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Vascular changes during calcium loading in experimental hypertension
55
Pharmacological Research Communications, Vol. 20, Supplement Ill, 1988
VASCULAR CHANGES DURING CALCIUM LOADING IN EXPERIMENTAL HYPERTENSION R.D. Bukoski, H. Xue and D.A. Mc Carron Division of Nephrology and Hypertension, Departments of Medicine and Physiology, Oregon Health Sciences University Portland, Oregon 97201 (U.S.A.)
Calcium loading (CaL) has been shown to lower blood pressure in the spontaneously hypertensive rat (SHR).
To determine whether CaL acts by
altering vascular function, diets with 1 or 2% Ca 2+ were fed to SHRs. Aortic responsiveness to KCI and membrane-associated reactivity were attenuated in the animals receiving the diet containing 2% Ca 2+. the effect of CaL on smaller vessels, mesenteric arteries from S ~ s
To assess and
normotensive Wistar Kyo~o (WKY) rats fed 0.5, 1 or 2% calclum were studied. Maximal contractile responses to KCI and norepinephrine (NE) as well &s membrane-associated reactivity were a~tenuated in mesenterlc arteries of SHRs on the I% Ca 2+ diets, indicating a nonlinear effect of Ca 2+ in the diet. Similar responses were observed in the WKY.
It has been proposed that
CaL acts via changes in 1,25 dlhydroxyvitamln D 3 (I,25D) or ionized Ca 2+. No acute affec~ of 1,25D on contractile responses of isolated mesenterlc arteries of the SFd% or WKY were obse~ed.
Chronic administration of 1,25D
to cultured aortic myocytes had no effect on 45Ca influx whereas altered ionized Ca 2+ in the growth medium did.
We conclude ~hat CaL alters arterial
muscle function and extracellular Ca 2+ may play a regulatory role in cell Ca 2+ metabolism.
0031-6989/88/201110057-16/$03.00/0
© 1968 The Italian PharmacologicalSociety
Pharmaco~g~alResearchCommunication~VoL 20, Supp~mentH/,1988
56 INTRODUCTION There l s a
growing body of evidence which indicates that oral calcium intake
lowers blood pressure in select humans with essential hypertension (Bellzan et el, 1983, Johnson etal, Hofman, 1986, Luft e t a l ,
1985, HcCarron and Morris, 1986, Grobbee and
1986) and in several models of experimental
hypertension (Ayachi 1979, Bukoski and HcCarron, 1986, Kageyama and Bravo, 1986, Peuler et el, 1986, Omatsu et el, 1986).
The mechanism of action of
calcium loading, however, is currently not well understood.
Postulated
pathways whereby oral calcium loading lowers blood pressure include induction of natrluresls with subsequent volume contraction (Ayaehi et el., 1979) a depressor effect on the autonomic nervous system (Hatton et el., 1986), a depletion of serum phosphate with consequent depression of general cardiovascular function (Lau et el., 1984) and an action on vascular smooth muscle whereby vascular reactivity is depressed, resulting in reduced peripheral resistance and consequently lower blood pressure (Bukoski and McCarron, 1986).
An earlier report from this laboratory indicates that increased calcium intake in the spontaneously hypertensive rat (SHR) is associated with altered aortic function and is consistent with an action of oral calcium on vascular smooth muscle (Bukoski and McCarron, 1986).
Among the proposed
mediators of the action of calcium loading on vascular smooth function are several known regulators or predictors of whole animal calcium homeostasis, 1,25 dihydroxyvitamin D3, parathyroid hormone, calcitonln and serum ionized calcium concentration (Resnick etal,
1986).
The present communication provides evidence that calcium intake alters reactivity of small mesenteric artery segments in both the SHR and its normotensive control, the Wistar Kyoto rat (WAY).
In addition, the results
PharmacQiogicalResea~h Communicalion&VoL2~ Supplementlll, 1988
57
o f initial experiments examining the effect o f 1,25 dihydroxyvitamin D3 and ionized extracellular calcium on vascular smooth muscle function and cellular calcium metabolism are presented.
MATERIALS and METHODS Male spontaneously hypertensive rats or normotensive Wista~ Kyoto rats were obtained from Charles River breeding farms at 6 weeks o f age and maintained
in an animal colony room with fixed llght/dark cycles and constant temperature and humidity.
The rats from which aortas were isolated were
maintained on diets containing either 1 or 2% Ca 2+ in a basic formulation that contained 0.45 Na +, 0.61% K +, 0.05% Mg 2+, 0.41% PO43°,
30% protein as
casein and no fiber. Mesenterlc arteries were isolated from ra~s fed diets with varied Ca 2+ content (0.5, i or 2%) as well as 0.45% Na +, 1% K +, 0.05% Mg 2+, 0.78%, PO43", 20% casein and 5% fiber.
Systolic blood pressure was
determined at fixed time intervals using t h e indirect pneumatic tail cuff
method (Narco-Bio, Houston).
For the experiments involving aortic segments, the animals were sacrificed by decapitation at an average age of either 14 or 21 weeks and the thoracic aorta was removed, cleaned of fat and connective tissue and mounted in an isolated organ chamber between a fixed support and a Grass FT.03 force o
transducer.
The artery was equilibrated for 90 minutes at 37 C in
physiologic salt solution (PSS) of the following composition in mM: NaCI, 130; KCI, 4.7; NaHCO 3, 14.9; MgSO4-TH2 O, 1.17; KH2P04,1.18; CaCl 2, 2.0. 02/5% CO 2.
dextrose, 5.0;
The PSS was maintained at pH 7.4 by constant gassing with 95% All protocols were carried out with the aortic segments
stretched to two times their initial length.
When mesenteric segments were
studied, they were isolated and prepared as described above with the exception that the PSS contained 1.25 mM CaCI 2 and each vessel was set to
Pharmaco~g~alResea~hCommunicationcVo~2~SupplementfiL1988
58
its optimal length for force development by construction of a length tension curve after mounting it in the chamber.
Concentration response relationships were determined by the cumulative addition of agonist as described by van Rossum and van den Brink, 1963. Mean effective concentrations of the agonlsts (EDso values) were calculated by problt analysis of plots of the percent of maximal response to an agonist against the log of the concentration of the agonlst.
Membrane associated
reactivity was defined as the response of the arterial segments to addition of 3mM Ca 2+ after incubation in Ca2+-free, K+-free PSS with or without 2 mM EGTA.
