692
[Ca ], Not Diacylglycerol, Is the Primary
Regulator of Sustained Swine Arterial Smooth
Muscle Contraction
Christopher M. Rembold and Barbara A. Weaver
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Sustained smooth muscle contraction has been proposed to be regulated by either 1) sustained
increases in intracellular Ca2+ concentration ([Ca 2+ ],)-dependent myosin phosphorylation or
2) diacylglycerol-dependent protein klnase C activation. We measured diacylglycerol mass with
the diacylglycerol kinase assay and myoplasmic [Ca2+] with aequorin in swine carotid medial
smooth muscle. Sustained and significant increases in [Ca 2+ ], myosin light chain phosphorylation, and isometric stress were observed with histamine or endothelin stimulation. Neither
stimuli, however, induced significant increases in diacylglycerol mass. Relaxation of hlstaminestimulated tissues was induced by removal of histamine or removal of extracellular CaCl2 in the
continued presence of histamine. The rate of decline of both [Ca2+] and force was similar in
both protocols, suggesting that removal of Ca2+ (without removing the stimulus) was equivalent
to removal of the stimulus. These data suggest that [Ca 2+ ], is the primary regulator of sustained
swine arterial smooth muscle contraction, whereas diacylglycerol has, at most, only a minor
role. {Hypertension 1990;15:692-698)
C
alcium-dependent myosin light chain phosphorylation appears to be the primary regulator of smooth muscle moysin's ATPase
activity,1 cross-bridge cycling rates [unloaded shortening velocity (Vo)2~4], and the rate of stress
development.5 Sustained contractile agonist stimulation, however, is frequently associated with maintenance of stress at peak values despite a decrease to
lower but suprabasal values of [Ca2+],4 phosphorylation,6 VQ,7 and energy consumption.8 There are several mechanisms proposed for the regulation of
sustained stress in smooth muscle.
Hai and Murphy9 proposed that both phosphorylated and dephosphorylated cross bridges can be
attached to the thin filament and produce force.
Attached, dephosphorylated cross bridges (which
were termed latch bridges) are posited to be formed
by dephosphorylation of attached, phosphorylated
cross bridges. Latch bridges are proposed to maintain
isometric force like a phosphorylated cross bridge but
have a slow detachment rate. Once a latch bridge
detaches, the lack of phosphorylation inhibits reatFrom the Division of Cardiology, Departments of Internal
Medicine and Physiology, University of Virginia Health Sciences
Center, Charlottesville, Virginia.
Supported by a grant from the Lucille P. Markey Charitable
Trust and grant RO1-HL-38918 from the National Institutes of
Health. C.M.R. is a Lucille P. Markey scholar.
Address for reprints: Christopher M. Rembold, MD, Box 146,
Division of Cardiology, University of Virginia Medical Center,
Charlottesville, VA 22908.
tachment. Isometric stress can be estimated as the
sum of attached phosphorylated cross bridges and
latch bridges.9 Small but significant elevations in Ca2+
concentration ([Ca2+])-dependent phosphorylation
are proposed to regulate steady-state stress by determining the number of phosphorylated cross bridges
that are available for dephosphorylation and resultant latch-bridge formation. At intermediate levels of
cross-bridge phosphorylation, latch bridges accumulate and maintain appreciable stress. Supporting this
hypothesis, there is a steep dependence of steadystate stress on increases in phosphorylation from
resting values of 0.07-0.30 mol inorganic phosphate
(Pi)/mol myosin light chain.1011 A model of crossbridge kinetics has quantitatively predicted high
stress at submaximal levels of [Ca2+], cross-bridge
phosphorylation, and energy consumption.9 This
model has also predicted the linear dependence of Vo
on phosphorylation.12
Other investigators have proposed that a system
not dependent on intracellular Ca2+ is necessary to
regulate sustained smooth muscle contraction. Phorbol diester stimulation (presumably by activation of
protein kinase C) produces slowly developing, sustained smooth muscle contraction.13 A number of
investigators hypothesized that agonist-dependent
activation of phospholipase C can induce formation
of diacylglycerol, thereby activating protein kinase
C.14 Protein kinase C-dependent phosphorylation
has been proposed to maintain stress by either
Rembold and Weaver Diacylglycerol and [Ca 2+ ] in Smooth Muscle
1) increasing the sensitivity of the contractile apparatus to [Ca ] 15 or 2) cross-linking cytoskeletal proteins (i.e., stress that is not cross-bridge dependent16). Supporting this hypothesis was the finding
that diacylglycerol mass increased in a smooth muscle
cell line17 and a tissue18 stimulated with contractile
agonists. Furthermore, in some laboratories, phorbol
diester-induced contractions19 and the sustained
phase of some agonist-induced contractions15'16 were
not associated with significant elevations in myoplasmic [Ca2+]. In our laboratory, however, agonist4 and
low-dose phorbol dibutyrate20 (PDB)-induced contractions are associated with small but significant
sustained increases in [Ca2+] and phosphorylation.
