Vol. 277, Issue 2, H749-H755, August 1999
Sarcoplasmic reticulum and endothelium independently regulate
venous smooth muscle
[Ca2+]i
and contraction
Régent
Laporte1 and
Ismail
Laher2
1 Division of Maternal-Fetal
Medicine, Department of Obstetrics and Gynecology, Beth Israel
Deaconess Medical Center and Harvard Medical School, Boston,
Massachusetts 02215; and
2 Department of Pharmacology and
Therapeutics, Faculty of Medicine, University of British Columbia,
Vancouver, British Columbia, Canada V6T 1Z3
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ABSTRACT |
In rings of rabbit facial vein (RFV), depletion
of sarcoplasmic reticulum (SR)
Ca2+ by caffeine abolished the
subsequent isometric contraction to 25 mM
K+ physiological salt solution
(25K-PSS). However, the associated steady-state increase of smooth
muscle intracellular free Ca2+
concentration
([Ca2+]i),
measured using fura PE3 and cuvette photometry, was not altered. Treatment with the specific SR
Ca2+ pump inhibitor cyclopiazonic
acid (30 µM) after caffeine-induced SR
Ca2+ depletion restored and
greatly augmented the 25K-PSS-induced contraction. This suggests that
SR Ca2+ depletion leads to a
dissociation of K+-induced
[Ca2+]i
increase from contraction that was dependent on
Ca2+ pump-mediated SR
Ca2+ uptake. Endothelium removal
augmented the 25K-PSS-induced
[Ca2+]i
increase after caffeine-induced SR
Ca2+ depletion. However, this was
associated with only a small and transient contraction. Exposure of
endothelium-denuded RFV to cyclopiazonic acid after caffeine-induced SR
Ca2+ depletion further amplified
the 25K-PSS-induced
[Ca2+]i
increase, which was associated with a large and sustained contraction. However, the latter
[Ca2+]i
increase was still higher than in endothelium-intact RFV. This suggests
that the endothelium dampens the
[Ca2+]i
rise associated with K+-induced
Ca2+ influx, but independently of
Ca2+ pump-mediated SR
Ca2+ uptake.
calcium uptake; caffeine; cyclopiazonic acid; potassium; rabbit
facial vein
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INTRODUCTION |
THE SARCOPLASMIC RETICULUM (SR) plays a crucial role in
the control of smooth muscle (SM) tone by regulating intracellular free
Ca2+ concentration
([Ca2+]i)
(12). In addition to its widely accepted functions as a sink and a
source of Ca2+, a third role for
the SR was proposed by van Breemen and colleagues (28), i.e., the
superficial buffer barrier. The authors argued that peripheral SR,
through its Ca2+ pump, diverts a
portion of Ca2+ that enters the
cell before it reaches deeper regions of the myoplasm to initiate
contraction. One important prerequisite for the peripheral SR to
function as a buffer to Ca2+
influx is maintenance of a storage reserve. That is, the peripheral SR
must have the ability to continuously pump at least some of the
incoming Ca2+ (2, 13). To explain
how such a reserve is maintained at rest and during stimulation, the
superficial buffer barrier model was modified to integrate an unloading
mechanism oriented toward the sarcolemma and able to operate
concomitantly with the Ca2+
pump-mediated uptake, i.e., the vectorial
Ca2+ release (28). Specifically,
the revised model states that SR Ca2+ release channels facing the
sarcolemma unload stored Ca2+ into
the subsarcolemmal space. This unloaded
Ca2+ would then be extruded by the
sarcolemma through the
Na+/Ca2+
exchanger and the Ca2+ pump. Van
Breemen and colleagues (28) recently reviewed the experimental evidence
for the SR superficial buffer barrier function. In this study we
examine a possible means of unloading
Ca2+ from superficial SR.