All responses are reported as either percent of maximal contractile
response to KCI, or absolute force normalized to cross sectional area of the vessel calculated as described previously (Bukoski and McCarron, 1986).
Primary cultures of aortic smooth muscle cells were prepared as follows. Male WKY or SHR rats, 12 weeks of age, were sacrificed by decapitation and the thoracic aorta was removed and placed in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with penicillin, neomycin sulfate and streptomycin.
Connective tissue was removed by blunt dissection,
the
endothelial layer removed by gently scraping the Intimal side of the vessel wlch a sterile razor blade and the advential layer peeled away using dissecting forceps.
The remaining medial layer was minced into small pieces
and digested overnight in DMEM containing O.1% collagenase (Cooper Biomedical).
The dispersed cells were then washed free of collagenase and
plated in 35 mm culture dishes in DMEM containing antibiotics and supplemented with 10% fetal calf serum (Gibco).
After the cells reached
confluency (approximately 2 weeks), they were either immediately used for determination of 45Ca influx or the growth medium was changed to a serumfree formulation containing insulin and transferrln.
In some experiments,
Pharmaco~gmalResearchCommunication~VoL 2~ Supp~mentlll, 1988
59
the serum-free medium was supplemented wlth i ng/ml 1,25 dlhydroxyvltamln 03 or the Ca 2+ concentration was modified to either 1.0, 1.25 or 1.50 mmoles/llter.
In all cases, the cells were kept in serum-free medium an
additional 7 days before measurement of ~Sca influx.
~bca influx was determined in cells that were prelncubated for 15 minutes at 37" with HEPES-buffered salt solution (pH 7.4) of the following composition in mM: NaCI, 140;, MgSO4-7H2 O, 1.19; KCI, 5.8; CaCI2, 1.25, HEPES, 5.0. Influx of 45Ca was initiated by addition of the isotope (1 ~Ci/ml) and the label was subsequently removed f£om the excraoellular compartment by sequential washing of the plates with ice cold Ca2+-free HEPES-buffered salt solution containing 2 mM EGTA over a 5 minute period.
Cells were ~hen
digested in 1 ml 1 N NaOH, an aliquot neutralized with HCI and radioactivity in it quantitated using liquid scintillation counting. Protein content was determined in another aliquot using the Bradford method (Sigma).
Results are expressed as nmol Ca2+/mg proteln/5 minutes.
RESULTS AND DISCUSSION Aortic responsiveness to KCI was examined in tissue isolated from SHRs maintained on diets containing 1% or 2% calcium for periods of either 8 or 15 weeks. Although there was a trend for a greater degree of maximal force
development in aortas from animals on the diet with 2% calcium, no significant differences were detected (Figure I).
There was however,
a
differential aging response of the animals maintained on the two diets in terms of sensitivity to KCI (Bukoskl and McCarron,
1986).
For example,
it
was observed that there was a significant decrease in the sensitivity to KCI of the vessels isolated from the rats on the diet with 2% calcium while no change between 8 and 15 weeks on the diets was observed in aortas from the animals maintained on the diet containing 1% calcium (13.6 ± 0.6 mM vs
Pha/macoiogical Research Communications, VoL 20. Supplement III, 1988
60
4.0.
3.0.
o x
~" 2.0 E z
1.0,
8 weeks 1% calcium
15 weeks ~
2 % calcium
Figure I: Effect of dietary calelum content on the maximal contractile response of isolated aortic segments to KCI. "8 or 15 weeks" indicates weeks on the diets. Values are mean ± s,e.m, and n = 8 in each group. No significant differences were detected. (From Bukoski and Mc Carron 1986, with permission).
17.1 ± 1.2 mM for SHRs on 2% Ca 2+, p < 0.05 and 13.8 ± 0.3 mM vs 14.8 ± 0.6 mM for SHRs on 1% Ca2+).
Hembrane-assoclated reactivity of the aortas, as assessed by examining the response to a challenge with 3 mM Ca 2+ after incubation in Ca2+-free, K +free PSS for i0 minutes, was found to be altered with respect to aging in the rats on the diet containing 2% Ca 2+.
As shown in Figure 2, there was no
difference in the magnitude of~the response of the aortic segments isolated from the SHRs on the diet with 1% calcium between 8 and 15 weeks on the diets, while those on the dlec with 2% calcium e x p e r i e n c e d a significant attenuation of the response over this time period.
This finding has been
Pharmaco/ogica/Research Communications, Voi, 20, Supplement///, 1988
61
100. 90.
T
80. O) to f.. 0 to
.E_
I
7060.
50,
X
E
40. 30. 20. 10. 0
,,,
--
8 weeks--
[ - - 1 1 % calcium
Figure 2:
.
- -
15 weeks--
2 % calcium
Effect of dietary calcium on the response of aortic segments to a
20 minute challenge with 3 mM Ca 2+ after a i0 minute incubation in Ca 2+free, K+-free PSS.
"8 or 15 weeks" indicates weeks on the diets.
are mean ± s.e.m, n - 8.
* indicates a significant difference in response
between 8 and 15 weeks on the diet containing 2% Ca 2+ at p < 0.05. Bukoskl and McCarron,
Values
(From
1986, with permission.)
interpreted to reflect a decre@se in the degree of membrane disruption caused by the preincubation in Ca2+-free, K+-free solution.
These resul~s
indicate that there are m~nor, but s~gnifleant effects of calcium
supplementation on aortic function in the SHR.
The demonstration of these actions of calcium loading on aortic function led to several other questions.
These included:
i) whether a greater effect of
calcium in the diet can be demonstrated on a smaller artery that is more likely to be involved in regulation of peripheral resistance;
2) whether
alterations in response to norepinephrine also occur; and 3) whether these effects of calcium loading are present in a normotenslve strain of the rat.
PharmacologicaI Research Communications, Vo/. 20, Supplement III, 1988
62
T
1.50
_
:::::::::::::::~ i~':::::::::::.': 7":,' ~,.-'o:Z~.'~,';.:~ ;'::::::::::::! -[ :;::..::::::::::: :;::::::,-':::: :t:.:'.:;' ,:::::::::::::::
1.00,
m
) l
" "-
N
;;:;..;*.,. .p.
A;:'.:::'
.:..'..'.;.;.;.;.;
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.':::::' @::::::::~ ."2"."2' ;,;:.';.V ;.;.:.;.;.;....; '.V::~:#; :"."E."~ :::::::::::::::?
