Minor role of a Ca2+-depleted
sarcoplasmic reticulum in heterologous desensitization of smooth
muscle to K+
Robert L.
Wardle and
Richard A.
Murphy
Department of Molecular Physiology and Biological Physics,
University of Virginia Health Sciences Center, Charlottesville,
Virginia 22906-0011
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ABSTRACT |
Exposure of
porcine carotid artery smooth muscle (PCASM) to histamine was followed
by a large reduction in the rate of force generation in response to 40 mM KCl. This was shown to be a manifestation of slow attainment of a
steady-state myoplasmic Ca2+
concentration
([Ca2+]i).
We hypothesized that if net transsarcolemmal
Ca2+ flux into the depolarized
PCASM cells is the same before and after a desensitizing histamine
treatment, then the transient attenuation of the increase in
[Ca2+]i
may be due to accelerated uptake of
Ca2+ by a partially depleted
sarcoplasmic reticulum (SR) acting as a
Ca2+ sink or superficial buffer
barrier. We tested this hypothesis by eliciting responses of
"desensitized PCASM" to 40 mM KCl in the presence of
cyclopiazonic acid (CPA), an SR
Ca2+-ATPase inhibitor.
Contractions of CPA-treated tissues were attenuated less than those of
tissues not treated with CPA, but they were not abolished.
CPA-insensitive mechanism(s) dominated the desensitization. We conclude
that histamine pretreatment reduced net transsarcolemmal Ca2+ flux into PCASM in response
to 40 mM KCl.
arterial smooth muscle; latch; excitation-contraction coupling; activation-contraction coupling; calcium transients; attenuation
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INTRODUCTION |
HETEROLOGOUS DESENSITIZATION of smooth muscle can be
defined as the reduced contractile response to a stimulus after a
limited exposure to a different contractile agonist. Presumably, this capacity to modulate sensitivity to different mediators (many of which
induce transsarcolemmal depolarization and L-channel Ca2+ conductance) enables vascular
smooth muscle to regulate blood pressure and regional blood flow
appropriately in vivo. For example, in the presence of chronic
periarteriolar inflammation and local high concentrations of vasoactive
mediators (such as histamine released from mast cells), desensitization
would prevent excessive vascular smooth muscle contraction that might
compromise adequate blood flow to tissues distal to the site of
inflammation.
In our experimental model of heterologous desensitization
(23), exposure of porcine carotid artery medial rings to
histamine was followed by long-lasting reductions in the force elicited by 40 mM KCl (Fig. 1). In contrast to the classic
interpretation of desensitization as a reduced activation involving
fewer cycling cross bridges, we concluded that the apparent reduction
in force-generating capacity was really a manifestation of a slowing in
the rates at which the myoplasmic
Ca2+ concentration
([Ca2+]i),
and consequently myosin regulatory light chain (MRLC) phosphorylation and cross-bridge recruitment (estimated from increases in stiffness), attained steady-state values (23). This retarded
stimulus-[Ca2+]i
coupling resulted in very slow cross-bridge cycling and rates of force
development (latch) (23). The steady-state 40 mM KCl-induced force was
not reduced, and there was no detectable change in the Ca2+ sensitivity of MRLC
phosphorylation or the dependence of cross-bridge cycling rate on
myosin phosphorylation (23).

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Fig. 1.
Desensitization of porcine carotid artery smooth muscle to
K+ depolarization.
A: relative force induced by 40 mM KCl
(arrow) before histamine and 20 min after removal of histamine
(10 3 M, 30 min). Note delay
of active force development and slower rate of force development after
exposure to histamine. B: attenuation
of relative force (from A) is
difference between time-matched responses to 40 mM KCl before and after
histamine (modified from Ref. 23).
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The retarded
stimulus-[Ca2+]i
coupling during K+ depolarization
could be due to attenuated Ca2+
influx into the myoplasm or to accelerated
Ca2+ extrusion from the myoplasm.
We hypothesized that if net transsarcolemmal Ca2+ flux into the depolarized
carotid medial cells is the same before and after a desensitizing
histamine treatment, then the transient attenuation of the increase in
[Ca2+]i
and MRLC phosphorylation may be due to accelerated uptake of Ca2+ by a partially depleted
sarcoplasmic reticulum (SR) acting as a
Ca2+ sink or superficial buffer
barrier (Fig. 2) [van Breeman et al. (22)].
