endothelium and in vitro sympathectomy after removal of endothelium clearly
depressed the contractile responses of vascular rings
in K+-free solution, but the contractions remained
more pronounced in the SHR than in the WKY rats.
Thus, in the WKY rats an intact endothelium depresses more and adrenergic nerve endings participate more in the K+-free contractile response than in
the SHR. The latter could reflect differences either
in the function of adrenergic nerve endings or
smooth muscle cell membrane. After sympathectomy, the greater K+-free contractions of endothelium-denuded preparations from the SHR most likely
reflect increased intracellular accumulation of ions
since cell membrane permeability has been found to
be increased in the SHR.69 The endothelium-denuded preparations were chosen to study ^-relaxation because they presented the greatest and fastest
contractions. However, it should be pointed out that
relaxation was always slower in SHR whether the
endothelium or functioning adrenergic nerve endings
were present or not.
The potassium-induced relaxation is an indirect
means of measuring the activity of the sodium pump.
When potassium is returned to the organ bath, the
activation of Na+,K+-ATPase repolarizes the cell
membrane,23-24 initiating smooth muscle relaxation
with calcium sequestration, extrusion, and contractile
protein dephosphorylation.25 Contrasting results on
the activity of Na+,K+-ATPase in hypertension have
been reported, demonstrating sometimes an increase,23 sometimes a decrease26 in sodium pump
activity. In the present study, potassium relaxation was
TABLE 3. Parameters of Smooth Mnsde Responses of Endotheliam-Intact, EndotheUum-Denuded, and EndotheUnm-Denaded and
Sympathectomized Mesenteric Arterial Rings in Spontaneously Hypertensive Rats and Wistar-Kyoto Rats
Variable
Potassium-free contraction
Time to maximum (min)
Maximal force related to 124 mM Kd (%)
Potassium relaxation rate
Related to 124 mM Kd (%/min)
Related to tissue dry weight (mg/mg/min)
Endothelium-intact
SHR
WKY
Endothelium-denuded
SHR
WKY
Endothelium-denuded and
sympathectomized
SHR
WKY
49.4±0.8
47.0±2^
27.2±2.7'
28.8±2.9*
41.6±2.2
66-5+5.0
25.4+1.8+
72J+4.6
75.9±2.2
54.1 ±4.0
99.1±8.6t
23.2±3.6+
8.6±1.1
382±61
22.2±1.8t
1,568+135t
11.3+0.6
548±91
27.6+2J>t
1,649+202+
10.0±0.7
403±75
24.0±2.4f
1,374±141+
Values are mean±SEM. KQ (124 mM) induces the maximal contractile response to high potassium. n=12-16 for SHR, JI=6-9 for WKY
rats. SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats.
•p<0.01, t/xO.001 compared with SHR using one-way analysis of variance.
Porsti et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
clearly attenuated in SHR when compared with WKY
rats. The present results support the conception of
reduced Na+,K+-ATPase activity in this type of genetic hypertension, although direct measurements of
enzyme activity or electrical events were not performed. The high calcium diet significantly augmented
the K+-relaxation response in the SHR; however, the
relaxation after 124 mM KQ-induced contraction was
not affected and remained prolonged. Thus, calcium
treatment appears not to alter relaxation per se but
augments the response to sodium pump blockade
followed by potassium repletion. This supports alterations in the recovery of ionic gradients (e.g., intracellular sodium) in the Ca-SHR group, and more active
Na+,K+-ATPase, which could directly reduce peripheral resistance by hyperpolarizing the cell membrane
and thus explain the blood pressure-lowering effect of
oral calcium supplementation.
The intracellular free calcium concentration is
elevated in lymphocytes and smooth muscle cells of
SHR,6-27 and a cytoplasmic calcium-elevating factor
has been found in the serum of SHR.28 Increased
intracellular calcium leads to augmented vascular
contraction, raised peripheral resistance, and elevated blood pressure. Platelet intracellular free calcium has been reported to be elevated also in essential hypertension and to correlate well with the fall in
blood pressure caused by antihypertensive therapy.29
On the other hand, increased extracellular calcium in
primary hyperparathyroidism has been associated
with reduced intracellular free calcium.30 In a recent
study, calcium supplementation reduced intracellular
free calcium in stroke-prone SHR, the proposed
mechanism being the correction of the generalized
membrane defect associated with genetic hypertension.6 This membrane defect leads to increased permeability of the plasma membrane to major ions,
which in turn enhances vascular contractions.9 We
have reported increased activity of red blood cell
Ca2+-ATPase and reduced blood pressure in calciumsupplemented SHR.13 In the present study, platelet
intracellular free calcium was clearly lower in the
WKY than in the SHR groups, and the high calcium
diet reduced it in the SHR. The activation of calcium
transport mechanisms or a direct effect on cell membrane could have contributed to the reduction in
intracellular calcium.