The goal of this study was to evaluate whether Ca2+
or diacylglycerol is the primary second messenger regulating sustained stress maintenance in smooth muscle.
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Methods
Swine common carotid arteries were obtained
from a slaughterhouse and transported at 2° C in
physiological salt solution (PSS). Dissection of
medial strips and mounting were performed as illustrated by Driska et al.6 The intimal surface was
mechanically rubbed to remove the endothelium.
PSS contained (mM) NaCl 140, KC1 5, 3[7V-morpholino]propanesulfonic acid (MOPS) 2,
CaCl21.6, MgCl21.2, Na2HPO< 1.2, and D-glucose 5.6
(pH adjusted to 7.4 at 37° C). The Ca2+-free solution
was PSS with 1 mM ethyleneglycol bisO-aminoethylethe^Af^W-tetraacetic acid (EGTA) without
added CaCl2. Agonist stimulation was performed by
injecting an appropriate volume of 10 mM stock
histamine into the tissue bath. Agonist stock solutions were prepared daily.
Aequorin (obtained from Dr. John Blinks, Mayo
Medical School, Rochester, Minnesota) was loaded
intracellularly by reversible isosmotic hyperpermiabilization.4 This procedure involves incubation of
free-floating tissues in a series of Ca2+-free solutions at
2° C. Solution 1 chelates extracellular Ca2+ with high
EGTA, solution 2 contains aequorin, solution 3 contains higher Mg2* concentration ([Mg2"1"]) that might
help to "reseal" the membrane, and solution 4 is
Ca*+-free PSS with high MgCl2. The tissues were
mounted isometrically and wanned to 22° C, extracellular CaCl2 was slowly restored, and the tissues were
equilibrated overnight at 37° C. The aequorin-loading
procedure did not affect maximal stress development
or the time course of myosin phosphorylation.4-5 21
Light measurements were made in a light-tight
enclosure that allows simultaneous measurement of
aequorin light, force, and length.21 Force was measured with a Cambridge Technology 300H servo
(Cambridge Technology, Inc., Watertown, Massachusetts) in the isometric mode, and stress was
calculated as force per cross-sectional area, which
was estimated from measured length, weight, and a
density of 1.050 g/cm3. Aequorin light signals are
presented in the form of log 'LfLmm, where L is the
photon count (in counts per second) and L ^ is an
693
estimate of total aequorin.22 Log L/Lnm, change was
calculated for each tissue by subtracting mean resting
log L/Ln,,, from each data point. Aequorin light
emission was calibrated in a series of Ca2+ per EGTA
buffers with 0.5 mM Mg2+ at 37° C.4
Myosin light chain phosphorylation in tissues frozen by immersion at -78° C was determined by the
method of Driska et al6 and Aksoy et al.3 Phosphorylation is reported as moles of P; per mole 20 kD
smooth muscle-specific myosin light chain isoform.
Diacylglycerol mass was measured by the diacylglycerol kinase assay.23 Swine carotid medial strips
were blotted and weighed before mounting for force
measurements for later normalization. After pharmacological manipulations, the tissues were frozen in
liquid nitrogen and stored at -70° C. Lipids were
extracted by homogenization on ice in 2.25 ml prechilled (-70° C) CHClj/methanol (MeOH) (1 = 2, vol/
vol) and 0.6 ml of 1 M NaCl.24 The homogenization
tube was rinsed with 0.75 ml of 1 M NaCl, and the
homogenate, rinse, and 0.75 ml CHC13 were combined, vortexed, and incubated for 30 minutes at
2° C. After centrifugation at l,400g for 5 minutes, the
CHC13 phase was aspirated, measured, and stored at
-70° C until assayed.