Ratz et al. (17) invoked this hypothesis to account for delayed
responsiveness of rabbit femoral artery smooth muscle to 23 mM KCl
after pretreatment with phenylephrine. We employed two different
strategies to test this hypothesis. First, we attempted to create a
superficial buffer barrier to increases in
[Ca2+]i
by exposing tissues to SR Ca2+
depletion protocols. Evidence that
Ca2+ depletion desensitized the
smooth muscle to K+ depolarization
would support the hypothesis. Second, we elicited responses of
"desensitized carotid media" to 40 mM KCl in the presence of
cyclopiazonic acid (CPA), a selective SR
Ca2+-ATPase inhibitor (1, 4, 5,
10, 12, 20, 25). If effective, CPA should prevent the SR from
accumulating Ca2+ on
K+ depolarization, and the
desensitization should be abolished or diminished.

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Fig. 2.
Schematic illustration of sarcoplasmic reticulum (SR) acting as a
Ca2+ sink or superficial buffer
barrier. We hypothesized that if net transsarcolemmal
Ca2+ flux into depolarized carotid
media smooth muscle cells is the same before
(left) and after
(right) a desensitizing histamine
treatment, then transient attenuation of increase in myoplasmic
Ca2+ concentration may be due to
accelerated uptake of Ca2+ by a
partially depleted SR acting as a
Ca2+ sink or superficial buffer
barrier. SL, sarcolemma; L, L-type voltage-dependent channel.
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Our objectives were to determine 1)
whether SR Ca2+ depletion
protocols or histamine pretreatment depleted the SR
Ca2+ stores,
2) whether contractile responses to
depolarization with 40 mM KCl were attenuated, and
3) whether CPA abolished or
diminished any desensitization to
K+ depolarization. Contractions
induced by phenylephrine
(10
4 M in
Ca2+-free solution, 5 min) were
employed as a rough index of the amount of releasable
Ca2+ in the inositol
1,4,5-trisphosphate receptor
(IP3R)-mediated SR
Ca2+ store. Contractions induced
by caffeine (20 mM, 90 s) were employed as a rough index of the amount
of releasable Ca2+ in the
ryanodine receptor (RyR)-mediated SR
Ca2+ store. In contrast to
phenylephrine, caffeine bypasses G protein-coupled, plasmalemmal
receptor-activated signaling and inositol 1,4,5-trisphosphate generation. Thus brief caffeine-induced contractions should be less
affected by any histamine-induced downregulation of signal transduction
mechanisms proximal to activation of
Ca2+ release from the SR.
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MATERIALS AND METHODS |
Tissue preparation.
Porcine carotid arteries obtained from a slaughterhouse were
transported to the lab and stored at 4°C in physiological saline solution (PSS) containing (in mM) 140.1 NaCl, 4.7 KCl, 1.2 Na2HPO4, 1.6 CaCl2, 5.6 D-glucose, 1.2 MgCl2, 2.0 MOPS buffer (pH 7.4 at 4 or 37°C), and 0.02 EDTA to chelate trace heavy metal ions.
Deendothelialized carotid artery medial rings were prepared as
described by Wingard et al. (24).
Drugs and solutions.
Histamine dihydrochloride, pyrilamine maleate, cimetidine,
phenylephrine hydrochloride, caffeine, CPA, DMSO, and EGTA were purchased from Sigma Chemical (St. Louis, MO).
Ca2+-free PSS (also referred to as
0 Ca2+ PSS) contained the
Ca2+ chelator EGTA (1.0 mM) and no
added CaCl2. Concentrated stock solutions of most drugs were prepared weekly with PSS containing ascorbic acid (0.1% wt/vol), and drugs were diluted in PSS immediately before use. A concentrated stock solution of CPA in DMSO was diluted in
PSS immediately before use. KCl was stoichiometrically substituted for
NaCl in depolarizing high-K+ PSS
(40 or 109 mM KCl).
Force.
Deendothelialized carotid artery medial rings (2-3 mm in width)
were mounted over two stainless steel posts. One post was connected via
a support rod to a micrometer used to adjust the length of the
preparation, and the other post was part of a 1-mm-diameter stainless
steel wire connected to a Grass FT 03 force transducer. Mounted rings
were immersed in water-jacketed baths containing PSS at 37°C and pH
7.4, continuously bubbled with compressed air. Medial rings were
adjusted to the length at which the active isometric force generated in
response to 109 mM KCl was maximized
(Lo). Isometric
force was continuously recorded on a polygraph. Stress was calculated
as force per tissue cross-sectional area estimated using tissue blotted
wet weight, ring length at
Lo, and tissue density of 1.055 g/cm3.
