Calcium Activation of Vascular Smooth Muscle
State of the Art Lecture
CORNELIS VAN BREEMEN, PAUL LEIJTEN, HIROMICHI YAMAMOTO, PHILIP AARONSON,
AND CYNTHIA CAUVIN
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SUMMARY Tension development in arterial smooth muscle is regulated by variations of calcium
concentration in the submicromolar range. The receptor for Ca 2+ is calmodulin, which through
stimulation of myosin light chain kinase can activate sequentially two apparently different contractile
states. A third possible contractile state may be related to C-kinase activation. These contractile states
are thought to have different Ca2+ sensitivities. Ca 2+ is supplied from two major sources: the
sarcoplasmic reticulum and the extracellular space. The release of sarcoplasmic reticulum Ca 2+ is
mediated by the intracellular messenger inositol-l,4,5-trisphosphate (IP3) and perhaps by Ca 2+ itself.
These two messengers have the potential for amplification; for example, IP 3 may release some Ca 2+
that may subsequently cause Ca2+-induced Ca 2+ release. The entry of Ca2+ from the extracellular
space into the cytoplasm is mediated by a Ca2+ leak and by excitable Ca 2+ channels and is modulated
by a Ca2+ buffer barrier consisting of the superficial sarcoplasmic reticulum. Two types of adenosine
5'-triphosphate-driven Ca 2+ pumps in the sarcoplasmic reticulum and plasmalemma are responsible
for returning the cytoplasmic Ca 2+ concentration to resting level after contraction and for maintaining Ca2+ homeostasis during the life of the cells. (Hypertension 8 [Suppl II]: II-89-II-95, 1986)
2+
KEY WORDS • actomyosin interactions • sarcoplasmic reticular Ca
release •
receptor-operated Ca 2+ channels • voltage-sensitive Ca 2+ channels
C
ALCIUM ions perform a central role in the
activation of most cells, including vascular
smooth muscle.1 This regulatory role can be
divided into two parts: activation of the enzymes that
initiate cross-bridge cycling between the myosin and
actin filaments, and Ca 2+ transport across cellular
membranes, which determines the fluctuating calcium
concentration in the cytoplasm ([Ca2*],). Nearly all
physiological, as well as pharmacological, control
over smooth muscle activity is exerted at the membrane level, which is the main subject of this paper,
after a brief section on myofilament activation.
transmit the tension developed by actomyosin crossbridge cycling to the cell periphery and also interconnect the very narrow sarcomere-like contractile units,
as do the Z lines in skeletal muscle. 2 Although the
basic sliding filament mechanism of contraction is
similar to that of skeletal and cardiac muscle, smooth
muscle myofilaments have a unique and still incompletely understood mechanism of activation (see
Kamm and Stull3 for a recent review). Sobieszek's 4
discovery that phosphorylation of smooth muscle
myosin is associated with an increase in actin-activated
Mg 2+ -adenosine triphosphatase (ATPase) activity has
led to the now widely accepted hypothesis that Ca 2+ calmodulin activation of myosin light chain kinase is
the initial step in myofilament activation.5 Murphy and
co-workers6 expanded this hypothesis by postulating
two states of actomyosin activation, characterized by
rapidly cycling phosphorylated cross bridges and
slowly cycling Ca2+-activated cross bridges. On the
basis of these theories and their own data on the effects
of smooth muscle phosphatase, Hoar et al.7 recently
presented the two-state Ca2+ activation model for
smooth muscle contraction illustrated in Figure 1.
Ca 2+ -calmodulin-activated myosin light chain kinase
catalyzes phosphorylation of the 20,000-dalton myo-
Myofilament Activation
The contractile apparatus of smooth muscle consists
of regular arrays of myosin and actin filaments, the
latter being attached to dense bodies. The dense bodies
From the Department of Pharmacology, University of Miami
School of Medicine, Miami, Florida.
