Angiotensin Increases Cytosolic Free Calcium
in Cultured Vascular Smooth Muscle Cells
TOMMY A.
BROCK, R. WAYNE ALEXANDER, LAURIE S. EKSTEIN,
WILLIAM J. ATKINSON, AND MICHAEL A. GIMBRONE, JR.
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
SUMMARY We used the calcium-sensitive fluorescent indicator quin 2 to monitor changes in
cytosolic free calcium concentration ([Ca2+],) associated with angiotensin II receptor activation in
cultured vascular smooth muscle cells isolated from rat aorta. Resting [Ca2+], in unstimulated vascular smooth muscle cells was 198 ± 7 nM. Angiotensin II induced concentration-dependent rapid
increases in [Ca2+], (threshold =*1()-" M; effective concentration, 50% =<5 x lO"10 M; maximum
=10~' M); the rate of increase in [Ca 2+ ], also appeared to be concentration dependent. The angiotensin Il-induced changes were completely blocked by the angiotensin II receptor antagonist [Sar1, Ile8]angiotensin II. In the presence of extracellular calcium, 10"' M angiotensin II induced an increase in
[Ca2+], that reached peak values offiveto six times the resting levels within 15 seconds, followed by a
gradual decline to a plateau at two to three times the resting level. When EGTA was added to chelate
external calcium, the angiotensin Il-induced increases in peak [Ca2+], were attenuated and the plateau
phase was abolished. These data show that (l).quin 2 can be used in cultured vascular smooth muscle
cells to study changes in calcium homeostasis induced by angiotensin II, and (2) angiotensin II acts on
cultured vascular smooth muscle cells to cause a rapid increase in [Ca 2+ ], that appears to depend on
both the mobilization of intracellular calcium and the influx of extracellular calcium.
(Hypertension 7 [Suppl I]: I-105-I-109, 1985)
• intracellular calcium
• cell culture
KEY WORDS
aorta
• fluorescent calcium indicator
T
HE importance of the renin-angiotensin system
in the maintenance of vascular tone is widely
recognized.' The principal effector peptide of
this system, angiotensin II (ANG II), initiates a sequence of biochemical events in vascular smooth muscle cells (VSMC) that result in contraction and increased peripheral resistance. Direct characterization
of ANG II binding sites in a wide variety of vascular
tissues has substantially added to our understanding of
the molecular interactions between this peptide and its
receptor.2 In contrast, there is little information regarding the mechanisms by which ANG II increases the
cytosolic free calcium concentration ([Ca2"1"],), a major
determinant of VSMC contractility.3
Direct measurements of [Ca 2+ ], in VSMC of intact
blood vessels have been technically difficult.4's Using
• quin 2 • rat
the bioluminescent calcium photoprotein aequorin,
Morgan and Morgan* have demonstrated in the isolated Amphiuma aorta that a single concentration of ANG
II induces a rapid, transient increase in light signal.
The introduction of this relatively large molecule
(==20,000 daltons) into living cells requires either microinjection or membrane permeabilization procedures. Recently, an alternative approach using quin 2,
a fluorescent calcium-sensitive tetracarboxylate dye,
has been developed to monitor stimulus-coupled
changes in [Ca 2+ ]| in several types of mammalian
cells.6"10 Cells can be efficiently and nondisruptively
loaded with this indicator by means of its lipophilic,
acetoxymethyl ester derivative (quin 2/AM), which
becomes hydrolyzed intracellularly to yield the impermeant free acid.
To facilitate the study of biochemical events associated with ANG II receptor activation in vascular
smooth muscle, we have developed an in vitro model
system utilizing cultured rat aortic VSMC. These cultured VSMC retain functional ANG II receptors"-13
and can be readily propagated in vitro to provide sufficient numbers of intact cells for biochemical analyses.
In the present study, we used quin 2 to monitor ANG
Il-induced changes in [Ca 2+ ], in cultured rat aortic
From the Vascular Pathophysiology Laboratory, Department of
Pathology and Cardiovascular Division, Department of Medicine,
Bngham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Supported by Grant HL-20054 from the National Institutes of
Health
Address for reprints Dr Tommy A. Brock, Department of Pathology, Bngham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115
1-105
1-106
1984 BLOOD PRESSURE COUNCIL
VSMC Our data indicate that ANG II causes a concentration-dependent increase in [Ca 2+ ], by stimulating the rapid mobilization of sequestered stores of
intracellular calcium, as well as the influx of extracellular calcium.