Contractile responses were normalized to a control 109 mM KCl-induced response (% of K109) elicited
before the experimental protocol.
General protocol.
Porcine carotid artery medial rings were contracted by
10
4 M phenylephrine in 0 Ca2+ PSS (PhE/0
Ca2+), 20 mM caffeine, or 40 mM
KCl both before and after intervening SR
Ca2+ depletion protocols (see Fig.
3; protocol modified from Ref. 16) or before and after an intervening
treatment with histamine (10
3 M in PSS, 30 or 40 min). The general protocol is illustrated by force tracings in which
caffeine-induced contractions were elicited both before and after an
intervening treatment with or without histamine (see Fig. 6,
top). After the intervening
treatment and immediately before the final exposure to PhE/0
Ca2+, caffeine, or KCl (with or
without the Ca2+-ATPase inhibitor
CPA), all tissues were washed three times over 30 min with PSS
(containing 1.6 mM CaCl2) with
or without CPA (10
5 M or 3 × 10
5 M) and
containing the H1- and
H2-receptor antagonists pyrilamine (10
6 M) and cimetidine
(10
5 M) to
1) facilitate rapid removal of
histamine from the histamine-treated tissues and
2) block response to any histamine
that remained after the first two solution changes. Treatment with PSS
containing histamine receptor antagonists had no effect on the final 40 mM KCl-induced responses in control experiments.
Data analysis.
We previously adopted an alternative form of data presentation, i.e.,
attenuation analysis, because it has advantages for understanding the
mechanisms underlying heterologous desensitization and the complex
temporal relationships among the key signaling events regulating smooth
muscle contraction (23). Because the hypothesis being tested in the
present study was proposed in the discussion section of Ref. 23 and
because we desired to readily integrate information gained from the two
studies, we performed the same type of data analysis in the present
study.
Medial rings that failed to generate
105
N/m2 active stress in response to
a 10-min 109 mM KCl exposure were excluded from further analyses. PhE/0
Ca2+-, caffeine-, or KCl-induced
active force, basal force, the delay between initiation of 40 mM KCl
exposure and onset of active force (see Fig.
1A), and the PhE/0
Ca2+- or 40 mM KCl-induced maximum
rate of force development (maximum dF/dt) were determined from chart
records. Attenuation of these indexes of medial ring activation was
calculated as the difference between values attained before and after
intervening treatment. For example, Fig. 1 illustrates how the
attenuation of KCl-induced active force was calculated by subtracting
time-matched values attained after histamine treatment from the values
attained before histamine treatment. The intervening treatment was
sometimes associated with increases in basal force. The increase in
basal force was calculated as the difference between the values
attained immediately before exposures to the contractile agonist. For
each contraction, agonist-induced active force was calculated from the
basal force immediately before an exposure to the contractile agonist.
Each of the paired rings from three to six different arteries received
one intervening treatment between two contractions elicited by one of
the contractile agonists (PhE/0
Ca2+, caffeine, or KCl), and the
second contraction was elicited in the presence or absence of CPA. The
effect of CPA on the intervening treatment-induced attenuation of
indexes of medial ring activation was assessed by comparing means or
medians of the attenuation for different treatment groups (paired
t-test, repeated measures ANOVA, or
analogous nonparametric procedures).
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RESULTS |
Effect of CPA on basal force.
Tissues were treated with CPA to selectively inhibit SR
Ca2+-ATPase activity after SR
Ca2+ depletion protocols or
exposure to histamine. Tissues treated with CPA exhibited a greater
increase in basal force before the final PhE/0
Ca2+-, caffeine-, or KCl-induced
contraction than did tissues that were not treated with CPA (Figs.
3-7,
bottom). This difference reflects the contribution of SR Ca2+-ATPase
activity to regulation of
[Ca2+]i.
The CPA-induced increase in basal force (<10% of the control 109 mM
KCl-induced active force elicited before the experimental protocol,
i.e., <10% of K109) was small
relative to PhE/0 Ca2+-,
caffeine-, or 40 mM KCl-induced peak active force (~55, 22, and 77%
of K109, respectively).
Nevertheless, the CPA-induced basal forces were probably large enough
to influence estimates of reductions in caffeine-induced peak active
force (see Fig. 6, top). Generally a
CPA-induced increase in basal force correlated with a subtle change in
the attenuation of agonist-induced force development (see Figs.