It has been suggested that calcium can regulate
sodium transport, an elevation in cytosolic calcium
reducing and a decline enhancing the activity of
Na+,K+-ATPase.31 Thus, in vascular tissue reduced
intracellular free calcium would increase the activity
of Na+,K+-ATPase and reduce smooth muscle tone
with this mechanism.
A circulating inhibitor of Na+,K+-ATPase has
been found in the plasma of both essentially hypertensive patients and rats with renovascular and saltinduced hypertension.32-34 This digjtalislike factor is
thought to augment contractile responses in vascular
smooth muscle and elevate peripheral resistance and
blood pressure.35 Increased dietary calcium has been
Calcium Diet and Potassium Relaxation
91
reported to prevent salt-induced hypertension and to
reduce plasma digitalislike immunoreactive factor in
rats.34 In the present study, calcium supplementation
could have increased the activity of vascular Na+,K+ATPase in SHR by reducing the amount of this
circulating sodium pump inhibitor.
In conclusion, the present results indicate that
calcium supplementation attenuates the development
of hypertension in SHR with concomitant alterations
in intracellular free calcium concentration and vascular smooth muscle responses. Reduced intracellular
free calcium indicates improved calcium regulation,
and augmented potassium-relaxation rate suggests
increased activity of smooth muscle Na+,K+-ATPase.
References
1. McCarron DA, Morris CD, Henry HV, Stanton JL: Blood
pressure and nutrient intake in the United States. Science
1984;224:1392-1398
2. Garcia-Palmieri MR, Costas R, Cruz-Vidal M, Sorlie PD,
Tillotson J, Harlik RJ: Milk consumption, calcium intake, and
decreased hypertension in Puerto Rico. Hypertension 1984;6:
322-328
3. McCarron DA: Is calcium more important than sodium in the
pathogenesis of essential hypertension? Hypertension 1985;7:
607-627
4. Saito K, Sano H, Furuta Y, Fukuzaki H: Effect of oral calcium
on blood pressure response in salt-loaded borderline hypertensive patients. Hypertension 1989;13:219-226
5. Lau K, Chen S, Eby B: Evidence for the role of PO4 deficiency
in antihypertensive action of high-Ca diet. Am J Physiol
1984;246:H324-H331
6. Furspan PB, Rinaldi GJ, Hoffiman K, Bohr DF: Dietary
calcium and cell membrane abnormality in genetic hypertension. Hypertension 1989;13:727-730
7. Capuccio FP, Nirmala DM, Singer DRJ, Smith SJ, Shore AC,
MacGregor GA: Does oral calcium supplementation lower
blood pressure? A double-blind study. / Hypertens 1987;5:
67-71
8. Luft FC, Ganten U, Meyer D, Steinberg H, Gless KH, Unger
Th, Ganten D: Effect of high calcium diet on magnesium,
catecholamines, and blood pressure of stroke-prone spontaneously hypertensive rats. Proc Soc Exp Bid Med 1988;187:
474-481
9. Bohr DF, Webb RC: Vascular smooth muscle function and its
changes in hypertension. Am J Med 1984;77:3-16
10. Stern N, Lee DBN, Silis V, Beck FWJ, Deftos L, Manolagas
SC, Sowers J: Effects of high calcium intake on blood pressure
and calcium metabolism in young SHR. Hypertension 1984;6:
639-646
11. Porsti I, Wuorela H, Arvola P, Mfimmi P, Nurmi A-K,
Koistinaho J, Laippala P, Vapaatalo H: Effects of calcium
supplementation and deoxycorticosterone on plasma atrial
natriurelic peptide and electrolyte excretion in spontaneously
hypertensive rats. Ada Physiol Scand 1991;141343-350
12. Bukoski RD, McCarron DA: Altered aortic reactivity and
lowered blood pressure associated with high calcium intake.