0,50.
:::::::::.:..::::: .V.'.V;:; .....,..;:..
;:.'.':#:4
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-
::;:,':::;:;:;:i
-
10 weeks
~
0.5 % calcium
~
16 weeks
1% calcium ~
~
2 % calcium
Figure 3: Effect of altered calcium intake on maximal contractile response of mesenteric arteries of the SHR to KCI. "10 or 16 weeks" indicates weeks on the diets. Values are mean + s.e.m, and n = 8 in each group. * indicates a significant effect of the diet at p < 0.05.
3.0.
T.. ~ , 2.0. Q .p-
x -L-...-.
Z
1.0.
o
iiiiiiii --10
weeks--
0,5 % calcium
I--11%
--16 calcium
weeks-~ _ 2 % calcium
Figure 4: Effect of altered calcium intake on maximal contractile response o f m e s e n t e r i c arteries of the SHR to norepinephrine. "10 or 16 weeks" indicates weeks on the diets. Values are mean + s.e.m, and n = 8 in each group. * indicates a significant effect of the~die~ at p < 0.05.
Pharmaco~gicalResea~hCommunication~VoL2~ Supp~mentl/I, 1988
63
To this end, SHR. and NKY rats were placed on diets containing 0.5%, 1% or 2% calcium and mesenteric artery segments were studied after i0 or 16 weeks on the diets.
When the responsiveness of the mesenterlc arteries to KCI was
determined it was found that vessel segments isolated from animals maintained on 1% calcium for I0 weeks had significantly attenuated maximal contractile responses compared with animpls maintained on 0.5 or 2% calcium (Figure 3)~
In contrast, r@sponses of mesenterlc arteries isolated from
rats maintained on the three diets for 16 weeks were not different.
When
maximal contractile responses to norepinephrine were examined, a similar pattern of response was observed (Figure 4), i.e. the vessels from rats maintained on the diet with 1% calcium for 10 weeks showed a slgnlficantly depressed capacity for maximal force development.
It is known that KCI induces contraction in vascular smooth muscle by activation of potential-dependent Ca 2+ influx pathway s whereas noreplnephrlne acts via receptorooperated channels and release of intracellular stones of Ca 2+ (Bolton, 1979).
The observation that slm£1ar effects of the diet
were seen on the responses to these two agonists which have different mechanisms on action indicates that the site
at which calcium loading
exerts its effect is not specific to either the potential- or receptoroperated channel.
This conclusion is further supported by the results of
the protocols which examined membrane-assoclated reactivity (Figures 5 and 6).
In these experiments, mesenterlc segments were exposed to Ca2+-free,
K+-free PSS for i0 minutes, then challenged with 3 mM Ca 2+ and the response recorded.
The protocol was then repeated
Ca2+-free, K+-free medium.
with the addition of EGTA to the
The differences in the magnitude of the
responses before and after treatment with EGTA are" illustrated in Figure%5. Mesenterlc artery segments isolated from the animals maintained on the diets containing I% calcium once again show an attenuated response after l0 weeks
PharmacologicaI Research Communications. Vol. 20. Supplement II!, 1988
64
SHR 0,9 •
0.8 ° Q.7 •
~.
0,6-
Ci
x
0.50,4-
z
0.3-
0.2, 0.1 '
o 16 weeks
10 weeks
0.5 % calcium [ ' - ] 1 % calcium ~
2 % calcium
Figure 5: Effect of altered calcium intake oD the difference in the response of mesenteric segments of the SHR to a 20 minute challenge with 3 mM Ca2+ after incubation in Ca2+-free, K + free PSS + EGTA. "10 or 16 weeks" indicates weeks on the diets. Values are mean + s.e~m., n = 8. * indicates a significant difference a t p < 0.05.
WKY 0.8.
0.7. 0.6v--
I
0.5-
X
0.4Z
0,3-
L
0,20.10
_
10 weeks - ~0.5
T
- - 16 weeks
% calcium l - - - I 1 % calcium ~--~2 % calcium
Figure 6: Effect of altered c a l c i u m i n t a k e on the difference in the response of mesenteric artery segments of the WKY to a 20 minute challenge with 3 m M Ca2+-free, K+-free PSS + EGTA. V a l u e s a r e mean + s.e.m, n = 8. * indicates a Significant" difference at p < 0.05,
Pharmaco~gicalReseamh Communication~VoL 2~ Supplementlll, 1988
65
on the diets whereas no differences are p~esent after 16 weeks on the diets. For comparative purposes, the response of mesenterlc artery segments isolated from Wistar Kyoto rats are shown in Figure 6 and the pattern of their responses was found to be similar to that of the SHR.
Although the molecular events underlying the membrane-assoclated reactivity responses are not understood, it seems likely that they are different in nature from those evoked by stimulation wilt KCI or noreplnephrlne and could possibly reflect some membrane associated property, such as cell membrane permeability to monovalent cations or stabilization of the cell membrane by bound calcium.
Whatever the mechanism, the level of Ca 2+ in the diet has a
significant effect.
Furthermore, it can be concluded that the mechanisms
responsible for the diet-lnduced effects are not predicated on some distinct primary defect of the spontaneously hypertensive rat since the effect is also present in the WKY.
An additional point to emphasize is that the effect of calcium on mesenteric artery function is nonlinear with respect to the calcium content of the diet.
Possible explanations for this alteration include a specific response
to the ratio of Ca 2+ in the diet to the content of some other nutrient or potential alterations in the relative proportions of hormonal r%gulators of calcium homeostasis.
Examples of the latter are 1,25 dihydroxyvitamin D3,
parathyroid hormone, calcltonin and serum ionized Ca 2+ content. of these may have an effect on vascular smooth muscle,
Since each
(Resnlck et al,
1986), one approach of this laboratory has been to study the effects that several of these factors have on intact vessel function and vascular smooth muscle cell Ca 2+ metabolism.
It has been proposed that 1,25 dihydroxyvitamln D 3 has an ionophore-like
PharmacologicaI Research Communications, Vol. 20, Supplement III, 1988
66
WKY I Vehicle b.~.',':l 1,25 (OH)2 D3
1.0.