The endothelium has the ability to influence vascular SM
[Ca2+]i
in a manner that reduces contraction (23, 24) by targeting sarcolemmal
Ca2+ influx (6, 7, 15, 18, 22, 26,
31) and efflux (20), as well as SR
Ca2+ uptake (16, 21, 30) and
release (11, 20, 30). This is accomplished through the action of
endothelium-derived relaxing factors, including nitrosyl radicals such
as nitric oxide (NO). First, endothelium-derived relaxing
factor-induced sarcolemmal hyperpolarization could decrease
Ca2+ influx occurring through
L-type voltage-gated Ca2+
channels. This usually proceeds through activation of
K+ channels (15, 18, 22, 31),
although inhibition of the Na+-K+-ATPase
pump (7) and inactivation of
Cl
channels (6) are
implicated in some instances. Alternately, NO-induced increase in cGMP
concentration could directly inhibit L-type voltage-gated
Ca2+ channels (26). Authentic NO
and NO donors can also activate sarcolemmal
Ca2+ efflux (20) and SR
Ca2+ uptake (16, 21, 30) and
inhibit SR Ca2+ release (11, 20,
30). Finally, the agonist-stimulated endothelium and NO donors can
reduce contraction of isolated arterial segments without proportionally
reducing spatially averaged
[Ca2+]i,
as detected by fura 2 (3, 6, 10). This latter observation suggests that
the endothelium can reduce the apparent
Ca2+ sensitivity of the
contractile process by altering the subcellular distribution of
[Ca2+]i
or by altering the Ca2+
sensitivity of Ca2+-regulated
proteins involved in the contractile process.
In this study we tested the hypothesis that prior SR
Ca2+ depletion would promote the
SR superficial Ca2+ buffer barrier
function. By increasing the Ca2+
storage reserve capacity of the SR, prior SR
Ca2+ depletion would dampen the
effect of an increase in Ca2+
influx on contraction. We also tested the hypothesis that, with prior
SR Ca2+ depletion, the endothelium
promotes the SR Ca2+ buffer
barrier function by enhancing Ca2+
pump-mediated SR Ca2+ uptake. Our
results support the idea that the buffer barrier is dependent on the
Ca2+ storage reserve capacity of
the SR. They also suggest that the endothelium dampens the
[Ca2+]i
rise during increased Ca2+ influx,
but not through Ca2+ pump-mediated
SR Ca2+ uptake.
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MATERIALS AND METHODS |
Tissue and solutions.
The mandibular segment of the facial vein was isolated from adult New
Zealand male rabbits (2-4 kg) after anesthesia with pentobarbital
sodium (50 mg/kg iv combined with 1,000 U/kg heparin) and subsequent
exsanguination. In some tissues, endothelium was removed by gentle
rubbing of the intimal surface of the venous segments with a human hair.
The physiological salt solution (PSS) was of the following composition
(in mM): 140 NaCl, 4.7 KCl, 1.6 CaCl2, 1.0 MgCl2, 5.0 HEPES, and 11.0 glucose. The pH of the PSS was adjusted to 7.4 at 30°C with NaOH,
and the solution was bubbled with air. Venous rings were always exposed
to propranolol (1 µM), phentolamine (1 µM), and prazosin (1 µM)
to prevent possible activation of SM adrenergic receptors by endogenous
norepinephrine during K+-induced
depolarization. High-K+ PSS (25, 80, and 125 mM) were made by equimolar substitution of NaCl by KCl.
Ca2+-free PSS was made by omitting
CaCl2 with or without addition of
0.2 mM EGTA, as indicated.
Experimental protocol.
The venous rings were initially stretched in an isometric configuration
to an optimal resting tension of 1 mN. The rings were allowed to
recover for 
90 min, initially at room temperature and then for at
least the last 30 min at 30°C. The temperature was maintained at
30°C to avoid myogenic tone that develops in the facial vein at
higher temperature (17).
The rings were then exposed to 25 mM
K+ PSS (25K-PSS) for 30 min. After
they were washed for 30 min in PSS, the rings were contracted two to
four times for 5- to 10-min periods with 80 mM
K+ PSS (80K-PSS) alternating with
15-min wash periods in PSS. Exposures to 80K-PSS were repeated to
obtain reproducible levels of contraction. This stimulation sequence
allowed for the SR Ca2+ content to
reach its steady-state value through repeated depolarization-induced Ca2+ influx (2). The
endothelium-mediated relaxation was then assessed in each experiment by
the presence of an ACh (1 µM)-induced relaxation of the penultimate
80K-PSS-induced contraction. The endothelium-denuded rings either did
not respond to ACh or produced a small contraction.