4-7) but the latter was not always statistically significant at
P < 0.05.

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Fig. 3.
SR Ca2+ depletion-repletion
protocols. Isometric force tracings. Medial rings were exposed to 1.0 mM EGTA-buffered Ca2+-free
physiological saline solution (0 Ca2+ PSS) changed every 5 min for
40 min total (bottom trace). Some
tissues were exposed 3 separate times to 0 Ca2+ PSS containing 0.1 mM
histamine (open bars beneath top
trace) to ensure depletion of inositol
1,4,5-trisphosphate receptor-releasable SR
Ca2+ stores. All tissues were
exposed to 20 mM caffeine (solid bars) for 2 min at end of these
protocols to ensure depletion of ryanodine receptor-releasable SR
Ca2+ stores. All tissues were
exposed to 1.6 mM Ca2+ PSS for 30 min to replete SR Ca2+ stores in
absence (top trace) or presence
(bottom trace) of 0.03 mM cyclopiazonic acid
(CPA; hatched bar).
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Fig. 4.
SR Ca2+ depletion protocols
depleted phenylephrine-releasable
Ca2+ store. Attenuation of peak
active force (top) and attenuation
of maximum rate of force development (max
dF/dt;
middle) in response to
10 4 M phenylephrine in 0 Ca2+ PSS (5 min) 30 min after
cessation of SR Ca2+ depletion
protocols without histamine
(Ca2+-free PSS; illustrated in
Fig. 3, bottom trace) or SR
Ca2+ depletion protocols with
histamine (Histamine/0 Ca2+ PSS;
illustrated in Fig. 3, top trace); 0 Ca2+ PSS contained the
Ca2+ chelator EGTA (1.0 mM) and no
added CaCl2. Phenylephrine-induced
contractions after depletion protocols were attenuated.
Phenylephrine-induced contractions of CPA-treated tissues (shaded bars)
were attenuated more than those of tissues not treated with CPA (open
bars; * P < 0.05 vs. CPA-treated tissues). Bottom:
CPA-treated tissues exhibited a greater increase in basal force before
final phenylephrine-induced contraction than tissues that were not
treated with CPA
(* P < 0.05 vs. CPA-treated tissues). Values are means + SE; paired rings for
n = 4-6 arteries in each group.
%K109, % of control 109 mM
KCl-induced response elicited before experimental protocol.
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Fig. 5.
SR Ca2+ depletion protocols
desensitized muscle to K+
depolarization. Top: attenuation of 40 mM KCl-induced active force 30 min after cessation of SR
Ca2+ depletion protocols without
( ) histamine (Ca2+-free PSS
alone; illustrated in Fig. 3, bottom
trace) or SR
Ca2+ depletion protocols with (+)
histamine (Histamine/0 Ca2+ PSS;
illustrated in Fig. 3, top trace); 0 Ca2+ PSS
(Ca2+-free PSS) contained the
Ca2+ chelator EGTA (1.0 mM) and no
added CaCl2.
* P < 0.05 vs.
non-CPA-treated tissues. Bottom:
KCl-induced contractions exhibited a delay of active force development
(# P < 0.05 only for Ca2+-free PSS
alone in absence of CPA group; other groups not different from 0) and
an attenuated maximum dF/dt. Note that
these KCl-induced contractions were attenuated less than those
following exposure to histamine in PSS (cf.
top and Fig. 7,
top; illustrated together in Fig. 8).
KCl-induced contractions of CPA-treated tissues (3 × 10 5 M CPA, filled symbols
and shaded bars) after depletion with histamine/0
Ca2+ PSS were attenuated less than
KCl-induced contractions of tissues not treated with CPA (open symbols
and open bars;
* P < 0.05 vs. non-CPA-treated tissues). CPA-treated tissues after
depletion with 0 Ca2+ PSS alone
exhibited a greater increase in basal force before final KCl-induced
contraction than non-CPA-treated tissues after depletion with
histamine/0 Ca2+ PSS
( P < 0.05). Values are
means ± SE; paired rings for n = 3-5 arteries in each group.
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Fig. 6.
Histamine and CPA depleted caffeine-releasable
Ca2+ store.