Supported by National Institutes of Health Grants HL3O412 and
HL29467 and by the Florida Affiliate of the American Heart
Association.
Address for reprints: Dr. Cornelis van Breemen, Department of
Pharmacology, University of Miami School of Medicine, Miami,
FL 33101.
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CaH • HLCK
A
2
-Ca *
•Ca 2 *
Ca-CaM'HLCK
(Relaxation)
H
M-P
I
-C. 2 *
)f •Ca 2 *
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(Contraction)
II
Phoaphataa*
Ca'CaM-HLCK
-A
•A
(A-M-P)
(Contraction)
I
FIGURE 1. Two-state Ca2+ activation model for smooth muscle contraction. CaM = calmodulin; MICK myosin light chain kinase; M = myosinfilament; M-P = phosphorylatedmyosinfilament; A = actin; n = number
ofCa2* ions. (Reprinted from Hoar et al.7 with permission.)
sin light chain initiating the contractile state associated
with rapid shortening. Subsequent dephosphorylation
catalyzed by smooth muscle phosphatase in the presence of Ca 2+ yields the second contractile state, which
is presumably characterized by slower cross-bridge
turnover and maintained tension. Removal of Ca 2+
then returns the actomyosin filaments to their original
resting state. Still unanswered, however, are many
important questions regarding the exact Ca 2+ sensitivity of the two contractile states in intact cells and how
this sensitivity may be regulated by cyclic nucleotides
and protein kinase C activation. 18
Ca 2+ Delivery During Activation
Two membrane systems function in the control and
rapid fluctuations of cytoplasmic Ca2+ concentrations:
the sarcolemma and the sarcoplasmicreticulum(SR).
In addition, Ca 2+ -binding molecules in the cytoplasm
exert Ca 2+ buffering action, which has not yet been
well quantified.
Both the plasmalemma and the SR function in Ca 2+
delivery and Ca 2+ removal. The former process is energetically downhill through leak and excitable channels, whereas the latter is uphill through adenosine 5'triphosphate (ATP)-dependent pumps and, perhaps to
a smaller extent, the Na + -Ca 2 + exchange carrier.
Since these subjects have been extensively reviewed
in the past, 9 "" this discussion will be limited to recent
developments in the field, with a bias toward work
from our laboratory.
Intracellular Ca 2+ Release
Ca release from intracellular organelles has been
measured in a number of ways: contraction12 13 and
enhanced Quin 2 fluorescence14 in the absence of extracellular Ca 2+ , electron probe x-ray microanalysis of
subcellular total Ca 2+ concentrations, 15 ' 16 transient
stimulation of 45Ca efflux from intact or skinned13-"
smooth muscle cells, and contraction and Ca2+ activity
increases measured with a calcium electrode in
skinned arterial smooth muscle.18 Although a minor
component of Ca 2+ release may be derived from the
inner plasmalemmal surface, 19 ' M there seems to be little doubt that the main component originates from the
SR. Somlyo and co-workers 13 ' l6 have demonstrated
that the number of "hot spots" containing high concentrations of Ca2+ ( > 10—12 mmol per kilogram of dry
weight), as measured by the electron probe and coinciding with the location of both superficial and deep
SR, decreased upon administration of maximally effective doses of norepinephrine (NE) in guinea pig
mesenteric veins and rabbit pulmonary artery. The
measured values for SR Ca 2+ content were 14.3 mmol
per kilogram of dry weight in the central SR of relaxed
pulmonary artery and 28 mmol per kilogram of dry
weight in the junctional SR of guinea pig portal
veins.15-l6 However, the real concentrations were estimated to be three times higher, since the probe analyzes a volume greater than that of the SR. The calculated release was 17 to 23 mmol per kilogram of dry
weight, or about 4 mmol per liter of SR. We measured
caffeine-induced 45Ca release of 74 /tmol per kilogram
2+
CA2+-ACTIVATED VASCULAR MUSCLE/van Breemen et al.
n-91
of wet weight in the rabbit aorta, which yields a remarkably similar value for caffeine-releasable SR
Ca2+ (5.3 mmol per liter of SR). 13 The observations
that caffeine releases Ca 2+ from isolated SR vesicles
and that in the rabbit aorta the caffeine- and NE-releasable Ca 2+ fractions are identical corroborate the
hypothesis that the SR is the main intracellular storehouse of releasable Ca 2+ .