One-half milliliter of the stored CHC13 phase was
evaporated under N2, and to the dried lipid, 20 /xl of
7.5% octyl-/3-D-glucoside, 5 mM cardiolipin, and 1
mM diethylenetriaminepentaacetic acid (DETA
PAC) were added. The sample was sonicated for 15
seconds in a bath sonicator (Branson 2200, Shelton,
Connecticut) and incubated at room temperature for
15 minutes. Fifty microliters of 2x buffer (100 mM
imidazole HC1, 100 mM NaCl, 25 mM MgCl2, and 2
mM EGTA) was added, followed by 10 /A of 20 mM
dithiothreitol and 10 fil diacylglycerol kinase (1.0
mg/ml). The reaction was started by adding 10 ill of
10 mM adenosine triphosphate (ATP) that had 15X105 cpm/nmol y-[32P]ATP in 100 mM imidazole
and 1 mM DETAPAC (pH 6.6). The reaction continued for exactly 30 minutes at 25° C and was
stopped by the addition of 3 ml CHCl3:MeOH (1:2,
vol/vol) and 0.7 ml of 1% HC1O4. After mixing, 1 ml
CHCI3 and 1 ml of 1% HC1O4 were added and the
mixture centrifuged for 5 minutes at l,000g. The
upper aqueous phase was aspirated to radioactive
waste, and the lower phase was washed twice with 2
ml of 1% HCIO4. The volume of the lower CHC13
phase was measured, and a 0.5-ml sample was
removed and dried under N2. The sample was reconstituted in 100 pi of 5% MeOH in CHC13, and 20 /il
of this solution was spotted on a 20 cm silica series-60
thin-layer chromatographic plate that had been prerun in acetone. The plates were developed in
CHCl3:MeOH:AcOH (acetic acid) (65:15 = 5, vol/
vol/vol), air dried, and autoradiographed. The radioactive spot corresponding to known standards of
diacylglycerol was scraped into a scintillation vial and
counted. Known standards of diacylglycerol were run
through an identical procedure, including thin-layer
694
Hypertension
Vol 15, No 6, Part 2, June 1990
600
o
E
100i
Sample
BO
y - 1.33 x + 258
r2 •= 0.87
60
_6
40
400
(0
rh
rh
-h -h
20
u
No Sample
D
£
rh
200!
£i
o
15
10
y - 0.99 x +1.7
r2 - 0.99
UJ
w
o
200
400
600
DAG Added (pmol)
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
FIGURE 1. A graph depicting the validation of the diacylgfycerol mass assay. Typical standard curve for diacylglycerol
kinase assay is shown in open circles and dashed regression
line (labeled No Sample). Second standard curve in presence
of tissue extract is also shown; known amounts of diacylglycerol were added to unstimulatcd smooth muscle tissues before
homogenization, and resultant diacylglycerol mass is shown in
filled circles and solid regression line. DAG, diacylglycerol.
2.0
chromatography, to obtain a reliable standard curve
(an example is shown in Figure 1).
Extraction of diacylglycerol from tissue samples was
evaluated by adding known quantities of diacylglycerol
to frozen unstimulated tissues before homogenization
in CHClj/MeOH. Added diacylglycerol was 99%
extracted from the tissues and produced an upward
parallel shift in the standard curve (Figure 1). We
concluded that the extraction procedure was relatively
complete and no substantial diacylglycerol was lost
during the assay.
Diacylglycerol mass was normalized to tissue wet
weight, which was calculated by subtracting the
weight of the tissue ends that remained in the muscle
holders after freezing from the wet weight measured
before mounting the tissue.
Statistical analysis was performed with Student's
unpaired t test.