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Methods
Cell Culture
The VSMC were isolated from rat thoracic aorta by
enzymatic dissociation using techniques similar to
those described previously from this laboratory.16 In
brief, aortas were removed aseptically from eight male
Sprague-Dawley rats (200-250 g), cleaned of adherent
connective tissue, and cut into small rings The tissue
was incubated in 12 ml of Eagle's Minimum Essential
Medium (MEM, M A. Bioproducts, Walkersville,
MD) with 0.2 mM CaCl2, 1 mg/ml of collagenase
(CLS type I; Worthington Biochemical Corp., Freehold, NJ), 0.375 mg/ml of soybean trypsin inhibitor
(Worthington Biochemical Corp.), 0.125 mg/ml of
elastase (Type III; Sigma Chemical Co., St. Louis,
MO), and 2.0 mg/ml of bovine serum albumin (Pentex; Miles Laboratories, Inc., Elkhart, IN) for 90 minutes at 37°C in a gyratory water bath. The partially
digested tissue fragments then were collected with a
coarse stainless steel mesh, resuspended in 20 ml of
MEM, titrated 10 times through a 14-g stainless steel
cannula, and sieved through a 100-/xm mesh to separate the dispersed cells from undigested tissue fragments. The resulting cell suspension was centrifuged
(200 X j , 5 minutes), resuspended in 20 ml of Dulbecco's Modified Eagle's Medium (DME) supplemented
with 10% calf serum and antibiotics (M.A. Bioproducts), and seeded into two 100-mm culture dishes.
Typically, these primary isolates had a 40 to 60%
plating efficiency and grew to form confluent monolayers within 7 to 10 days. Thereafter, cells were harvested twice a week with trypsin-versene and passaged
at a 1:4 ratio in 75-cm2 culture flasks (Coming, Elmira,
NY) For experiments, cells at passage levels 4 to 25
were rephcate-plated into 100-mm culture dishes (2 X
104 cells/cm2), refed every other day, and used after 4
to 6 days.
Measurement of Quin 2 Fluorescence
To harvest cells for experimental use, eight replicate-plated 100-mm cultures were incubated for 20
minutes at 37°C in Hanks Balanced Salt Solution
(HBSS, 0.5 mM CaCl2:1.0 mM MgCl2) with 0 1
mg/ml of collagenase, 0.1 mg/ml of soybean trypsin
inhibitor, and 0.3 mg/ml of bovine serum albumin
(BSA). Cell suspensions were collected by gentle titration through a wide-bore pipette followed by centrifugation. The total yield was approximately 4.5 to 6.0 X
107 cells. The cells were resuspended in HBSS (5 x
10*/ml) and incubated with 50 (iM quin 2/AM (Lancaster Synthesis, Morecambe, Lancaster, UK) for 20
minutes at 37°C. The quin 2-loaded cells were then
washed twice by centrifugation, resuspended in a
modified balanced salt solution (BSS; 130 mM NaCl,
5 mM KC1, 1.5 mM CaCl2, 1.0 mM MgCl2, 10 mM
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HYPERTENSION, VOL
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1985
glucose, 20 mM HEPES, buffered to pH 7.4 with Tris
base) containing 1 mg/ml of BSA, divided into 1-ml
aliquots (4 x 10* cells/ml), aerated with 95% O 2 :5%
CO2, and kept at room temperature for up to 2 hours.
Immediately before use, each cell suspension was centrifuged in a Beckman microfuge (model B, Palo Alto,
CA), resuspended in 2 ml of BSS, and placed in a
disposable plastic cuvette (Evergreen Scientific Co.,
Los Angeles, CA). Measurements of fluorescence (excitation, 339 nm; slit, 4 nm; emission, 492 nm; slit, 10
nm) were made at 37°C in a Perkin-Elmer MPF 44A
Spectrofluonmeter (Norwalk, CT) equipped with a
thermostated cuvette holder, stirring apparatus, and
chart recorder.