After the above series of 80K-PSS exposures, rings were allowed to
recover for 25 min in PSS and then exposed to 25K-PSS for 5 min (Fig.
1). They were subsequently washed in a 0.2 mM EGTA Ca2+-free PSS for another
5 min. For the last 2 min in this
Ca2+-free PSS, they were exposed
to 20 mM caffeine, a maximally effective concentration, as determined
from preliminary experiments. However, the declining
[Ca2+]i
baseline, the short time to peak, and the apparent small amplitude of
the
[Ca2+]i
increase induced by caffeine in
Ca2+-free PSS (Fig. 1) precluded
the estimation of the size of the caffeine-sensitive SR
Ca2+ store by measure of the
associated
[Ca2+]i
transient. The amplitude of the associated contraction was used
instead. This contraction was used as an index of the total amount of
releasable Ca2+ stored by the SR
(14), since the caffeine-sensitive SR
Ca2+ store functionally included
the inositol 1,4,5-trisphosphate-sensitive SR
Ca2+ store, as demonstrated in
preliminary experiments with use of histamine as an inositol
1,4,5-trisphosphate-producing agonist.
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Fig. 1.
Experimental protocol illustrated by a typical experiment done with
intact endothelium. Bars 1, 2, and
3, between force and intracellular
Ca2+ concentration
([Ca2+]i)
traces, indicate time periods plotted in Figs. 2 and 3. Arrows on
[Ca2+]i
trace indicate when measurements reported in Table 1 were taken. CPA,
cyclopiazonic acid.
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The Ca2+ chelator EGTA was used
during Ca2+-free PSS exposure to
prevent reentry of Ca2+ released
from the SR by favoring Ca2+
extrusion from the SM cells. The venous rings were then washed for 20 min in a nominal Ca2+-free PSS to
prevent sarcolemmal ionic permeability alterations associated with EGTA
and vascular effects of caffeine other than the SR
Ca2+ release. SR reloading with
Ca2+ during this last period was
undetectable, inasmuch as additional exposure to caffeine (20 mM) or
histamine (1 µM) during this period in
Ca2+-free PSS failed to induce any contraction.
After depletion of the SR, venous rings were exposed to
Ca2+-containing 25K-PSS for 5 min,
and the developed force was monitored (Fig. 1). Tissues were then
washed in Ca2+-free PSS containing
EGTA for 5 min, and refilling of the SR with Ca2+ was assessed with caffeine.
The rings were then incubated in
Ca2+-free PSS (without EGTA)
containing 30 µM cyclopiazonic acid (CPA) for 20 min (Fig. 1). CPA is
a specific inhibitor of the SR
Ca2+ pump (5), and the
concentration used was maximally effective, as demonstrated in
preliminary experiments. The rings were then reexposed to 25K-PSS for 5 min in the presence of 30 µM CPA and washed in CPA-free
EGTA-containing Ca2+-free PSS for
5 min. The refilling of the SR with
Ca2+ was then assessed with caffeine.
Measurement of
[Ca2+]i.
Ring segments (2 mm long) were incubated for 6 h at 30°C in a
lateral shaker set at 1 Hz in PSS containing fura PE3-AM (15 µM),
Cremophor EL (0.36% vol/vol), and Pluronic F-127 (0.02% vol/vol) to
emulsify and solubilize fura PE3-AM, neostigmine (250 nM) to inhibit
extracellular esterases, and DMSO (0.18% vol/vol) to dissolve fura
PE3-AM and Pluronic F-127. Fura PE3 is an analog of fura 2 putatively
resistant to cellular leakage and compartmentalization into organelles
(29).
Fura PE3-loaded venous rings were mounted in an isometric configuration
in a continuously perfused (1.5 ml/min) cuvette in a modified
fluorometer (Scientific Instruments, Heidelberg, Germany). Excitation
light was obtained from a xenon high-pressure lamp (75 W) equipped with
a rotating filter wheel (125 Hz) with narrow-band (10-nm) interference
filters (340, 360, and 380 nm). The excitation light beam illuminated
the venous ring's outer surface at an angle of ~45° in the
horizontal plane, and the emitted light was collected vertically by a
photomultiplier, at right angles from the excitation light beam,
following its passage through a 510-nm low-pass filter. The emission signals produced at the three exciting wavelengths [fluorescence at 340, 360, and 380 nm
(F340,
F360, and
F380)] were collected
every 200 ms.