Control
NE
Post D3
NE
1st
2nd
challenge challenge
SHR
m --
2.0 •
;t
~ ~
Vehicle 1,25 (OH)2 D3
1.0. ~N
0
Control NE
Post 03 NE
1st
challenge
2 nd challenge
Figure 7: Effect of 1,25 dihydroxyvitamin D 3 on the reactivity of isolated mesenteric artery segments from SHR and WKY rats, 12 weeks of age. A control response to I pM NE was ellc~ted, then 1,25 dihydroxyvitamin D 3 (I ng/ml) was added for 3 hours and the challenge with NE repeated. No effects on either parameter were observed. "Ist" and "2nd challenge" indicate responses of the arteries to NE after subsequent 10 minute incubations in Ca2+-free PSS. No differences were detected. Values are mean + s.e.m., and ~,= 6 for each group,
action which can increase basal tone or reactivity in the vascular system (Resnick et el., 1986).
We have preliminarily tested this thesis by
determining the action of 1,25 dihydroxyvitamin D 5 (i ng/ml) on basal vascular tone and contractile responsiveness to l ~M norepinephrine in mesenteric artery segments isolated from SHR and WKY.rats,
12 weeks of age.
The results indicate that 1,25 dlhydroxyvltamin D3 has no acute effect (3 hours) on basal tone, on responsiveness to norepinephrine or on residual
67
Pharmacologica/ Research Communications, Vo/. 20, Supplement/11, 1988 1000
"1~ II
ol=45Ca ,rom primary cultures of aortic myocytes
Washout
,WKY oSHR
100.
,9. °
el.
E m
0
o
E
I::
10.
| a
i
,
. _ _ |
|
. i
0
5
10
15
20
Time
FiEure
(rain)
8
Ca 2+ stores within the cell, determined by giving the tissue successive challenges with noreplnephrlne after incubation in Ca2+-free medium (Figure
7),
These results cannot, however, exclude a chronic action of 1,25 dihydroxyvi~amin D 3 on vascular smooth muscle.
In an attempt Co address
this issue, primary cultures of aortic smooth muscle cells were exposed to 1,25 dlhydroxyvltamln D 5 (1 ng/ml) for 7 days, then 45Ca influ~ was measured using the technique illustrated regulating hormone on
45Ca
in
Figure 8.
No effect of this calcium-
influx was detected (Table I).
Such was not the
68
Pharmacological Research Communications, VoL 20, Supplement/I/, 1988 ~ab~e ~
:
Effect of 1,25 dihydroxyvitamin D3 and Ca ~'+ on 4sCa influx Condition
n
4SCa influx (nmol Ca/rag protein 5 min.)
Control
n = 7
6.90:1:1.50
1,25 Vit D
n = 8
7.60 :i: 1.60
1.0 m M Ca 2+
n = 6
3.03 :t: 0 . 2 3
1.25 m M C a 2+
n = 6
4.90 :t: 0 . 5 9 *
1.50 m M C a 2+
n = 8
3.54:1:0.29
45Ca influx into primary cultures of aortic smooth muscle cells exposed to I ng/ml 1,25 dihydroxyviCamin D 3 (1,25 vitamin D) or grown in medium containing varied extracellular (Ca 2+) for 7 days. Values are mean + s.e.m.; n is indicated. * denotes a significant difference at p < 0.05.
case however, when the effect of altered ionized Ca 2+ concentration in the growth medium of aortic myocytes was examined.
As shown in Table I, when
45Ca influx was determined in cells that were grown in the presence of 1.0, 1.25 or 1.50 mM extracellular Ca 2+, 45Ca influx was found to be elevated in the ceils grown in the medium containing 1.25 mM Ca 2+.
These results are
consistent with an effect of extracellular Ca 2+ concentration on cellular Ca 2+ metabolism.
The precise mechanism of action, however, can only be
speculated upon at this time, since it is not known whether 45Ca influx reflects basal membrane permeability to Ca 2+ or some balance of influx, efflux and sequestration within internal Ca 2+ storage pools.
I n conclusion
these studies have demonstrated that oral calcium loading
alters functional properties of aortic and mesenteric arterial muscle.
The
observed changes include alterations in maximal contractile ability, sensitivity to KC1 and membrane-assoclated reactivity.
The effect of oral
Pharmaco/ooicalResearchCommunication~Vo~ 2~ Supplementlll, 1988 calcium loading is not specific
to the h y p e r t e n s i v e
state
normotenslve control responded in a similar fashion.
69
as t h e
Furthermore,
our
results indicate tha= the effects o f dietary calcium are not specific =o either the potential- or receptor-operated channel.
When the effect of 1,15
dlhydroxyvltamln D 3 and extracellular ionized Ca 2+ on vascular smooth muscle were examined, only extracellular ionized Ca 2+ appeared to modify cellul.r Ca 2+ metabolism, using 45Ca influx as an index.
The mechanism by which
calcium loading alters vascular function will require further investigation and the question of whether these alterations are associated with basal or stimulated systolic and diastolic blood pressures in the intact animal remains to be delineated.
Acknowledgements:
The authors wish to thank Mike Pressley for his expert
technical assistance and Gloria Ellis for her assistance during the preparation of this manuscript.
The work was funded by a Grant from the
National Dairy Board for Promotion and Research administered by the National Dairy Council.
REFERENCES
Ayachl S.
Metabolism,
28:1234-1238,
1979.
Bellzan J.M., Villar J., Pideda 0., Gonzalez A.B., Salnz E., Garrera G., Sldrlan R.
J.Am.Med.Assoc.
249:1161-I165,
Boland A.R., Gallego S., Boland R. Bolton T.B.
Physiol.Rev.
Grobbee D.E., Hofman A.
Biochlm.Biophys.Acta.
39:606-718,
Bukoskl R.D., McCarron D.A.
1983. 733:264-273,
1979.
Am.J.Fhyslol.
251:H976-H983,
Lancet, Sept. 27:703-707,
1986.
1986.
1983.
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Hatcon D.C., Huie P.B., Muntzel H.S., Mecz J.A., McCarron D.A. A m . J . P h y s £ o l , in p r e s s . Johr~son N . F . , Smith E . L . , Freudenheim J . L .
Am,J.Clln.Nutr. 42:12-17,
1985. Kaseymna Y., Bravo E.L.
Hypertension:
Lau K., Chen S . , Eby B.
am.J.Physlol,
in press, 1987. 246:H324-H329,
1984.
Luft F.C., Aronoff G.R., Slosh R.S., Fineberg N.S., Welnberger M.H. Pharmacol.Ther. 39:414-419,
1986.