There was a slow, spontaneous leakage of quin 2
while the samples were kept at room temperature
(measured as an increase in supernatant quin 2 fluorescence); however, there appeared to be very little quin 2
carryover when the samples were washed and resuspended for fluorescence measurements. Multiple
washes did not significantly reduce resting [Ca 2 + ] r
The calibration of quin 2 fluorescence and calculation
of cytosolic free calcium concentration were determined as described by Tsien et al. 6 Maximum fluorescence intensity was obtained by permeabilizing the
cells with 50 ju,M digitonin, which exposed the dye to
the calcium concentration of the assay buffer. Minimum fluorescence intensity was obtained by chelating
calcium with 2 mM EGTA and enough Tris base to
increase the pH above 8 3. When the cell suspensions
had been preincubated with EGTA, 5 mM CaCl2 was
added to obtain maximum fluorescence. The intracellular concentration of quin 2 was measured by monitoring the cellular uptake of [3H]-quin 2/AM (Amersham/Searle, Chicago, IL) under conditions similar to
those described above.
Measurement of Na+ and K+ Contents and Cell Volume
Intracellular Na + and K + contents of control and
quin 2-loaded suspensions of VSMC were determined
by quickly washing the cells five times with isotonic,
ice-cold 0.1 M MgCl2. Electrolytes were extracted
with 0.1 N HNO3 and quantitated by atomic absorption
spectroscopy (Perkin Elmer 5000). The equilibrium
distribution of 3-0-[methyl-MC]-D-glucose was used to
measure cellular water space.12 The cell volume of
cultured VSMC was about 5.5 /u.L/mg of protein. Protein was determined by the method of Lowry et al 17
and standardized to BSA (Biorad Laboratories, Richmond, CA).
Results
Effect of Quin 2 on Cell Viability
Quin 2-loaded cells consistently maintained greater
than 95% viability by trypan blue exclusion for up to 2
hours. In addition, as illustrated in Figure 1, quin 2loaded cells did not exhibit significantly altered Na +
and K + contents. In two experiments using [3H]-quin
2, the intracellular concentration of quin 2 was 1.2 ±
0.2 and 1.8 ± 0.3 mM (n = 6 samples).
1-107
ANGIOTENSIN-INDUCED CHANGES IN CYTOSOLIC FREE CALCIUM/Broc* et al.
100--
10-' 0
It
M Anu-ll
^
^
—
—
•
—
_ _ _ _ _ _ ^
-
420
220
50 •
i
i
i
i
i
i
i
8
10"*
-
| 100 6 irtn
30 mln
6 0 min
+
FIGURE 1. Effect of quin 2 on cell electrolyte content. Na
and K+ contents were measured at various time intervals after
the standard quin 2 loading procedure Initial Na+ and K+
contents of unloaded cells were 25 ± 4 and 134 ± 10 mM
respectively Bars represent mean values for six determinations
in two separate experiments
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Angiotensin I I Effects on [Ca 2+ ], in the Presence of
Extracellular Calcium
When VSMC were suspended in buffer containing
1.5 mM Ca 2+ , the resting cytosolic free Ca 2+ level was
198 ± 7 nM (mean ± SEM, 51 determinations). Figure
2 depicts representative tracings of the effects of two
different ANG II concentrations on [Ca 2 + ] r In this
particular experiment, 10" l0 M ANG II caused a transient increase in [Ca 2+ ], after a brief, initial lag period
(Figure 2, upper trace). The [Ca 2+ ], reached a maximum value (about twofold) within 1 to 2 minutes and
subsequently returned to prestimulus levels within 8
minutes. In contrast, 10" 8 M ANG II induced a rapid
increase in [Ca 2+ ], that reached peak values of five to
six times the resting level within 15 seconds, followed
by a gradual decline to a new plateau at two to three
times the prestimulus level (Figure 1, lower trace).
Figure 3 summarizes the maximal increases in
[Ca 2+ ], induced by various ANG II concentrations in
several experiments. Threshold and half-maximal increases in cytosolic free Ca 2+ occurred at 10"" M and
5 x 10"10 M respectively. The quin 2 fluorescence
response was nearly saturated ([Ca 2+ ], > 1 juM) at
maximal doses of ANG II (1O" 8 -1O- 7 M). Therefore,
calculated changes in [Ca 2+ ], at higher ANG II concentrations may represent an underestimation. Although
not routinely quantitated in these experiments, the rate
of increase in [Ca 2+ ], also appeared to be related to
ANG II concentration (see Figure 2). Pretreatment of
suspensions of VSMC with the ANG II antagonist
[Sar\ Ile8]-ANG II completely blocked ANG H-induced changes in [Ca 2+ ], (data not shown). When
[Sar1, Ile8]-ANG II was added 3 to 5 minutes after the
addition of agonist, however, there was no immediate
effect on the shape of the calcium transient.