An in vivo calibration of the fluorescence signals was made at the end
of each experiment. Autofluorescence signals were subtracted from total
fluorescence signals before estimation of the minimum and maximum
ratios (Rmin and
Rmax).
[Ca2+]i
was estimated from the ratio,
R340/380 = F340/F380,
as follows
F380 max
and F380 min are expressed
as percentage of F360, i.e., fura
PE3 fluorescence on excitation at its isosbestic wavelength (362 nm)
(29). Kd, the
fura PE3-Ca2+ complex dissociation
constant, is 209 nM at 37°C and 204 nM at 20°C (in vitro) (29)
and was interpolated to be 206 nM at 30°C (25).
Rmax was 7.32 ± 0.63, Rmin was 0.93 ± 0.06, and
F380 max/F380 min was 4.11 ± 0.35 (n = 7);
these values were not significantly influenced by the endothelium.
Drugs.
The following drugs were dissolved in DMSO as stock solutions: CPA (10 mM; Sigma Chemical, St. Louis, MO), 4-bromo-A-23187 (4 mM; Sigma
Chemical), and ionomycin (10 mM; Calbiochem, La Jolla, CA). Fura PE3-AM
(Teflabs-Texas Fluorescence Laboratories, Austin, TX) was prepared in
DMSO as described above. The final concentration of DMSO to which the
tissues were exposed was <0.1%. All other chemicals were prepared in
double-distilled deionized water and were of analytic grade.
Data analysis.
Values are means ± SE. Comparisons between groups were made using a
paired or unpaired bilateral Student's
t-test or a one-way ANOVA for repeated
measures. A significant repeated-measures ANOVA was followed by a
Student-Newman-Keuls multiple comparison test. P 
0.05 was considered significant.
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RESULTS |
Effect of SR
Ca2+ depletion.
At the beginning of every experiment, repeated exposures to 80K-PSS
ensured repletion of the rabbit facial vein rings' SR Ca2+ store. Subsequent exposure of
these rings to 25K-PSS induced a rise in
[Ca2+]i
associated with contraction (Fig. 1, minutes
36-41 of typical traces, and Fig.
2, averaged data). To assess the effect of
SR Ca2+ depletion on the response
to 25K-PSS, the SR Ca2+ store was
depleted by removing extracellular
Ca2+ and exposing the rings to 20 mM caffeine (Fig. 1, minutes
41-66). This procedure led to reduced resting
[Ca2+]i
(Table 1). Reexposure of the venous rings
to 25K-PSS in the presence of extracellular
Ca2+ raised
[Ca2+]i
to a steady-state level similar to that reached previously with an
initially replete SR Ca2+ store
(Fig. 1, minutes 66-71, and Fig.
2, bottom). However, this [Ca2+]i
rise was not accompanied by contraction (Fig. 2,
top).
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Fig. 2.
Effect of sarcoplasmic reticulum (SR)
Ca2+ store initial content and CPA
(30 µM) on
[Ca2+]i
increase and associated force production induced by 25 mM
K+ physiological salt solution
(25K-PSS) in presence of endothelium. Data for time periods of
experimental protocol indicated by numbered bars between force and
[Ca2+]i
traces in Fig. 1 are summarized: "undepleted SR" plot
corresponds to bar 1, "depleted
SR" plot corresponds to bar 2,
and "depleted SR + CPA" plot corresponds to bar
3.
# Significant difference
between depleted SR + CPA and depleted SR and between depleted SR + CPA
and undepleted SR [P 0.05, Student-Newman-Keuls test (SNK) after repeated-measures ANOVA
(RM-ANOVA)]. * Significant difference between depleted SR
and undepleted SR (P 0.05, SNK
after RM-ANOVA).
& Significant difference
between depleted SR + CPA and undepleted SR
(P 0.05, SNK after RM-ANOVA).