Results
Diacylglycerol mass in unstimulated swine carotid
media was 72±6 pmol/mg wet wt (mean±SEM,
n=26). There was no significant difference in diacylglycerol mass measured 30 minutes after histamine or
endothelin stimulation when compared with unstimulated controls (Figure 2). Considering the standard
error of the unstimulated diacylglycerol determinations, a 40% change in diacylglycerol mass would be
required for a significant change. R59022, an inhibitor of diacylglycerol kinase, was added to tissues in an
attempt to increase diacylglycerol mass.23 Administration of 10 IJM R59022 and 100 /xM histamine for
30 minutes did not significantly change diacylglycerol
mass (73±9 pmol/mg wet wt). However, R59022
inhibited 100 fiM histamine contractions (stress,
0.77±0.09xl05 N/m2) potentially because R59022
has known histamine HI antagonist activity.25
4,4,
1.51.0
0.5
0.0
OJ
1 3 10 100
[Hitamlna]
0 0.1
uM
[EndothiSn]
FIGURE 2. Bar graphs showing sustained changes in diacylglycerol mass (pmol/mg wet wt), Ca2+ concentration ([Ca2+])
(shown as log L/Lmcx change), myosin phosphorylation, and
isometric active stress on stimulation with various doses of
histamine or endothelin for 30 minutes. The diacylglycerol
mass, [Ca2+], and phosphorylation assays are each destructive, so comparison is between tissues treated with identical
protocols. Each bar contains number of tissues examined, and
stress data is from tissues processed for diacylglycerol mass;
similar values were obtained in measurements of [Ca2*] and
phosphorylation. ^Significant difference from the control (no
added agonist). Some of the phosphorylation and [Ca2+] data
have been previously published4-5 and are shown for comparison. DAG, diacylglycerol; MLC, myosin light chain; P,,
inorganic phosphate.
In contrast to the diacylglycerol data, histamine or
endothelin stimulation was associated with significant increases in aequorin-estimated myoplasmic
[Ca2+] and myosin phosphorylation levels, which correlated well with isometric stress production (Figure
2, and References 4 and 10). These data suggest that
agonist-dependent changes in diacylglycerol mass
are, at most, small and do not appear to correlate
with sustained stress as well as sustained elevations in
[Ca2+] and phosphorylation.
Staurosporine is a putative inhibitor of protein
kinase C.26 We evaluated the effect of preincubation
with 1 IJM staurosporine on contractions induced by
histamine or PDB (phorbol dibutyrate, a watersoluble activator of protein kinase C). The initial
phases of 1 or 100 /iM histamine-induced contrac-
Rembold and Weaver Diacylglycerol and [Ca2+] in Smooth Muscle
695
Histamine 1 uM
FIGURE 3.
0.12
0.07
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
0.0'
-10
10
20
30 - 1 0
0
Time (min)
10
20
tions were not affected by staurosporine preincubation;
however, the sustained contraction was partially inhibited (Figure 3). Sustained stress (measured 30 minutes
after stimulation) was decreased 44 ±4% by staurosporine in the group administered 100 fjM histamine and
46 ±16% in the group administered 1 pM histamine. In
contrast, 0.1 uM PDB-induced sustained contractions
were 82±8% inhibited and 1.0 /xM PDB-induced
sustained contractions were 59±4% inhibited by stauu
c
o
s:
u
Stauroaporine 1 uM
1.2
istamine 100 uM
0.9 <
Graphs of the change in
active stress observed on preincubation
of tissues in 1 \iM staurosporine ( • ) or
vehicle (dimethylsulfoxide) (o) for 10
minutes, and subsequent stimulation
with 100 uM histamine, 1 uMhistamine,
1 uMphorbol dibutyrate (PDB), or 0.1
fiMPDB (n=4). Values to right of each
stress trace are phosphorylation results
from tissues frozen after 30 minutes of
stimulation. SEM ofphosphorylation did
not exceed 0.04 mol inorganic phosphate
(PJ/mol myosin light chain for any phosphorylation determinations.
KCI
rosporine (p<0.05 for the comparison of 100
histamine vs. 1 fjM PDB, p< 0.10 for 1 fiM. histamine
vs. 0.1 fiM PDB). Staurosporine appeared to more
effectively block PDB-induced than histamine-induced
sustained contractions.