Top: isometric force tracings for 4 carotid medial rings from 1 artery. Tissues were exposed to caffeine
(20 mM, 90 s; arrowheads) before and after intervening treatment with
PSS alone (trace a), CPA (3 × 10 5 M;
trace b), histamine
(10 3 M;
trace c) or both histamine and CPA
(trace d). Force scale (vertical
bar) above trace a is applicable for
caffeine-induced contractions only (traces
a-d). Note that
in traces c and
d amplification of force signal was
halved during histamine activation (early spike) to resolve maximum
force on chart record. Amplification of force signal was returned to
original sensitivity after response to histamine (apparent sudden
increase in basal force) to resolve caffeine-induced force.
Bottom: attenuation of
caffeine-induced peak active force 30 min after cessation of either PSS
alone (a and
b) or PSS with histamine
(c and
d; letters in parentheses correspond
to traces in top). Caffeine-induced
contractions after histamine were attenuated
( P < 0.05 vs. PSS
alone). Caffeine-induced contractions of CPA-treated tissues (shaded
bars) were attenuated more than those of tissues not treated with CPA
(open bars; * P < 0.05 vs.
CPA-treated tissues). CPA-treated tissues exhibited a
greater increase in basal force before final caffeine-induced
contraction than tissues that were not treated with CPA
(* P < 0.05 vs. CPA-treated
tissues). Values are means + SE; paired rings for
n = 4-5 arteries in each group.
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Fig. 7.
CPA diminished histamine-induced desensitization to
K+ depolarization.
Top: attenuation of 40 mM KCl-induced
active force 30 min after cessation of histamine
(10 3 M, 30 min;
left) or histamine
(10 3 M, 40 min;
right).
* P < 0.05 vs.
non-CPA-treated tissues. Bottom:
KCl-induced contractions exhibited a delay of active force development
and an attenuated maximum dF/dt as
illustrated in Fig. 1. KCl-induced contractions of CPA-treated tissues
(10 5 M CPA,
left; 3 × 10 5 M CPA,
right) were attenuated less than
those of tissues not treated with CPA
(* P < 0.05 vs. non-CPA-treated tissues;
P < 0.05 vs. non-CPA-,
non-DMSO-treated tissues). CPA-treated tissues
exhibited a greater increase in basal force before final KCl-induced
contraction than tissues that were not treated with CPA
(* P < 0.05 vs. non-CPA-treated tissues). Values are
means ± SE; paired rings for
n = 6 arteries in each group.
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Effects of SR
Ca2+ depletion
protocols and CPA on sensitivity to
K+
depolarization.
Phenylephrine (PhE/0
Ca2+)-induced contractions
before and after intervening treatments were used to assess the amount
of releasable Ca2+ in the
IP3R-mediated
Ca2+ store. PhE/0
Ca2+-induced contractions were
attenuated 30 min after cessation of SR
Ca2+ depletion protocols (Fig. 4),
consistent with partial depletion of the
IP3R-mediated
Ca2+ store. CPA should inhibit SR
Ca2+-ATPases and refilling of SR
Ca2+ stores during the 30-min
repletion by incubation of tissues in 1.6 mM
Ca2+-PSS (illustrated in Fig.
3,
bottom). This was manifested by a greater attenuation of SR
Ca2+-dependent (PhE/0
Ca2+-induced) contractile
responses of CPA-treated tissues than of responses of tissues not
treated with CPA (Fig. 4). Although the greater attenuation of PhE/0
Ca2+-induced peak active force in
CPA-treated tissues was not statistically significant
(P > 0.05; Fig. 4,
top), attenuation of the maximum rate of PhE/0 Ca2+-induced force
development (maximum dF/dt) of
CPA-treated tissues was greater than that exhibited by tissues not
treated with CPA (P < 0.05; Fig. 4,
middle).
Having indirect evidence that our protocol effectively reduced the
amount of PhE/0 Ca2+-releasable
Ca2+ in the SR, we exposed another
group of tissues to SR Ca2+
depletion protocols to assess their impact on contractile responses to
K+ depolarization. These
contractions were strictly dependent on extracellular
Ca2+ concentration and
transsarcolemmal Ca2+ influx.
Ca2+-depleted tissues exhibited an
attenuated maximum dF/dt in response to 40 mM KCl (Fig. 5, bottom). A
modest attenuation of active force relative to the histamine-induced
attenuation was observed (cf. Fig. 5,
top, and Fig. 7,
top; illustrated together in Fig. 8). CPA should inhibit SR
Ca2+ uptake during exposure to 40 mM KCl and abolish any attenuation of force due to SR sequestering
Ca2+ (see Fig. 2). Indeed, in two
of the five tissues that were exposed to histamine during the
Ca2+ depletion protocol, CPA
abolished attenuation of 40 mM KCl-induced maximum
dF/dt and active force. The
KCl-induced contractions of the other three CPA-treated tissues were
37-67% less attenuated than KCl-induced contractions of tissues
that were not treated with CPA (P < 0.05; Fig. 5, top and
bottom).