McCarron D.A., Young N.N., Ugorecz B.A., Krotzlk S. 3:I162-I167,
Hypertension
1981.
McCarron D.A., Morris C.D. Ann. Inc.Medtcine, Nichols G.A., Mecz M.A., Cllne W.H.
103:825-831,
1985.
J.Fharmacol. Expcl.Therap.
236:414-
423, 1986. Omatsu T., Sano H., Suzuki H., Motoyama T., Kawaguchi K., Salto K., Yamanlshl J., Nishimura Y., Shao-Hing H., Furulca Y., Fukuzakl H. Jap. Circ.J. 50(6):481, 1986. Peuler J.D., Morgan D.S., Mark A.L.
Clln.Res.
Resnlck L.~., Muller, F.B., Laragh J.H.
34(4):A919,
1986.
Ann. lnt.Med. 105:649-654,
1986. Van Rossum J.M., van den Brink F.G. 1963.
Arch.Inu.Pharmacodyn. 143:240-246,
Clin.
Pharmacological Research Communications, Vol. 20, Supplement Ill, 1988
VASCULAR CHANGES DURING CALCIUM LOADING IN EXPERIMENTAL HYPERTENSION R.D. Bukoski, H. Xue and D.A. Mc Carron Division of Nephrology and Hypertension, Departments of Medicine and Physiology, Oregon Health Sciences University Portland, Oregon 97201 (U.S.A.)
Calcium loading (CaL) has been shown to lower blood pressure in the spontaneously hypertensive rat (SHR).
To determine whether CaL acts by
altering vascular function, diets with 1 or 2% Ca 2+ were fed to SHRs. Aortic responsiveness to KCI and membrane-associated reactivity were attenuated in the animals receiving the diet containing 2% Ca 2+. the effect of CaL on smaller vessels, mesenteric arteries from S ~ s
To assess and
normotensive Wistar Kyo~o (WKY) rats fed 0.5, 1 or 2% calclum were studied. Maximal contractile responses to KCI and norepinephrine (NE) as well &s membrane-associated reactivity were a~tenuated in mesenterlc arteries of SHRs on the I% Ca 2+ diets, indicating a nonlinear effect of Ca 2+ in the diet. Similar responses were observed in the WKY.
It has been proposed that
CaL acts via changes in 1,25 dlhydroxyvitamln D 3 (I,25D) or ionized Ca 2+. No acute affec~ of 1,25D on contractile responses of isolated mesenterlc arteries of the SFd% or WKY were obse~ed.
Chronic administration of 1,25D
to cultured aortic myocytes had no effect on 45Ca influx whereas altered ionized Ca 2+ in the growth medium did.
We conclude ~hat CaL alters arterial
muscle function and extracellular Ca 2+ may play a regulatory role in cell Ca 2+ metabolism.
0031-6989/88/201110057-16/$03.00/0
© 1968 The Italian PharmacologicalSociety
Pharmaco~g~alResearchCommunication~VoL 20, Supp~mentH/,1988
56 INTRODUCTION There l s a
growing body of evidence which indicates that oral calcium intake
lowers blood pressure in select humans with essential hypertension (Bellzan et el, 1983, Johnson etal, Hofman, 1986, Luft e t a l ,
1985, HcCarron and Morris, 1986, Grobbee and
1986) and in several models of experimental
hypertension (Ayachi 1979, Bukoski and HcCarron, 1986, Kageyama and Bravo, 1986, Peuler et el, 1986, Omatsu et el, 1986).
The mechanism of action of
calcium loading, however, is currently not well understood.
Postulated
pathways whereby oral calcium loading lowers blood pressure include induction of natrluresls with subsequent volume contraction (Ayaehi et el., 1979) a depressor effect on the autonomic nervous system (Hatton et el., 1986), a depletion of serum phosphate with consequent depression of general cardiovascular function (Lau et el., 1984) and an action on vascular smooth muscle whereby vascular reactivity is depressed, resulting in reduced peripheral resistance and consequently lower blood pressure (Bukoski and McCarron, 1986).
An earlier report from this laboratory indicates that increased calcium intake in the spontaneously hypertensive rat (SHR) is associated with altered aortic function and is consistent with an action of oral calcium on vascular smooth muscle (Bukoski and McCarron, 1986).
Among the proposed
mediators of the action of calcium loading on vascular smooth function are several known regulators or predictors of whole animal calcium homeostasis, 1,25 dihydroxyvitamin D3, parathyroid hormone, calcitonln and serum ionized calcium concentration (Resnick etal,
1986).
The present communication provides evidence that calcium intake alters reactivity of small mesenteric artery segments in both the SHR and its normotensive control, the Wistar Kyoto rat (WAY).
In addition, the results
PharmacQiogicalResea~h Communicalion&VoL2~ Supplementlll, 1988
57
o f initial experiments examining the effect o f 1,25 dihydroxyvitamin D3 and ionized extracellular calcium on vascular smooth muscle function and cellular calcium metabolism are presented.
MATERIALS and METHODS Male spontaneously hypertensive rats or normotensive Wista~ Kyoto rats were obtained from Charles River breeding farms at 6 weeks o f age and maintained
in an animal colony room with fixed llght/dark cycles and constant temperature and humidity.
The rats from which aortas were isolated were
maintained on diets containing either 1 or 2% Ca 2+ in a basic formulation that contained 0.45 Na +, 0.61% K +, 0.05% Mg 2+, 0.41% PO43°,
30% protein as
casein and no fiber. Mesenterlc arteries were isolated from ra~s fed diets with varied Ca 2+ content (0.5, i or 2%) as well as 0.45% Na +, 1% K +, 0.05% Mg 2+, 0.78%, PO43", 20% casein and 5% fiber.
Systolic blood pressure was
determined at fixed time intervals using t h e indirect pneumatic tail cuff
method (Narco-Bio, Houston).
For the experiments involving aortic segments, the animals were sacrificed by decapitation at an average age of either 14 or 21 weeks and the thoracic aorta was removed, cleaned of fat and connective tissue and mounted in an isolated organ chamber between a fixed support and a Grass FT.03 force o
transducer.