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To assess the role of Ca2+
pump-mediated SR Ca2+ uptake in
this effect of SR Ca2+ depletion,
the SR was again depleted of Ca2+
by removal of extracellular Ca2+
and exposure to 20 mM caffeine (Fig. 1, minutes
71-96). The amplitude of the caffeine-induced
contraction was only about two-thirds that of the preceding
caffeine-induced contraction (Table 2). After this exposure to caffeine, the venous rings were exposed to the
specific SR Ca2+ pump inhibitor
CPA (30 µM) for 20 min in the absence of extracellular Ca2+ (Fig. 1,
minutes 76-96). An immediate
but transient increase in
[Ca2+]i,
without concomitant contraction, was observed. The venous rings were
then exposed a last time to 25K-PSS in the presence of extracellular
Ca2+ and 30 µM CPA (Fig. 1,
minutes 96-101). In contrast to
the previous exposure to 25K-PSS determined in the absence of CPA, the
rise in
[Ca2+]i
produced under these conditions was accompanied by a contraction (Fig.
2).
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Table 2.
Effect of initial SR Ca2+ store filling state and
endothelium on amplitude of caffeine-induced contraction after exposure
to 25K-PSS
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Influence of endothelium removal on the effect of SR
Ca2+ depletion.
The same experimental protocol was then applied to venous rings
stripped of their endothelium to assess endothelial influence on the
effect of SR Ca2+ depletion in
response to 25K-PSS. First, we observed that the contraction of
endothelium-denuded rings induced by 80K-PSS was larger than the
corresponding contraction of endothelium-intact rings, despite the fact
that the associated increase in
[Ca2+]i
was not different (Table 3). However, in
the presence of a replete SR Ca2+
store (Fig. 1, minute 35), resting
[Ca2+]i
was higher than in endothelium-intact rings (Table 1). The subsequent
exposure to 25K-PSS (Fig. 1, minutes
36-41) induced an increase in
[Ca2+]i
and an associated contraction not different from that observed in
endothelium-intact rings (Table 3).
The SR Ca2+ store was then
depleted, and the absolute amplitude of the caffeine-induced
contraction was larger than in endothelium-intact rings (Table 2, in
mN). The depletion procedure led to a reduction of resting
[Ca2+]i
(Fig. 1, minute 65) to a value
similar to that measured in endothelium-intact rings (Table 1). With a
now-depleted SR Ca2+ store,
reexposure to 25K-PSS in the presence of extracellular Ca2+ led to a larger increase in
[Ca2+]i
than observed in endothelium-intact rings (Fig.
3A,
bottom). This was accompanied by a transient
contraction that was not observed in endothelium-intact rings (Fig.
3A, top).
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Fig. 3.
A: effect of endothelium on
[Ca2+]i
increase and associated force production induced by 25K-PSS with an
initially depleted SR Ca2+ store.
B: effect of endothelium on
[Ca2+]i
increase and associated force production induced by 25K-PSS with an
initially depleted SR Ca2+ store
and in presence of 30 µM CPA. In A,
data are summarized for time period of experimental protocol indicated
by bar 2 in Fig. 1; in
B, data are summarized for time period
indicated by bar 3.
* Significant differences (P 0.05, unpaired bilateral Student's
t-test). Data obtained with
endothelium are replotted from Fig. 2.
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The SR Ca2+ store was depleted
again, and as observed in endothelium-intact rings, the amplitude of
the caffeine-induced contraction was only about two-thirds of the
preceding caffeine-induced contraction (Table 2). After a 20-min
incubation with 30 µM CPA in the absence of extracellular
Ca2+, the last exposure to 25K-PSS
(in the presence of extracellular Ca2+ and 30 µM CPA) induced a
rise in
[Ca2+]i
larger than in endothelium-intact rings (Fig. 3B,
bottom). However, the associated contraction was not
larger than in endothelium-intact rings (Fig. 3B,
top).
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DISCUSSION |
Effect of SR
Ca2+ depletion.
We hypothesized that prior Ca2+
depletion of the SR would promote the superficial
Ca2+ buffer barrier function by
increasing the SR Ca2+ storage
reserve capacity. This increased capacity would allow the superficial
SR to intercept a greater fraction of stimulated Ca2+ influx, resulting in a
dampened contraction. We tested this hypothesis by comparing the fura
PE3-detected
[Ca2+]i
increase and associated contraction caused by 25K-PSS-induced Ca2+ influx in isolated rings of
the rabbit facial vein before and after caffeine-induced SR
Ca2+ depletion (Fig. 1,
minutes 36-41 vs.
minutes 66-71). Our results show that SR Ca2+ depletion
abolished the subsequent 25K-PSS-induced contraction, although the
increase in steady-state
[Ca2+]i
was not attenuated (Fig. 2).