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Fig. 8.
Comparison of SR Ca2+
depletion-induced and histamine pretreatment-induced desensitization to
K+ depolarization. Attenuation of
40 mM KCl-induced active force 30 min after SR
Ca2+ depletion protocols with
histamine and caffeine in 0 Ca2+
PSS (40 min; from Fig. 5, top) or 30 min after histamine (10 3 M
in 1.6 mM Ca2+ PSS, 40 min; from
Fig. 7, top right); 0 Ca2+ PSS contained
the Ca2+ chelator
EGTA (1.0 mM) and no added CaCl2.
CPA (3 × 10 5 M)
appeared to be as effective in diminishing histamine
pretreatment-induced desensitization to 40 mM KCl as it was in
abolishing or diminishing SR Ca2+
depletion-induced desensitization to 40 mM KCl.
* P < 0.05 vs. non-CPA-treated
tissues.
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Effects of histamine pretreatment and CPA on SR
Ca2+ stores and
sensitivity to
K+
depolarization.
Caffeine-induced contractions were used to assess the amount of
releasable Ca2+ in the
RyR-mediated Ca2+ store.
Caffeine-induced contractions were not attenuated 30 min after
cessation of PSS alone but were attenuated 30 min after cessation of
histamine (10
3 M, 30 min;
Fig. 6). This result is consistent with histamine-induced partial
depletion of the RyR-mediated Ca2+
store. CPA should inhibit SR Ca2+
uptake, and CPA alone should deplete the RyR-mediated
Ca2+ store. Such was the case:
caffeine-induced contractions of CPA-treated tissues were attenuated
more than those of tissues not treated with CPA, regardless of whether
they had an intervening histamine treatment (Fig. 6). This implies that
CPA alone or after histamine was more effective than histamine alone in
depleting the RyR-mediated Ca2+
store.
Indirect evidence indicates that histamine pretreatment effectively
reduced the amount of caffeine-releasable
Ca2+ in the SR. Another group of
tissues exposed to histamine exhibited a delay in active force
development and an attenuated maximum dF/dt in response to 40 mM KCl (Fig.
7, bottom). This attenuated the
active force (Fig. 7, top), as we
described previously (23). CPA should prevent the SR from accumulating
Ca2+ on
K+ depolarization, and the
desensitization should be abolished or diminished. The KCl-induced
contractions of CPA-treated tissues were attenuated up to 44% less
than KCl-induced contractions of tissues that were not treated with CPA
(Fig. 7, top), but CPA did not
abolish desensitization to K+
depolarization.
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DISCUSSION |
The SR is a primary regulator of the cytosolic free
Ca2+ concentration
([Ca2+]i)
in vascular smooth muscle cells and is thereby critically involved in
controlling vascular smooth muscle tone. Our current understanding of
how the SR contributes to maintenance of homeostasis of
[Ca2+]i
includes a role as a "superficial buffer barrier" or sink for Ca2+ entering the cell when the SR
Ca2+ store is depleted and a role
extruding Ca2+ when the SR
Ca2+ store is filled
[reviewed by Daniel et al. (3) and by van Breeman et al.
(22)]. Filling of the SR
Ca2+ store is coupled to
regulation of transsarcolemmal
Ca2+ flux, thereby providing
mechanisms whereby depleted stores may be readily refilled
["capacitative Ca2+
entry" reviewed by Putney (13); Daniel et al. (3);
"Ca2+ sparks" described by
Nelson et al. (9)]. The maintenance of homeostasis of
[Ca2+]i
in porcine carotid artery smooth muscle subjected to various treatments
(18, 19) supports both the buffer barrier hypothesis and the
capacitative Ca2+ entry hypothesis
(which are not mutually exclusive).
Prior in vitro exposure and withdrawal of contractile agonists such as
histamine, phenylephrine, and carbachol may desensitize smooth muscle
to depolarizing stimuli for prolonged periods (6, 8, 14, 15, 17, 23).