Histamine-induced changes in myoplasmic [Ca2+]
were not significantly affected by staurosporine pretreatment (Figure 4). Staurosporine pretreatment
gradually attenuated levels of myosin phosphorylaDMSO
Histamine 100 uM
:
KCI
o.e
300
0J
200
0.0
100
+
CM
0
10 2 0 3 0 4 0 5 0 8 0
70 8 0 9 0 0
10 2 0 3 0 4 0 5 0 6 0
70
8
8 0 9 0
Time (min)
FIGURE 4. Left panel: The change in log L/L^ [intracellular Ca2+ concentration ([Ca2+])J, myosin phosphorylation, and active
stress observed on preincubation of tissues in 1 \iM staurosporine at 10 minutes, stimulation with 100 iiM histamine at 20 minutes,
and addition of 109 mM KCI at 80 minutes. Right panel: 77K control experiments in which the staurosporine vehicle (60 \d
dimethylsulfoxide [DMSO] in 60-ml bath) is substituted for staurosporine. Results are shown as mean (—) ±SEM (• • •) with
n=4. Symbols without error bars reflect SEM smaller than the size of the symbol
696
Hypertension
Vol 15, No 6, Part 2, June 1990
300
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
10
<
20
30 0
Time (min)
10
20
30
FIGURE 5. Left panels: Change in log UL^ ([Ca2+]) and active stress observed on stimulation with 10 yM histamine at 10
minutes and washout of histamine with physiological salt solution at 20 minutes. Results are shown as mean (—) ±2 SEM (• • •)
with n=5. Right panels: Change in log L/Lmaz ([Ca2+]) and active stress observed on stimulation with 10 yM histamine at 10
minutes and removal of extracellular CaCl2 without removal of histamine at 20 minutes.
tion and stress during the course of the 60-minute
histamine contraction, suggesting that staurosporine
uncoupled [Ca2+] increases from increases in phosphorylation. Additionally, 109 mM KC1 depolarization-induced [Ca2+] increases were also uncoupled
from increases in both phosphorylation and stress.
If an agonist-dependent second messenger other
than Ca2+ is important in the regulation of sustained
stress, then removal of Ca2+ without removal of the
agonist should result in agonist-dependent, Ca2+independent stress maintenance. One group of tissues
was stimulated with 10 fiM histamine for 10 minutes,
then the histamine was removed by washing the tissues
with Ca2+-containing PSS (Figure 5, left panels).
Washout of histamine induced a prompt decrease in
[Ca2+] and relaxation. A second group of tissues was
also stimulated with 10 ^tM histamine for 10 minutes,
and then, extracellular Ca2+ was removed by washing
the tissues in Ca2+-free PSS that still contained histamine (Figure 5,rightpanels). Washout of extracellular
Ca2+ in the continued presence of histamine resulted
in a decrease in [Ca2+] and relaxation, although both
started after an approximate 100-second delay. These
two relaxations are plotted together in Figure 6. The
second set of data (continued histamine in Ca2+-free
PSS) was shifted 100 seconds to the left, so that the
time course of [Ca2+] decrease was similar in both
protocols. The resulting relaxation was also similar in
both protocols regardless of whether agonist or Ca2+
was removed (Figure 6).
Discussion
Receptor-dependent activation of phospholipase
C results in hydrolysis of phosphatidylinositol
bisphosphate into 1,4,5-inositol trisphosphate (1,4,5IP3) and diacylglycerol. 1,4,5-IP3 is thought to release
intracellular Ca2+ stores27-28 and contribute to the
rate of stress development in smooth muscle
(although demonstration of substantial agonistdependent 1,4,5-IP3 increases in intact smooth muscle has been difficult29). A role for diacylglycerol as a
second messenger mediating sustained force maintenance in smooth muscle is appealing. The present
study, however, was not able to demonstrate significant or reproducible sustained increases in diacylglycerol concentration in correlation with sustained
contractions (Figure 2). We did not, however, investigate the time course of diacylglycerol concentration; therefore, an early transient elevation in diacylglycerol concentration might occur. In contrast,
sustained significant increases in aequorin-estimated
myoplasmic [Ca2+] and myosin phosphorylation were
associated with sustained levels of isometric stress
(Figure 2, and References 4 and 5).