The artery was equilibrated for 90 minutes at 37 C in
physiologic salt solution (PSS) of the following composition in mM: NaCI, 130; KCI, 4.7; NaHCO 3, 14.9; MgSO4-TH2 O, 1.17; KH2P04,1.18; CaCl 2, 2.0. 02/5% CO 2.
dextrose, 5.0;
The PSS was maintained at pH 7.4 by constant gassing with 95% All protocols were carried out with the aortic segments
stretched to two times their initial length.
When mesenteric segments were
studied, they were isolated and prepared as described above with the exception that the PSS contained 1.25 mM CaCI 2 and each vessel was set to
Pharmaco~g~alResea~hCommunicationcVo~2~SupplementfiL1988
58
its optimal length for force development by construction of a length tension curve after mounting it in the chamber.
Concentration response relationships were determined by the cumulative addition of agonist as described by van Rossum and van den Brink, 1963. Mean effective concentrations of the agonlsts (EDso values) were calculated by problt analysis of plots of the percent of maximal response to an agonist against the log of the concentration of the agonlst.
Membrane associated
reactivity was defined as the response of the arterial segments to addition of 3mM Ca 2+ after incubation in Ca2+-free, K+-free PSS with or without 2 mM EGTA.
All responses are reported as either percent of maximal contractile
response to KCI, or absolute force normalized to cross sectional area of the vessel calculated as described previously (Bukoski and McCarron, 1986).
Primary cultures of aortic smooth muscle cells were prepared as follows. Male WKY or SHR rats, 12 weeks of age, were sacrificed by decapitation and the thoracic aorta was removed and placed in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with penicillin, neomycin sulfate and streptomycin.
Connective tissue was removed by blunt dissection,
the
endothelial layer removed by gently scraping the Intimal side of the vessel wlch a sterile razor blade and the advential layer peeled away using dissecting forceps.
The remaining medial layer was minced into small pieces
and digested overnight in DMEM containing O.1% collagenase (Cooper Biomedical).
The dispersed cells were then washed free of collagenase and
plated in 35 mm culture dishes in DMEM containing antibiotics and supplemented with 10% fetal calf serum (Gibco).
After the cells reached
confluency (approximately 2 weeks), they were either immediately used for determination of 45Ca influx or the growth medium was changed to a serumfree formulation containing insulin and transferrln.
In some experiments,
Pharmaco~gmalResearchCommunication~VoL 2~ Supp~mentlll, 1988
59
the serum-free medium was supplemented wlth i ng/ml 1,25 dlhydroxyvltamln 03 or the Ca 2+ concentration was modified to either 1.0, 1.25 or 1.50 mmoles/llter.
In all cases, the cells were kept in serum-free medium an
additional 7 days before measurement of ~Sca influx.
~bca influx was determined in cells that were prelncubated for 15 minutes at 37" with HEPES-buffered salt solution (pH 7.4) of the following composition in mM: NaCI, 140;, MgSO4-7H2 O, 1.19; KCI, 5.8; CaCI2, 1.25, HEPES, 5.0. Influx of 45Ca was initiated by addition of the isotope (1 ~Ci/ml) and the label was subsequently removed f£om the excraoellular compartment by sequential washing of the plates with ice cold Ca2+-free HEPES-buffered salt solution containing 2 mM EGTA over a 5 minute period.
Cells were ~hen
digested in 1 ml 1 N NaOH, an aliquot neutralized with HCI and radioactivity in it quantitated using liquid scintillation counting. Protein content was determined in another aliquot using the Bradford method (Sigma).
Results are expressed as nmol Ca2+/mg proteln/5 minutes.
RESULTS AND DISCUSSION Aortic responsiveness to KCI was examined in tissue isolated from SHRs maintained on diets containing 1% or 2% calcium for periods of either 8 or 15 weeks. Although there was a trend for a greater degree of maximal force
development in aortas from animals on the diet with 2% calcium, no significant differences were detected (Figure I).
There was however,
a
differential aging response of the animals maintained on the two diets in terms of sensitivity to KCI (Bukoskl and McCarron,
1986).
For example,
it
was observed that there was a significant decrease in the sensitivity to KCI of the vessels isolated from the rats on the diet with 2% calcium while no change between 8 and 15 weeks on the diets was observed in aortas from the animals maintained on the diet containing 1% calcium (13.6 ± 0.6 mM vs
Pha/macoiogical Research Communications, VoL 20. Supplement III, 1988
60
4.0.
3.0.
o x
~" 2.0 E z
1.0,
8 weeks 1% calcium
15 weeks ~
2 % calcium
Figure I: Effect of dietary calelum content on the maximal contractile response of isolated aortic segments to KCI. "8 or 15 weeks" indicates weeks on the diets. Values are mean ± s,e.m, and n = 8 in each group. No significant differences were detected. (From Bukoski and Mc Carron 1986, with permission).
17.1 ± 1.2 mM for SHRs on 2% Ca 2+, p < 0.05 and 13.8 ± 0.3 mM vs 14.8 ± 0.6 mM for SHRs on 1% Ca2+).
Hembrane-assoclated reactivity of the aortas, as assessed by examining the response to a challenge with 3 mM Ca 2+ after incubation in Ca2+-free, K +free PSS for i0 minutes, was found to be altered with respect to aging in the rats on the diet containing 2% Ca 2+.
As shown in Figure 2, there was no
difference in the magnitude of~the response of the aortic segments isolated from the SHRs on the diet with 1% calcium between 8 and 15 weeks on the diets, while those on the dlec with 2% calcium e x p e r i e n c e d a significant attenuation of the response over this time period.
This finding has been
Pharmaco/ogica/Research Communications, Voi, 20, Supplement///, 1988
61
100. 90.
T
80. O) to f.. 0 to
.E_
I
7060.
50,
X
E
40. 30. 20. 10. 0
,,,
--
8 weeks--
[ - - 1 1 % calcium
Figure 2:
.
- -
15 weeks--
2 % calcium
Effect of dietary calcium on the response of aortic segments to a
20 minute challenge with 3 mM Ca 2+ after a i0 minute incubation in Ca 2+free, K+-free PSS.
"8 or 15 weeks" indicates weeks on the diets.
are mean ± s.e.m, n - 8.
* indicates a significant difference in response
between 8 and 15 weeks on the diet containing 2% Ca 2+ at p < 0.05. Bukoskl and McCarron,
Values
(From
1986, with permission.)
interpreted to reflect a decre@se in the degree of membrane disruption caused by the preincubation in Ca2+-free, K+-free solution.