Our findings regarding the putative involvement of a
Ca2+-depleted SR in heterologous
desensitization of carotid media to depolarizing stimuli can be
summarized as follows. Thirty minutes after cessation of SR
Ca2+ depletion protocols,
1) the
IP3R-releasable
Ca2+ store was partially depleted
and appeared to be depleted further by CPA;
2) 40 mM KCl-induced contractile
responses of tissues not treated with CPA were attenuated, although
much less than was observed after histamine treatment; and
3) 40 mM KCl-induced contractile
responses of CPA-treated tissues were not attenuated or were attenuated
less than those of tissues not treated with CPA. Thirty minutes after
cessation of histamine treatment, 1) the RyR-releasable Ca2+ store was
partially depleted and was depleted further by CPA; and
2) 40 mM KCl-induced contractile
responses of CPA-treated tissues were attenuated less than those of
tissues not treated with CPA, but CPA did not abolish desensitization
to K+ depolarization. These
results are consistent with our hypothesis that accelerated uptake of
Ca2+ by a partially depleted SR
acting as a Ca2+ sink or
superficial buffer barrier contributes to heterologous desensitization
to submaximal K+ depolarization.
However, CPA-insensitive mechanism(s) appear to be responsible for the
majority of the attenuated carotid medial response to 40 mM KCl (see
Fig. 8). This suggests that net transsarcolemmal Ca2+ flux into the depolarized
smooth muscle cells is not the same before and after a desensitizing
histamine treatment.
The contribution of SR Ca2+-ATPase
activity to regulation of
[Ca2+]i
in unstimulated, resting carotid media was revealed by the CPA-induced
increase in basal force. These results are consistent with CPA-induced
increase of basal force in rat pulmonary artery, aorta, and some
superior mesenteric artery smooth muscle (4, 5, 20) and with
CPA-induced increase of both basal force and
[Ca2+]i
in ferret portal vein and rat femoral artery smooth muscle (1, 10). The
CPA-induced increase of basal force is likely due to the increase in
[Ca2+]i
rather than to an increase in the
Ca2+ sensitivity of the
contractile elements, since CPA did not modify the pCa-tension
relationship of
-escin-skinned rat pulmonary artery smooth muscle
(5).
After cessation of SR Ca2+
depletion protocols, the extracellular tissue space of the carotid
media should be Ca2+ replete
within 10 min of exposure to PSS (16). The
IP3R-releasable Ca2+ store of the carotid media
was partially depleted (attenuated PhE/0
Ca2+-induced contractions, Fig.
4), coincident with desensitization to
K+ depolarization (attenuated 40 mM KCl-induced contractions, Fig. 5) 30 min after cessation of SR
Ca2+ depletion protocols. However,
this desensitization to K+
depolarization was small compared with that after histamine
pretreatment, and it was nearly completely abolished by CPA (cf. Figs.
5 and 7; Fig. 8). The attenuated carotid media contractile responses to
K+ depolarization after SR
Ca2+ depletion were similar to
those of rabbit ear artery and dog mesenteric artery smooth muscle (2,
7). Furthermore, canine mesenteric artery treated with the SR
Ca2+-ATPase inhibitor thapsigargin
was not desensitized to K+
depolarization after SR Ca2+
depletion (7). These results support at least a minor role for a
Ca2+-depleted SR in heterologous
desensitization of carotid media to submaximal
K+ depolarization.
The attenuation of caffeine-induced contractions of the carotid media
30 min after cessation of histamine (Fig. 6) was consistent with the
attenuation of both caffeine-induced stress and
[Ca2+]i
transients of rabbit femoral artery smooth muscle 10 min after cessation of phenylephrine (17) and the attenuation of caffeine-induced contractions of guinea pig taenia cecum smooth muscle after carbachol exposure (6). These results imply that the RyR-mediated SR Ca2+ store was partially depleted
after agonist pretreatment, possibly due to impairment of mechanism(s)
required for refilling this store (e.g., reduced transsarcolemmal
Ca2+ influx). When we deliberately
impaired refilling of the SR Ca2+
store by treating tissues with CPA, caffeine-induced contractions were
greatly attenuated regardless of whether tissues were pretreated with
histamine (Fig. 6). This was consistent with attenuation of both
caffeine-induced tension and
[Ca2+]i
transients of rat femoral artery smooth muscle after CPA (10). Alternative explanations for the attenuated caffeine-induced
contractions of the carotid media are impaired mechanism(s) coupling
caffeine to RyR-mediated Ca2+
release from the SR or a decreased
Ca2+ sensitivity of the
contractile elements. Ratz et al. (15) and Oike et al. (11) reported
that agonist pretreatment of vascular smooth muscles modulated the
Ca2+ sensitivity of the
contractile machinery and thereby impaired contractility to subsequent
KCl exposure. However, we did not detect a change in the
Ca2+ sensitivity of cross-bridge
phosphorylation during 40 mM KCl-induced contractions of
histamine-pretreated carotid media (23).