Staurosporine, a putative inhibitor of protein kinase
C, had no effect on the initial phase but partially
inhibited the sustained phase of histamine-induced
contractions (Figure 3). PDB-induced contractions
were more effectively inhibited by staurosporine. This
result could be interpreted as suggesting that
Rembold and Weaver Diacylglycerol and [Ca2+] in Smooth Muscle
en
c
D
— Remove Histamine
.. ... — Remove Extracellular Ca
0.3
0.2
x
o 0.1
E
0.0
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
18
19
20
21
22
23
24
25
Time (min)
FIGURE 6. Graphs depicting the comparison of change in
log L/Lnu and relaxation induced by removal of histamine
alone ( ) and removal of extracellular CaCl2 alone (histamine still present) (
). Data is replotted from Figure 5.
Dotted lines represent ±1 SEM as in Figure 4. EGTA-treated
data (both light and stress) was shifted 100 seconds to the left
such that the time course of decrease in myoplasmic Ca2+
concentration ([Ca2*]) coincided.
staurosporine-sensitive protein kinase C activity is
partially responsible for the sustained phase of histamine contractions. Staurosporine incubation, however, uncoupled [Ca2+] from myosin phosphorylation
in 109 mM KG-depolarized tissues. Depolarization
does not release intracellular Ca2+ stores in the swine
carotid30; therefore, it should not activate phospholipase C, and consequently, no increases in diacylglycerol or activation of protein kinase C should be
expected with depolarization. This result suggests that
staurosporine is probably a nonspecific kinase inhibitor and that the staurosporine inhibition of sustained
histamine contraction might be at least partially
caused by myosin light chain kinase inhibition.
A clear result of the staurosporine experiments is
that staurosporine had no substantial effect on
histamine-induced [Ca2+] changes. This suggests that
protein kinase C is not involved in histaminestimulated increases in Ca2+ influx. A potential role
for protein kinase C might be in receptor desensitization. Prolonged phorbol diester treatment reportedly inhibits agonist-induced smooth muscle
activation.31 Staurosporine can inhibit protein kinase
C-dependent receptor desensitization in the swine
697
carotid, and this effect might explain the slightly
larger histamine-induced [Ca2+] signals in the presence of staurosporine (Figure 4).
Swine carotid artery exhibited similar rates of
decline in [Ca2+] and relaxation, independent of
whether the stimulus (histamine) or extracellular
Ca2+ (without removal of the stimulus) was removed
(Figure 6). If another second messenger, aside from
Ca , was responsible for sustained stress, then
removal of extracellular CaCl2 in the continued presence of histamine should have resulted in much
slower or absent relaxation. Thisfindingsuggests that
Ca2+ is the primary regulator of sustained stress in
the histamine-stimulated swine carotid artery. Other
second messengers that regulate sustained stress in
the swine carotid either 1) have a minor effect,
2) might also be dependent on myoplasmic [Ca2+] to
a degree similar to stress, or 3) regulate sustained
stress by modulating myoplasmic [Ca2+] per se.
Another form of evidence for physiological
increased protein kinase C activity in smooth muscle
can be obtained from two-dimensional electrophoresis of [32P]PO4-loaded tissues. In the bovine carotid
artery, the initial phase of agonist contraction is
associated with increased phosphorylation of a number of proteins including the 20 kD light chain of
myosin.32 This pattern differed from that induced by
phorbol diesters, suggesting that the initial agonistinduced pattern was not dependent on protein kinase
C. With sustained stimulation, the phosphorylation
pattern switched to that observed with phorbol
esters.32 This study has yet to be duplicated; more
data is required to determine whether protein kinase
C is functionally activated during sustained smooth
muscle contraction.
Cross-linking cytoskeletal proteins is one of the
proposed mechanisms for protein kinase Cdependent stress maintenance.16 Singer et al33 rapidly stretched depolarized swine arterial smooth muscle during both the initial phase of contraction (when
phosphorylation was high) and the sustained phase
(when phosphorylation was lower but stress was
similar). Stiffness and peak resistance to stretch were
similar during the initial and sustained phases of
contraction. If cytoskeletal cross-links were bearing a
substantial portion of stress in the sustained phase,
then the mechanical characteristics of these postulated cross-links should have been detected. The data
of Singer et al33 suggest that stress at both high and
low levels of phosphorylation is borne by mechanically similar structures. Either stress is maintained by.
cross bridges per se, as proposed by Hai and
Murphy,9 or these cytoskeletal cross-links have
mechanical characteristics identical to cross bridges,
an unlikely prospect.