These resul~s
indicate that there are m~nor, but s~gnifleant effects of calcium
supplementation on aortic function in the SHR.
The demonstration of these actions of calcium loading on aortic function led to several other questions.
These included:
i) whether a greater effect of
calcium in the diet can be demonstrated on a smaller artery that is more likely to be involved in regulation of peripheral resistance;
2) whether
alterations in response to norepinephrine also occur; and 3) whether these effects of calcium loading are present in a normotenslve strain of the rat.
PharmacologicaI Research Communications, Vo/. 20, Supplement III, 1988
62
T
1.50
_
:::::::::::::::~ i~':::::::::::.': 7":,' ~,.-'o:Z~.'~,';.:~ ;'::::::::::::! -[ :;::..::::::::::: :;::::::,-':::: :t:.:'.:;' ,:::::::::::::::
1.00,
m
) l
" "-
N
;;:;..;*.,. .p.
A;:'.:::'
.:..'..'.;.;.;.;.;
x
':'.'//q.;
.':::::' @::::::::~ ."2"."2' ;,;:.';.V ;.;.:.;.;.;....; '.V::~:#; :"."E."~ :::::::::::::::?
0,50.
:::::::::.:..::::: .V.'.V;:; .....,..;:..
;:.'.':#:4
,--.::::::'(: ~::::::::.i'.::::
"~:::::::::::
't;:44;':
..::'#.;:.. .::..=..::;
N;':?:
....:#.;.:, 't;::;#:: t::.*.'*':#
".'.:::::::::::: o
-
::;:,':::;:;:;:i
-
10 weeks
~
0.5 % calcium
~
16 weeks
1% calcium ~
~
2 % calcium
Figure 3: Effect of altered calcium intake on maximal contractile response of mesenteric arteries of the SHR to KCI. "10 or 16 weeks" indicates weeks on the diets. Values are mean + s.e.m, and n = 8 in each group. * indicates a significant effect of the diet at p < 0.05.
3.0.
T.. ~ , 2.0. Q .p-
x -L-...-.
Z
1.0.
o
iiiiiiii --10
weeks--
0,5 % calcium
I--11%
--16 calcium
weeks-~ _ 2 % calcium
Figure 4: Effect of altered calcium intake on maximal contractile response o f m e s e n t e r i c arteries of the SHR to norepinephrine. "10 or 16 weeks" indicates weeks on the diets. Values are mean + s.e.m, and n = 8 in each group. * indicates a significant effect of the~die~ at p < 0.05.
Pharmaco~gicalResea~hCommunication~VoL2~ Supp~mentl/I, 1988
63
To this end, SHR. and NKY rats were placed on diets containing 0.5%, 1% or 2% calcium and mesenteric artery segments were studied after i0 or 16 weeks on the diets.
When the responsiveness of the mesenterlc arteries to KCI was
determined it was found that vessel segments isolated from animals maintained on 1% calcium for I0 weeks had significantly attenuated maximal contractile responses compared with animpls maintained on 0.5 or 2% calcium (Figure 3)~
In contrast, r@sponses of mesenterlc arteries isolated from
rats maintained on the three diets for 16 weeks were not different.
When
maximal contractile responses to norepinephrine were examined, a similar pattern of response was observed (Figure 4), i.e. the vessels from rats maintained on the diet with 1% calcium for 10 weeks showed a slgnlficantly depressed capacity for maximal force development.
It is known that KCI induces contraction in vascular smooth muscle by activation of potential-dependent Ca 2+ influx pathway s whereas noreplnephrlne acts via receptorooperated channels and release of intracellular stones of Ca 2+ (Bolton, 1979).
The observation that slm£1ar effects of the diet
were seen on the responses to these two agonists which have different mechanisms on action indicates that the site
at which calcium loading
exerts its effect is not specific to either the potential- or receptoroperated channel.
This conclusion is further supported by the results of
the protocols which examined membrane-assoclated reactivity (Figures 5 and 6).
In these experiments, mesenterlc segments were exposed to Ca2+-free,
K+-free PSS for i0 minutes, then challenged with 3 mM Ca 2+ and the response recorded.
The protocol was then repeated
Ca2+-free, K+-free medium.
with the addition of EGTA to the
The differences in the magnitude of the
responses before and after treatment with EGTA are" illustrated in Figure%5. Mesenterlc artery segments isolated from the animals maintained on the diets containing I% calcium once again show an attenuated response after l0 weeks
PharmacologicaI Research Communications. Vol. 20. Supplement II!, 1988
64
SHR 0,9 •
0.8 ° Q.7 •
~.
0,6-
Ci
x
0.50,4-
z
0.3-
0.2, 0.1 '
o 16 weeks
10 weeks
0.5 % calcium [ ' - ] 1 % calcium ~
2 % calcium
Figure 5: Effect of altered calcium intake oD the difference in the response of mesenteric segments of the SHR to a 20 minute challenge with 3 mM Ca2+ after incubation in Ca2+-free, K + free PSS + EGTA. "10 or 16 weeks" indicates weeks on the diets. Values are mean + s.e~m., n = 8. * indicates a significant difference a t p < 0.05.
WKY 0.8.
0.7. 0.6v--
I
0.5-
X
0.4Z
0,3-
L
0,20.10
_
10 weeks - ~0.5
T
- - 16 weeks
% calcium l - - - I 1 % calcium ~--~2 % calcium
Figure 6: Effect of altered c a l c i u m i n t a k e on the difference in the response of mesenteric artery segments of the WKY to a 20 minute challenge with 3 m M Ca2+-free, K+-free PSS + EGTA. V a l u e s a r e mean + s.e.m, n = 8. * indicates a Significant" difference at p < 0.05,
Pharmaco~gicalReseamh Communication~VoL 2~ Supplementlll, 1988
65
on the diets whereas no differences are p~esent after 16 weeks on the diets. For comparative purposes, the response of mesenterlc artery segments isolated from Wistar Kyoto rats are shown in Figure 6 and the pattern of their responses was found to be similar to that of the SHR.
Although the molecular events underlying the membrane-assoclated reactivity responses are not understood, it seems likely that they are different in nature from those evoked by stimulation wilt KCI or noreplnephrlne and could possibly reflect some membrane associated property, such as cell membrane permeability to monovalent cations or stabilization of the cell membrane by bound calcium.
Whatever the mechanism, the level of Ca 2+ in the diet has a
significant effect.