The histamine-induced desensitization of the carotid media to 40 mM KCl
was significantly diminished but not abolished by CPA (Fig. 7). We
interpret this finding as evidence that some Ca2+ entering the cells through
L-channels was diverted from the myofilaments by
Ca2+-ATPases of the partially
depleted superficial SR, consistent with the superficial buffer barrier
hypothesis [reviewed by van Breeman et al. (22)]. Ratz et
al. (17) invoked this hypothesis to account for delayed responsiveness
of rabbit femoral artery smooth muscle to 23 mM KCl after pretreatment
with phenylephrine. Shima and Blaustein (20) suggested the buffer
barrier hypothesis could account for enhanced 30 mM
K+-evoked contractions of rat
mesenteric artery smooth muscle in the presence of CPA. Yoshikawa et
al. (25) tested the buffer barrier hypothesis in guinea pig urinary
bladder myocytes using voltage-clamp techniques and intracellular
Ca2+ microfluorometry. CPA
modulated caffeine-induced or depolarization-induced [Ca2+]i
transients and the Ca2+-modulated
membrane currents, L-channel current and
Ca2+-activated
K+ current, implying that
Ca2+ sequestration by the SR can
buffer part of the Ca2+ influx
during slow depolarizations (25).
Although CPA nearly abolished the small desensitization of carotid
media to K+ depolarization after
SR Ca2+ depletion protocols,
CPA-insensitive mechanism(s) appear to be responsible for the majority
of the attenuated carotid media response to 40 mM KCl after histamine
pretreatment (see Fig. 8). The inability of CPA to completely abolish
this desensitization to K+
depolarization could be a manifestation of some CPA-insensitive isoform(s) of SR Ca2+-ATPase, or
compartmentalized SR Ca2+-ATPases
protected from CPA. We found no evidence in the literature to support
either of these explanations. The involvement of intracellular compartments other than the SR seems unlikely, given that mitochondria contribute significantly to
[Ca2+]i
homeostasis only under pathophysiological conditions (21) and that the
role of the nucleus and/or nuclear membrane in
[Ca2+]i
homeostasis has not been established for vascular smooth muscle (12).
Finally, we cannot rule out the possibility that histamine pretreatment
induces changes in other biochemical signaling pathways that may
directly or indirectly affect myofilament activation and force
transduction. However, the temporal correlation of the attenuations of
aequorin-estimated
[Ca2+]i,
MRLC phosphorylation, and tissue stiffness (biochemical and mechanical
indexes of cross-bridge activation) during desensitization to 40 mM KCl
indicate that Ca2+-dependent
cross-bridge recruitment per se is not altered significantly (23).
Furthermore, as stated previously (23), "the dramatic temporal
dissociation between stiffness and stress implies that slow
cross-bridge cycling must contribute significantly to reduced mechanical performance and the appearance of desensitization." Consequently we conclude that histamine pretreatment reduced net transsarcolemmal Ca2+ flux into
carotid medial smooth muscle cells. This reduced net transsarcolemmal
Ca2+ influx impaired both
1) refilling of the SR
Ca2+ store upon removal of
histamine and 2) the rate of
increase of Ca2+ concentration at
the myofilaments in response to 40 mM KCl. Putative mechanisms by which
this could be accomplished include altered function or number of
sarcolemmal proteins involved in
Ca2+ fluxes and transient
redistribution of ions across the sarcolemma (see Ref. 23).
 |
ACKNOWLEDGEMENTS |
We thank Drs. Paul H. Ratz, Christopher J. Wingard, John D. Strauss, and John S. Walker for thoughtful discussions. Arteries were
donated by the Smithfield Company of Smithfield, VA.
 |
FOOTNOTES |
The work was supported by National Heart, Lung, and Blood Institute
Grant 5P01-HL-19242 and an American Heart Association (Virginia
Affiliate) fellowship to R. L. Wardle.
Address for reprint requests: R. L. Wardle, Molecular Physiology and
Biological Physics, University of Virginia, PO Box 10011, Charlottesville, VA 22906-0011.
Received 22 August 1997; accepted in final form 7 July 1998.
 |
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