The cross-bridge model of Hai and Murphy9 is
intrinsically simple and can quantitatively explain the
dependence of stress and Vo on phosphorylation and
Vo on phosphorylation.12 Furthermore, it does not
require the postulation of additional currently undescribed protein kinase cascades. Nevertheless, it is
698
Hypertension
Vol 15, No 6, Part 2, June 1990
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
possible that other systems, including protein kinase
C, might have a role in modulation of smooth muscle
contraction. When compared with contractile agonist
stimulation, depolarization with high KC1 is associated with a decrease in the [Ca2+]-sensitivity of the
contractile apparatus.15 This effect is caused by a
decrease in the [Ca2+]-sensitivity of phosphorylation4; there is no stimulus-dependent shift in the
dependence of stress on phosphorylation. Potentially, protein kinase C or another regulatory system
might be responsible for either 1) a KCl-induced
desensitization or 2) an agonist-induced sensitization
of the myosin phosphorylation system to [Ca2+].
Agonist-dependent increases in diacylglycerol concentration could not be detected in sustained swine
arterial smooth muscle contraction. Functionally,
small but significant increases in [Ca2+] appear to be
the primary regulators of sustained stress maintenance in smooth muscle.
Acknowledgment
We would like to thank Robert Michell for his
helpful discussions.
References
1. Hartshome DJ, Siemankowski RF: Regulation of smooth
muscle actomyosin. Annu Rev Physiol 1981;43:519-53O
2. Kamm KE, Stull JT: The function of myosin and myosin light
chain kinase phosphorylation in smooth muscle. Annu Rev
Pharmacol Toxicol 1985;25:593-620
3. Aksoy MO, Mras S, Kamm KE, Murphy RA: Ca2+, cAMP, and
changes in myosin phosphorylation during contraction of
smooth muscle. Am J Physiol 1983;245:C255-C270
4. Rembold CM, Murphy RA: Myoplasmic [Ca2+] determines
myosin phosphorylation in agonist-stimulated swine arterial
smooth muscle. Ore Res 1988;63:593-603
5. Rembold CM, Murphy RA: Histamine concentration and
Ca2+ mobilization in arterial smooth muscle. Am J Physiol
1989;257(Cetf Phys 2<5):C122-C128
6. Driska SP, Aksoy MO, Murphy RA: Myosin light chain
phosphorylation associated with contraction in arterial smooth
muscle. Am J Physiol 1981;240:C222-C233
7. Dillon PF, Aksoy MO, Driska SP, Murphy RA: Myosin
phosphorylation and the cross-bridge cycle in arterial smooth
muscle. Science 1981;211:495-497
8. Krisanda JM, Paul RJ: Phosphagen and metabolite content
during contraction in porcine carotid artery. Am J Physiol
1983;244:C385-C390
9. Hai C-M, Murphy RA: Crossbridge phosphorylation and
regulation of the latch state in smooth muscle. Am J Physiol
1988;254:C99-C106
10. Rembold CM, Murphy RA: Myoplasmic [Ca2+] determines
myosin phosphorylation and isometric stress in agoniststimulated swine arterial smooth muscle. / Cardiovasc Pharmacol 1988;12(suppl 5):S38-S42
11. Ratz PH, Hai C-M, Murphy RA: Dependence of stress on
crossbridge phosphorylation in vascular smooth muscle. Am J
Physiol 1989;256:C96-C100
12. Hai CM, Murphy RA: Regulation of shortening velocity by
cross-bridge phosphorylation in smooth muscle. Am J Physiol
1988;255:C86-C94
13. Danthuluri NR, Deth RC: Phorbol-ester-induced contractions
of arterial smooth muscle and inhibition of a-adrenergic
response. Biochem Biophys Res Common 1984;125:1103-1109
14. Nishizuka Y: Perspectives on the role of protein kinase C in
stimulus-response coupling. / Natl Cancer Inst 1986;