Furthermore, it can be concluded that the mechanisms
responsible for the diet-lnduced effects are not predicated on some distinct primary defect of the spontaneously hypertensive rat since the effect is also present in the WKY.
An additional point to emphasize is that the effect of calcium on mesenteric artery function is nonlinear with respect to the calcium content of the diet.
Possible explanations for this alteration include a specific response
to the ratio of Ca 2+ in the diet to the content of some other nutrient or potential alterations in the relative proportions of hormonal r%gulators of calcium homeostasis.
Examples of the latter are 1,25 dihydroxyvitamin D3,
parathyroid hormone, calcltonin and serum ionized Ca 2+ content. of these may have an effect on vascular smooth muscle,
Since each
(Resnlck et al,
1986), one approach of this laboratory has been to study the effects that several of these factors have on intact vessel function and vascular smooth muscle cell Ca 2+ metabolism.
It has been proposed that 1,25 dihydroxyvitamln D 3 has an ionophore-like
PharmacologicaI Research Communications, Vol. 20, Supplement III, 1988
66
WKY I Vehicle b.~.',':l 1,25 (OH)2 D3
1.0.
Control
NE
Post D3
NE
1st
2nd
challenge challenge
SHR
m --
2.0 •
;t
~ ~
Vehicle 1,25 (OH)2 D3
1.0. ~N
0
Control NE
Post 03 NE
1st
challenge
2 nd challenge
Figure 7: Effect of 1,25 dihydroxyvitamin D 3 on the reactivity of isolated mesenteric artery segments from SHR and WKY rats, 12 weeks of age. A control response to I pM NE was ellc~ted, then 1,25 dihydroxyvitamin D 3 (I ng/ml) was added for 3 hours and the challenge with NE repeated. No effects on either parameter were observed. "Ist" and "2nd challenge" indicate responses of the arteries to NE after subsequent 10 minute incubations in Ca2+-free PSS. No differences were detected. Values are mean + s.e.m., and ~,= 6 for each group,
action which can increase basal tone or reactivity in the vascular system (Resnick et el., 1986).
We have preliminarily tested this thesis by
determining the action of 1,25 dihydroxyvitamin D 5 (i ng/ml) on basal vascular tone and contractile responsiveness to l ~M norepinephrine in mesenteric artery segments isolated from SHR and WKY.rats,
12 weeks of age.
The results indicate that 1,25 dlhydroxyvltamin D3 has no acute effect (3 hours) on basal tone, on responsiveness to norepinephrine or on residual
67
Pharmacologica/ Research Communications, Vo/. 20, Supplement/11, 1988 1000
"1~ II
ol=45Ca ,rom primary cultures of aortic myocytes
Washout
,WKY oSHR
100.
,9. °
el.
E m
0
o
E
I::
10.
| a
i
,
. _ _ |
|
. i
0
5
10
15
20
Time
FiEure
(rain)
8
Ca 2+ stores within the cell, determined by giving the tissue successive challenges with noreplnephrlne after incubation in Ca2+-free medium (Figure
7),
These results cannot, however, exclude a chronic action of 1,25 dihydroxyvi~amin D 3 on vascular smooth muscle.
In an attempt Co address
this issue, primary cultures of aortic smooth muscle cells were exposed to 1,25 dlhydroxyvltamln D 5 (1 ng/ml) for 7 days, then 45Ca influ~ was measured using the technique illustrated regulating hormone on
45Ca
in
Figure 8.
No effect of this calcium-
influx was detected (Table I).
Such was not the
68
Pharmacological Research Communications, VoL 20, Supplement/I/, 1988 ~ab~e ~
:
Effect of 1,25 dihydroxyvitamin D3 and Ca ~'+ on 4sCa influx Condition
n
4SCa influx (nmol Ca/rag protein 5 min.)
Control
n = 7
6.90:1:1.50
1,25 Vit D
n = 8
7.60 :i: 1.60
1.0 m M Ca 2+
n = 6
3.03 :t: 0 . 2 3
1.25 m M C a 2+
n = 6
4.90 :t: 0 . 5 9 *
1.50 m M C a 2+
n = 8
3.54:1:0.29
45Ca influx into primary cultures of aortic smooth muscle cells exposed to I ng/ml 1,25 dihydroxyviCamin D 3 (1,25 vitamin D) or grown in medium containing varied extracellular (Ca 2+) for 7 days. Values are mean + s.e.m.; n is indicated. * denotes a significant difference at p < 0.05.
case however, when the effect of altered ionized Ca 2+ concentration in the growth medium of aortic myocytes was examined.
As shown in Table I, when
45Ca influx was determined in cells that were grown in the presence of 1.0, 1.25 or 1.50 mM extracellular Ca 2+, 45Ca influx was found to be elevated in the ceils grown in the medium containing 1.25 mM Ca 2+.
These results are
consistent with an effect of extracellular Ca 2+ concentration on cellular Ca 2+ metabolism.
The precise mechanism of action, however, can only be
speculated upon at this time, since it is not known whether 45Ca influx reflects basal membrane permeability to Ca 2+ or some balance of influx, efflux and sequestration within internal Ca 2+ storage pools.
I n conclusion
these studies have demonstrated that oral calcium loading
alters functional properties of aortic and mesenteric arterial muscle.
The
observed changes include alterations in maximal contractile ability, sensitivity to KC1 and membrane-assoclated reactivity.
The effect of oral
Pharmaco/ooicalResearchCommunication~Vo~ 2~ Supplementlll, 1988 calcium loading is not specific
to the h y p e r t e n s i v e
state
normotenslve control responded in a similar fashion.
69
as t h e
Furthermore,
our
results indicate tha= the effects o f dietary calcium are not specific =o either the potential- or receptor-operated channel.
When the effect of 1,15
dlhydroxyvltamln D 3 and extracellular ionized Ca 2+ on vascular smooth muscle were examined, only extracellular ionized Ca 2+ appeared to modify cellul.r Ca 2+ metabolism, using 45Ca influx as an index.
The mechanism by which
calcium loading alters vascular function will require further investigation and the question of whether these alterations are associated with basal or stimulated systolic and diastolic blood pressures in the intact animal remains to be delineated.
Acknowledgements:
The authors wish to thank Mike Pressley for his expert
technical assistance and Gloria Ellis for her assistance during the preparation of this manuscript.
The work was funded by a Grant from the
National Dairy Board for Promotion and Research administered by the National Dairy Council.
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