M AnflHI
1
-1100
J
- 160
50 -
0 1
2
TIME
3
4
5
6
7
8
(minutes)
FIGURE 2. Changes m [Ca2+], induced by ANG 11 in the presence of extracellular calcium. Top. Typical quin 2 fluorescence in response to 10~'° M ANG II. Bottom. Typical quin 2
fluorescence in response to 10~8 M ANG 11. Calculated values
for [Ca2+}l are indicated at the right.
11 10
9
8
7
ANGIOTENSIN II (-log M)
FIGURE 3 Concentration-response curve for ANG ll-induced
changes in [Ca2*],. Data points (mean ± SEM, 3-5 experiments) represent the peak change in calculated [Ca2*],, expressed as percentage of control (prestimulus level), induced by
different doses of ANG II. Control [Ca2*], averaged 173 ±
10 nM.
Angiotensin I I Effects of [Ca 2+ ], in the Absence of
Extracellular Calcium
Exposure to EGTA (2 mM) for 5 minutes produced a
small decrease in resting [Ca 2+ ],. In the presence of
EGTA, however, ANG II still caused a twofold increase in [Ca 2+ ],, although the peak [Ca 2+ ], was attenuated. In addition, the sustained plateau phase was abolished and [Ca 2+ ], returned quickly to prestimulus
levels. Qualitatively similar results were obtained in
zero-calcium buffer (no added EGTA or calcium; data
not shown).
To determine whether cultured VSMC contained an
intracellular pool of calcium that could be mobilized
by ANG II, these experiments were repeated in the
presence of EGTA to chelate external Ca 2+ (Figure 4).
Discussion
In the current study, we have developed methods
using the calcium-sensitive fluorescent dye quin 2 to
1-108
1984 BLOOD PRESSURE COUNCIL
100
640
I
140
o
,100
EQTA 10-* M
I I,
210
50
100
0
1 2
3 4
TIME (minutes)
5
6
7
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FIGURE 4. Effect of EGTA on ANG II-induced changes in
[Cat+Ji. Top Control response to 10~9M ANG II Bottom.
Responsiveness of ANG II after chelatton of extracellular calcium with EGTA EGTA (2 mM) was added 5 minutes before
ANG II.
monitor ANG II-induced changes in [Ca 2+ ], in cultured
rat aortic VSMC. Quin 2 has been successfully employed to monitor stimulus-coupled changes in [Ca 2+ ],
in a number of non-smooth muscle cell types.6"'0 Viability of cultured VSMC was not affected by the relatively high concentrations of quin 2 that were required
to obtain measurable calcium signals. The resting level
of [Ca 2+ ], in suspensions of cultured VSMC was about
200 nM (range, 100-300 nM); these values are well
within the range reported for other cell types with quin
2 6-io Previous reports in which aequorin was used to
monitor cytosolic ionized calcium in intact blood vessels did not provide values for resting [Ca 2 + ],. 4 5
Angiotensin II increased [Ca 2+ ],, as well as the rate
of increase in [Ca 2+ ], in a concentration-dependent
manner (see Figures 2-4). These changes were totally
blocked by the ANG II receptor antagonist [Sar1, Ile8]ANG II. The ANG II concentrations that caused
threshold and half-maximal increases in [Ca 2+ ], were
about 10"" M and 5 X 10"'° M, respectively. These
concentrations are comparable to those that stimulate
45
Ca2+ efflux in surface-attached monolayer cultures of
rat aortic VSMC (unpublished observations, 1984),
which indicates that quin 2 did not significantly alter
the ANG II responsiveness of these suspensions of
VSMC. Angiotensin II (10~8 M) induced a rapid increase in [Ca 2+ ], that reached a peak value in less than
15 seconds followed by a gradual decline to a plateau
phase two to three times above resting levels. In contrast, the time required to reach peak [Ca 2+ ], in response to 10"10 M ANG II was two to three minutes,
and [Ca 2+ ], subsequently returned to prestimulus levels
(see Figure 2). These data are consistent with the concentration and time dependency of ANG II contractile
effects on isolated vascular tissue1 and ANG IIinduced phosphorylation of the myosin light chain, a
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HYPERTENSION, VOL.