76:363-370
15. Morgan JP, Morgan KG: Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein.
J Physiol (Lond) 1984;351:155-167
16. Rasmussen H, Takuwa Y, Park S: Protein kinase C,in the
regulation of smooth muscle contraction. FASEB J 1987;
1:177-185
17. Griendling KK, Rittenhouse SE, Brock TA, Ekstein LS,
Gimbrone MA Jr, Alexander RW: Sustained diacylglycerol
formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. / Biol Chem 1986;
261:5901-5906
18. Takuwa Y, Takuwa N, Rasmussen H: Carbachol induces a
rapid and sustained hydrolysis of polyphosphoinositide in
bovine tracheal smooth muscle measurements of the mass of
polyphosphoinositides, 1,4-diacylglycerol, and phosphatidic
acid. / Biol Chem 1986;261:14670-14675
19. Jiang MJ, Morgan KG: Intracellular calcium levels in phorbol
ester-induced contractions of vascular muscle. Am J Physiol
1987;253:H1365-H1371
20. Rembold CM, Murphy RA: [Ca2+]-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle
contraction. Am J Physiol 1988;255:C719-C723
21. Rembold CM, Murphy RA: Myoplasmic calcium, myosin
phosphorylation, and regulation of the crossbridge cycle in
swine arterial smooth muscle. Ore Res 1986;58:803-815
22. Allen DG, Blinks JR: The interpretation of light signals from
aequorin-injected skeletal and cardiac muscle cells: A new
method of calibration, in Ashley CC, Campbell AK (eds):
Detection and Measurements of Free Ca2+ in Cells. Amsterdam,
Elsevier/North-Holland Biomedical Press, 1979, pp 159-174
23. Preiss JE, Loomis CR, Bell RM, Niedel JE: Quantitative
measurement of m-l,2-diacylglycerols. Methods Enzymol 1987;
141:294-300
24. Bligh EG, Dyer WJ: A rapid method of total lipid extraction
and purification. Can J Biochem Physiol 1959;37:911-917
25. Lippe IT, Holzer P: The diacylglycerol kinase inhibitor, R
59022, suppresses contractility of intestinal smooth muscle.
EwJ Pharmacol 1989;159:l-8
26. Laher I, Bevan JA: Staurosporine, a protein kinase C inhibitor, attenuates Ca2+-dependent stretch-induced vascular tone.
Biochem Biophys Res Commun 1989;158:58-62
27. Suematsu E, Hirata M, Hashimoto T, Kuriyama H: Inositol
1,4,5-trisphosphate releases Ca2+ from intracellular store sites
of skinned single cells of porcine coronary artery. Biochem
Biophys Res Commun 1984;120:481-485
28. Somlyo AV, Bond M, Somlyo AP, Scarpa A: Inositol
trisphosphate-induced calcium release and contraction in vascular smooth muscle. Proc Natl Acad Sci USA 1985;
82:5231-5235
29. Miller-Hance WC, Miller JR, Wells JN, Stull JT, Kamm KE:
Biochemical events associated with activation of smooth muscle contraction. J Biol Chem 1988;263:13979-13982
30. Rembold CM: Desensitization of swine arterial smooth muscle
to transplasmalemmal Ca2+ influx. / Physiol (Lond) 1989;
416:273-290
31. Brock TA, Rittenhouse SE, Powers CW, Ekstein LS, Gimbrone MA Jr, Alexander RW: Phorbol ester and 1-oleoyl2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle. / Biol Chem
1985;260:14158-14162
32. Takuwa Y, Kelley G, Takuwa N, Rasmussen H: Protein
phosphorylation changes in bovine carotid artery smooth
muscle during contraction and relaxation. Mol Cell Endocrinol
1988;60:71-86
33. Singer HA, Kamm KE, Murphy RA: Estimates of activation in
arterial smooth muscle. Am J Physiol 1986;251:C465-C473
KEY WORDS • calcium • vascular smooth muscle • endothelin
• aequorin • histamine • myosin • phosphorylation
[Ca2+], not diacylglycerol, is the primary regulator of sustained swine arterial smooth
muscle contraction.
C M Rembold and B A Weaver
Hypertension. 1990;15:692-698
doi: 10.1161/01.HYP.15.6.692
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1990 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://hyper.ahajournals.org/content/15/6_Pt_2/692
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located, click
Request Permissions in the middle column of the Web page under Services. Further information about this
process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/