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Mechanism of Ca J+ Release
Although some early reports claimed that agonists
were able to release Ca 2+ directly from subcellular
organelles, these claims have been refuted by subsequent experiments. It is quite clear that the primary
vascular neuromuscular transmitter NE loses its ability
to release intracellular Ca 2+ upon saponin skinning of
arterial smooth muscle cells. When the fibers are
skinned with 50 fig of saponin per milliliter for 20
minutes and incubated in a mildly buffered (0.1 mM
ethylene glycol bis03-aminoethyl eiher)-N fl JJ'fl'tetraacetic acid) cytoplasm replacement solution, it is
possible to maintain the SR in a functional state while
permeabilizing the plasmalemma. Figure 2 illustrates
that the SR of saponin-skinned rabbit mesenteric artery
can be loaded with Ca2+ and that caffeine releases this
Ca 2+ . In addition, it shows that [Ca 2+ ] exceeding
2 x 10" 6 M causes release of SR Ca 2+ (Ca 2+ -induced
Ca2+ release) and that this release is enhanced by
10 fiM of cyclic adenosine 3',5'-monophosphate
(cAMP). The latter result is not due to a direct effect of
cAMP on the myofilaments, since it has also been
shown for 45Ca release from the SR.17 That cAMPmediated potentiation of SR Ca 2+ release also occurs
in the intact tissue can be concluded from the observation that propranolol inhibits contractions in a Ca 2+ free medium."
This role of cAMP in enhancing the initial phase of
NE contraction is rapidly overshadowed by its stimulatory effect on Ca 2+ uptake, which inhibits the tonic
contractile phase.
Although the evidence for Ca 2+ -induced Ca 2+ release is clear, it does not explain a number of other
observations. For example, during high-K + -induced
contraction, the cytoplasmic [Ca 2+ ], is certainly elevated, but the SR accumulates Ca 2+ instead of releasing
it.13 However, it is now becoming questionable from
studies using intracellular [Ca 2+ ], indicators whether
cytoplasmic [Ca2+]j during depolarization would reach
levels high enough to cause Ca 2+ -induced Ca 2+ release.21 We have some evidence suggesting that NE, in
addition to releasing SR Ca 2+ , may release Ca 2+ bound
to the inner plasmaJemmal surface. This could result in
transient increases in the [Ca 2+ ] near the superficial SR
that would be large enough to cause Ca 2+ -induced
Ca2+ release. Thus, this process might result in only an
initial discharge of SR Ca2+ during agonist activation.
Another process proposed for intracellular Ca 2+ release involves the formation of inositol-1,4,5-trisphosphate (IP3) and its subsequent release of SR Ca 2 + . In
considering the evidence for its role as an intracellular
Cat
±
1 mtn
p
cAMP
1.0
tt
CO
CD
£ 0.5
CO
OP
co
O
6
pCa
FIGURE 2. Effect of cAMP on Ca2*-induced Ca2* release
from the sarcoplasmic reticulum (SR). The upper graph shows
the experimental procedure. The saponin-skinned mesenteric
artery was loaded with 10~6 M Ca2+ for 3 minutes and then
exposed to various concentrations ofCa (pCa) with or without
10'5 M cAMP for 1 minute. The amount ofCa2+ remaining in
the SR was estimated by the contraction induced by 25 mM
caffeine (Caf) after washing with relaxing solution containing
0.5 mM EGTA (G). The lower graph shows Ca2+ remaining in
the SR as a function ofpCA. The preparation was loaded with
10'6 M Ca2*, with (solid circles) or without (open circles)
10~s M cAMP. Points are means ± SEM (a = 5).