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MAY-JUNE
1985
(Ca2+-calmodulin)-dependent process, in cultured rat
mesenteric artery VSMC. 18 It should be noted, however, that extremely rapid [Ca 2+ ], transients may be
slowed and/or blunted because of the buffering properties of quin 2. 5
Our observations showing two distinct phases of the
[Ca 2+ ], transient induced by relatively high concentrations of ANG II are in agreement with a previous report
by Morgan and Morgan,4 who demonstrated similar
ANG II (2 x 10" 7 M) effects in Amphiuma aorta. The
decay of ANG II-induced quin 2 fluorescence most
likely reflects the action of cellular homeostatic
mechanisms that normally function to maintain low,
resting levels of ionized Ca 2+ . In vascular smooth
muscle, these may include (1) binding or sequestration
by intracellular organelles, such as mitochondria, sarcoplasmic reticulum, or plasma membrane3; (2) activation of a Mg 2+ -dependent, (Ca2+-calmodulin)-activated ATPase located in the plasma membrane19; and (3)
stimulation of Na + -Ca 2+ exchange.20 We found that
EGTA abolished the plateau phase in [Ca 2+ ], in response to ANG II, which suggests that extracellular
Ca2+ influx was increased during this phase. These
data are consistent with data obtained in intact aortic
segments that demonstrate that the slower, tonic phase
of ANG II-induced contraction is dependent on extracellular Ca 2+ . 21
Angiotensin II caused a significant elevation of
[Ca 2+ ], in the absence of external Ca 2+ (see Figure 4).
Previous studies utilizing isolated segments of rabbit
aorta have shown that ANG II causes a rapid, phasic
increase in tension that is independent of extracellular
calcium. 2122 In addition, kinetic analyses of 45Ca2+
efflux in intact vascular segments,23 as well as our unpublished observations (1984) with cultured rat aortic
VSMC, indicate that ANG II causes an increase in
cellular 45Ca2+ efflux in the presence of EGTA. Thus,
our observations indicate that cultured VSMC contain
sequestered pools of intracellular calcium that are sensitive to ANG II receptor activation. As mentioned
previously, mitochondria, sarcoplasmic reticulum,
and the plasma membrane are potential sites for intracellular calcium storage and release; however, recent
studies in VSMC24 and other non-smooth muscle
cells25-** suggest that mitochondria may not sequester
or release calcium at physiologically low resting levels
of ionized calcium (10~7 M). It has been postulated
that the endoplasmic reticulum is the major site of
intracellular calcium release.27"30
The mechanism by which ANG II causes the mobilization of intracellular calcium is not known. In the
case of several other Ca 2+ mobilizing hormones, it has
been shown that rapid hydrolysis of a membrane inositol phospholipid (phosphatidyl inositol-4,5-bisphosphate) by a specific phospholipase C is an early event
in receptor activation.31 One product of this reaction,
inositol-1,4,5-trisphosphate (IP3), can stimulate the release of calcium from a nonmitochondrial, intracellular compartment, presumably the endoplasmic reticulum, in saponin-treated VSMC from porcine coronary
ANGI0TENSIN-1NDUCED CHANGES IN CYTOSOLIC FREE CALCIUM/BrocA: et al.
artery.30 Creba et al.32 have demonstrated with hepatocytes that ANG II stimulates the hydrolysis of phosphatidyl inositol-4,5-bisphosphate and production of
IP3. We have also observed that ANG II causes the
rapid production of IP3 in cultured rat aortic
VSMC.33 Further studies are necessary to establish the
exact location of this ANG H-sensitive intracellular
calcium pool in VSMC, as well as to clarify the role of
inositol phospholipid degradation products in intracellular calcium mobilization. The experimental model
system described in this report should facilitate further
study of the biochemical events associated with ANG
II receptor activation in vascular smooth muscle.
Acknowledgments
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We thank Dr. Robert Powers for his valuable advice in setting up
the quin 2 assay. We also thank Crystal DeVance for her assistance
in preparing this manuscript.
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Angiotensin increases cytosolic free calcium in cultured vascular smooth muscle cells.
T A Brock, R W Alexander, L S Ekstein, W J Atkinson and M A Gimbrone, Jr
Hypertension. 1985;7:I105
doi: 10.1161/01.HYP.7.3_Pt_2.I105
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