Am J Physiol 1986;251:H976-H983
13. Pdrsti I, Arvola P, Wuorela H, Ilkka M, SaynSvalammi P,
Huhtala H, Metsa-Ketela T, Vapaatalo H: Effects of a high
calcium diet and deoxycorticosterone on vascular smooth
muscle responses in spontaneously hypertensive rats. J Hypertens 1990-^:835-841
14. Tsien RY, Pozzan T, Rink TJ: Calcium homeostasis in intact
lymphocytes: Cytoplasmic free calcium monitored with a new,
intracellularry trapped fluorescent indicator. J Ceil Siol 1982;
94:325-334
15. Aprigliano O, Hermsmeyer K: In vitro denervation of the
portal vein and caudal artery of the rat / Pharmacol Exp Ther
1976;198:568-577
92
Hypertension
Vol 19, No 1 January 1992
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
16. Ferrell F, Dreith AZ: Calcium appetite, blood pressure and
electrolytes in spontaneously hypertensive rats. Physiol Behav
1986^7:337-343
17. Resnick LM, Laragh JH, Sealey JE, Alderman MH: Divalent
cations in essential hypertension: Relations between serum
ionized calcium, magnesium and plasma renin activity. N Engl
J Med 19S3;3O9:888-&9\
18. McCarron DA, Yung NN, Ugoretz BA, Krutzik S: Disturbances of calcium metabolism in the spontaneously hypertensive rat. Hypertension 1981;3(suppl I):I-162—1-167
19. Kageyama Y, Suzuki H, Arima K, Saruta T: Oral calcium
treatment lowers blood pressure in renovascular hypertensive
rats by suppressing the renin-angiotensin system. Hypertension
1987;lft375-382
20. Murvany MI: Changes in sodium pump activity and vascular
contraction. J Hypertens 1985;3:429-436
21. Blaustein MP: Sodium ions, calcium ions, blood pressure
regulation, and hypertension: A reassessment and a hypothesis. Am J Physiol 1977;232:C165-C173
22. Vanhoutte PM, Lorenz RR: Na+,K+-ATPase inhibitors and
the adrenergic neuroeffector interaction in the blood vessel
wall. / Cardiovasc Pharmacol 1984;6(suppl I):S88-S94
23. Webb RC, Bohr DF: Potassium relaxation of vascular smooth
muscle from spontaneously hypertensive rats. Blood Vessels
1979;16:71-79
24. Bonaccorsi A, Hermsmeyer K, Aprigliano O, Smith CB, Bohr
DF: Mechanisms of potassium relaxation of arterial muscle.
Blood Vessels 1977;14:261-276
25. Johns A, Leijten P, Yamamoto H, Hwang K, van Breemen C:
Calcium regulation in vascular smooth muscle contractility.
Am J Cardiol 1987;59:18A-23A
26. Overbeck HW, Derifield RS, Pamnani MB, S6zen T: Attenuated vasodilator responses to K+ in essential hypertensive
men. / Clin Invest 1974^3:678-686
27. J dicks LA, Gupta K: NMR measurement of cytosolic free
calcium, free magnesium and intracellular sodium in the aorta
of the normal and spontaneously hypertensive rat. J Biol Chem
1990;265:1394-1400
28. Sugiyama T, Yoshizumi M, Kurihara H, Komuro I, Takaku F,
Yazaki Y: Cytoplasmic calcium ion elevating factor(s) in
spontaneously hypertensive rat serum. J Hypertens 1990,8:
919-925
29. Erne P, Bolli P, Burgisser E, Buhler FR: Correlation of
platelet calcium with blood pressure. N Engl J Med 1984^10:
1084-1088
30. Dominiczak AF, Lyall F, Morton JJ, Dargie HJ, Boyle IT,
Tune TT, Murray G, Scmple PF: Blood pressure, left ventricular mass and intracellular calcium in primary hyperparathyroidism. Clin Sci 1990;78:127-132
31. Shiftman FH, Bose R: A role of calcium in altered sodium ion
transport of hypertensives? Life Sci 1988;42:1573-1581
32. Hamryn JM, Ringel R, Schaeffer J, Levinson PD, Hamilton
BP, Kowarski AA, Blaustein MP: A circulating inhibitor of
(Na++K+)ATPase associated with essential hypertension.
Natuie 1982^300:650-652
33. Magargal WW, Overbeck HW: Effect of hypertensive rat
plasma on ion transport of cultured vascular smooth muscle.
Am J Physiol 1986;251:H984-H990
34. Doris PA: Digoxin-like immunoreactive factor in rat plasma:
Effect of sodium and calcium intake. Life Sci 1988;42:783-790
35. Haddy FJ: The role of humoral Na+,K+-ATPase inhibitor in
regulating precapillary vessel tone. / Cardiovasc Pharmacol
1984;6(suppl H):S439-S456
KEY WORDS • blood pressure • calcium • potassium
vascular smooth muscle • spontaneously hypertensive rats
High calcium diet augments vascular potassium relaxation in hypertensive rats.
I Pörsti, P Arvola, H Wuorela and H Vapaatalo
Hypertension. 1992;19:85-92
doi: 10.1161/01.HYP.19.1.85
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1992 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://hyper.ahajournals.org/content/19/1/85
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/