messenger in vascular smooth muscle, this chain of
events may apply22:
A + R—»A-R—»A-R - N protein—•activation
of PLC in
phosphoinositide breakdown:
PI
PIP
PLC
IP,
PLC
PIP2
D G ^ y | PLC
IP3 -•Ca, release
where A = agonist, R = receptor, N protein =
guanosine 5'-triphosphate-binding protein, PLC
phospholipase C, PI = phosphatidyl inositol, PIP
phosphatidyl inositol phosphate, and DG
diacylglycerol.
a
=
=
=
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1985 BLOOD PRESSURE COUNCIL
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Evidence from studies of smooth muscle indicates
that agonist activation of its membrane receptor raises
IP3 and the intracellular [Ca 2+ ] over similar time
courses and that IP3 does release Ca 2+ from intact SR
of skinned smooth muscle. All these events appear to
occur at least as fast as the rate of tension increases.
Alexander et al.23 have shown that IP, IP 2 , and IP3
increase upon the addition of angiotensin to cultured
rat aortic cells. Moreover, a transient increase in free
cytosolic [Ca 2+ ] has been observed upon administration of angiotensin.24 We have found that IP3 releases
Ca2+ from the SR in primary cultures of skinned aortic
smooth muscle cells (Figure 3). M Within 10 seconds,
200 /Ltmol of Ca 2+ is released per liter of cells, which is
more than sufficient to explain the tension development seen in aortae incubated and activated by NE in
Ca2+-free medium. Studies by Suematsu and Somlyo
and their colleagues 182 * have also demonstrated IP3induced contractions in skinned arterial fibers in Ca 2+ free medium. Our evidence raises an interesting question about SR heterogeneity with regard to Ca 2+
release, since IP3 releases a much larger Ca 2+ store
than does a maximum dose of caffeine. It is possible
that caffeine releases one SR fraction and that IP3 releases that and an additional fraction.
Cellular Ca 2+ Cycle
As described above, one continuous application of
NE or caffeine in the absence of Ca 2+ influx is sufficient to deplete the releasable SR Ca 2 + , suggesting that
it is lost to the outside and under physiological conditions is again replenished from the extracellular space.
Bond et al., 13 however, argued that if the muscle is
o
1.0
o
-
0.5
c
o
u
a)
o
2
3
Time
4
10
( mln )
FIGURE 3. Time course of
inositol-J,4,5-trisphosphate
(IP})-induccd Ca release from cultures of skinned aortic
smooth muscle cells. The skinned cells were preloaded with
45
Ca labeled in 1 x lO'6 M free Ca for 20 minutes and then
exposed to 1 x 10~5 M IP3 (solid circles) in the same solution
for various time periods (abscissa). In the control experiment
(open circles) the solution was changed to the same Ca solution
except for IP}. The skinned cells preloaded with I x 10'6 M
free Ca in the absence ofMg ATP were also treated in the same
manner (open squares, control; solid squares, I x JO'5 M
IP3). Ca content at time 0 means Ca content after Ca loading for
20 minutes.
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1986
exposed to NE for a much shorter period, repeated
contractions can be obtained, suggesting that Ca 2+
movements between the SR and cytoplasm are similar
to those thought to occur in skeletal muscle. We have
specifically tested this possibility by measuring both
45
Ca efflux and force development in the absence of
extracellular Ca2 + during diminishing periods of exposure to NE. Figure 4 shows that regardless of how short
the NE exposure is, the release of Ca 2+ into the extracellular space always accompanies the contraction.
This parallel activation of Ca2+ extrusion and contraction in smooth muscle may be due to the fact that both
processes are stimulated by the Ca 2+ -calmodulin complex. In any case, the Ca 2+ that is released from the SR
to the extracellular space must be restored through
Ca2+ entry. Our data do not exclude the possibility that
a fraction of the released activating Ca 2+ may also be
directly pumped back into the SR during relaxation.
Ca 2+ Entry
In 1979 two separate types of excitable Ca 2+ channels were postulated 9 ' v — namely, those activated by
receptor occupation by agonists (receptor-operated
channels), and those activated by a change in membrane potential (potential-sensitive channels). In addition, our Ca 2+ influx measurements indicate a considerable Ca 2+ influx in the absence of excitation, which
is termed the Ca2* leak. All three of these Ca 2+ entry
mechanisms contribute to contractile activity, but they
differ widely in their sensitivity to the inhibitory effects of Ca 2+ antagonists. The separate identity of the
two activation pathways (i.e., receptor-operated and
potential-sensitive channels) is further confirmed by
the fact that their 43Ca fluxes are additive.28 The 45Ca
influxes stimulated by various agonists, on the other
hand, are not additive and do not show appreciable
differentiated sensitivity to Ca 2+ antagonists in the
same tissue.29 These observations suggest that they
activate Ca 2+ entry through a common mechanism.
Recent studies using the whole-cell voltage clamp
suggest that smooth muscle may be heterogenous with
respect to populations of voltage-sensitive Ca 2+ channels. For example, rat aortic clonal cells30 and azygous
vein and mesenteric artery cells31-32 exhibit two types
of Ca2+ current. In addition to a rapidly inactivating
current evoked over a wide range of potentials, a sustained current is elicited with strong depolarizations.
The pattern appears to be similar to that reported by
Bean33 in canine atrial cells. Channels with similar
characteristics have also been reported in sensory neurons of the chick dorsal root ganglion34 and have been
called T (transient) and L (long-lasting). However,
Aaronson et al.33 did not observe a sustained current in
the rabbit ear artery. Instead, they found that depolarization induces a Ca 2+ current that is inactivated with
biexponential kinetics. Inactivation of both components is enhanced by depolarization. Therefore, functional differences in blood vessels may be associated
with varying populations of Ca 2+ channel subtypes.
Few studies have attempted to identify receptoroperated Ca 2+ channels using the voltage clamp technique. Benham and Bolton and colleagues 3637 have
CA 2 +-ACTIVATED VASCULAR MUSCLE/van Breemen et al.
•—•
•—•
segments of submucosal arterioles from the guinea pig
small intestine. These excitatory junction currents
(produced by the sympathetic neurotransmitter, presumably NE) are not voltage-sensitive. The reversal
potential for these currents is near 0 mV, suggesting
that, like the acetylcholine receptor-operated channels
in jejunal cells, these channels are nonselective cation
channels. Further studies using the whole-cell voltage
clamp and patch clamp are needed to confirm the existence of receptor-operated channels in vascular smooth
muscle. From these early studies, however, it appears
likely that such receptor-operated channels are not
Ca2+ channels in the strict sense but nonselective cation channels.
45
Ca Efflux
T«nston
20
1
2
3
4
DURATION (mhO of
5
15
NE APPLICATION
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FIGURE 4. The effect of decreasing periods of exposure to
norepinephrine (NE) on force and stimulated 45Ca efflux. After
JO minutes in Ca2 + -free solution, rabbit mesenteric artery rings
were exposed to NE (3 x 10~5 M) for various periods of time
(from 5 to 900 seconds), and the area under the tension transient and stimulated 45Ca efflux were calculated.
reported that acetylcholine induces an inward current
in voltage-clamped single smooth muscle cells of the
rabbit jejunum. The channels responsible for this current do show voltage dependence, but they appear to
be nonselective with respect to ion conductance (i.e.,
the current appears to be carried by a mixture of ions,
including K + , Na + , and/or Ca 2+ ). Hence, these receptor-operated channels appear to be nonselective cation
channels.
Finkel et al.3* have studied currents elicited by perivascular nerve stimulation of voltage-clamped short
Sarcoplasmic Reticulum Buffering of
Entering Ca 2+
The first indication that the SR could act as a "buffer
barrier" for incoming Ca 2+ was the observation that
tension development resulting from the net entry of
Ca2+ was variable, depending upon the rate of net
uptake.39 This observation suggested that a Ca 2+ accumulating system could compete with the myofilaments
for the Ca2+ entering the cells. A similar conclusion
was based on the reported correlation between Ca 2+
influx and tension development stimulated by Bay
K8644 in the rabbit aorta.40 A low rate of stimulated
Ca2+ entry by Bay K8644 does not produce tension but
does cause net Ca 2+ gain in the caffeine-releasable
Ca2+ store.40 Thus, it appears that Ca 2+ is diverted into the SR before it can bind to calmodulin associated
with the myofilaments. This effect may be responsible
for the threshold phenomenon of the tension-influx
curve (Figure 5). Only if the rate of Ca 2+ entry exceeds
NE + DSOO
/
K + 0000
KE + La
o-1
15
48
11-93
30
Ca Influx (pnot*«/kg/n*i)
FIGURE 5. Relationship between tonic tension development (ordinate) and45Ca influx (abscissa) during stimulationby6 x I0'5 M norepinephrine (NE) and 80 mM KCl, in the presence ofCa2+ entry blockers. Rings of rabbit
aorta were exposed to high K+ or NE levels in the absence and presence of various concentrations of D600 or
lanthanum. The maintained contractions were measured at 5 minutes. At this time, parallel aortic rings from the
same animals were exposed for 90 seconds to 45Ca, to measure Ca influx.28 Open circles = D600 (10~'-W~6
M) + 80 mM KCl; asterisks = D600 (W-'-W-6 M) + NE; solid circles = La3* (3 X 10-} to 3 X 10~3
M) + NE. Vertical dashed line represents basal 45Ca influx through intrinsic Co2 + "leak."
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1985 BLOOD PRESSURE COUNCIL
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a certain value does some of it bypass the Ca2+ pump
of the SR to cause contractile activation. Since NE
depletes this Ca2+ store (see above), it would be expected to affect the relationship between tonic tension
and Ca2+ influx. Initially, the NE-induced tension development depends to a large extent on Ca2+ release;
however, the tonic contractile phase is dependent on
Ca2+ entry. As shown in Figure 5, the threshold for
Ca2+ influx required to maintain the NE-induced contraction is reduced to a third of that required for highK+ activation of tension development.
We have also recently observed a rightward shift of
the threshold for tension development to higher Ca2+
influx values with agents that increase cAMP and stimulate SR Ca2+ uptake (K. Hwang and C. van Breemen,
unpublished data). Although the above data could also
be explained by the hypothesis that various agonists
are able to alter myofilament sensitivity to Ca2+,21 indirect evidence for a buffer function of the superficial SR
has also been obtained in the absence of agonist
stimulation.41
We thus propose that superficial SR takes up Ca2+
that has just entered the cells and thereby controls the
Ca2+ that is added to the cytoplasm. It thus acts as a
superficial buffer barrier, regulating tonic tension and
perhaps protecting small cells, with high surface to
volume ratios, from relatively high Ca2+ leaks. Maintenance of the barrier function of superficial SR requires that it discharge excessive Ca2+ loads toward
the extracellular space. One suggestion of how this
may be accomplished is presented in Figure 6.
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HEMB.
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FIGURE 6. A hypothetical scheme explaining the buffer barrier function of the superficial sarcoplasmic reticulum (SR).
Ca2* pumps of the superficial SR are shown to accumulate
Ca2*, which has just entered the cell through either the leak or
stimulated Ca2* channels (upper diagram). However, iftheSR
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Hypertension. 1986;8:II89
doi: 10.1161/01.HYP.8.6_Pt_2.II89
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