We verified that prior SR Ca2+
depletion facilitated the maintenance of the SR
Ca2+ storage reserve during the
period of 25K-PSS-induced Ca2+
influx (i.e., the maintenance of a submaximal steady-state load of
Ca2+, despite a persistent
Ca2+ influx). To do so, we
assessed the influence of SR Ca2+
depletion before exposure to 25K-PSS on the amplitude of the caffeine-induced contraction obtained immediately after the exposure to
25K-PSS (Fig. 1, minutes 44-46
vs. minutes 74-76). This
contraction was reduced by about one-third if the SR
Ca2+ was depleted before exposure
to 25K-PSS (Table 2). Because the maximal amplitude of this contraction
was attained within the 1st min of exposure to 25K-PSS (data not
shown), an insufficient refilling period cannot explain the observed reduction.
To demonstrate that the dissociation between 25K-PSS-induced
[Ca2+]i
increase and force production caused by prior SR
Ca2+ depletion was dependent on
Ca2+ pump-mediated SR
Ca2+ uptake, we repeated the
experimental procedure in the presence of the specific SR
Ca2+ pump inhibitor CPA (5) (Fig.
1, minutes 66-71 vs.
minutes 96-101). CPA not only
restored, but also greatly augmented, the 25K-PSS-induced contraction
(Fig. 2, top) to levels attained
during exposure to 80 mM K+, a
maximally effective K+
concentration. Others have determined that, in 
-escin-permeabilized vascular (9) and nonvascular (27) SM, CPA does not alter the
sensitivity of the contractile apparatus to
Ca2+. Thus the amplified
25K-PSS-induced contraction obtained in the presence of CPA would
correspond to a greater
[Ca2+]i
in the vicinity of the myofilaments.
Together, the results suggest that caffeine-mediated SR
Ca2+ depletion leads to a
dissociation of 25K-PSS-induced
[Ca2+]i
increase from force production that is dependent on
Ca2+ pump-mediated SR
Ca2+ uptake. They also suggest
that SR Ca2+ depletion increases
SR Ca2+ storage reserve during the
subsequent period of 25K-PSS-induced Ca2+ influx.
Influence of endothelium removal on the effect of SR
Ca2+ depletion.
We then tested the hypothesis that the endothelium modulates the
dissociation between
[Ca2+]i
and force by amplifying the SR superficial
Ca2+ buffer barrier function. We
did so by exposing endothelium-denuded rings to the same experimental
protocol used with endothelium-intact rings. Our results show that
endothelium removal amplified the increase in
[Ca2+]i
induced by 25K-PSS (Fig. 3A, bottom)
but only transiently restored contraction (cf. Fig.
3A, top with Table 3). This
amplification of the 25K-PSS-induced
[Ca2+]i
increase produced by endothelium removal was still evident in the
presence of CPA (Fig. 3B, bottom).
However, the associated contraction was not amplified compared with the
corresponding contraction of endothelium-intact rings (Fig.
3B, top).
The endothelium can dampen stimulation-induced
[Ca2+]i
increase in three ways, all potentially applicable to the present case, by 1) decreasing sarcolemmal
Ca2+ influx (6, 7, 15, 18, 22, 26,
31), 2) increasing SR
Ca2+ uptake (16, 21, 30), and
3) increasing sarcolemmal
Ca2+ efflux (20). It is unlikely
that the endothelium reduced 25K-PSS-induced Ca2+ influx in the present case,
because the contraction of intact venous rings was not smaller than the
contraction of denuded rings once CPA inhibited the SR
Ca2+ pump (Fig.
3B, top). This line of reasoning
presumes that the endothelium did not increase the apparent
Ca2+ sensitivity of the
contractile process. Also, the endothelium could not have minimized the
25K-PSS-induced
[Ca2+]i
increase by augmenting SR Ca2+
uptake, because CPA did not block this effect (Fig.
3B, bottom). Thus the endothelium
likely dampened the 25K-PSS-induced
[Ca2+]i
increase by augmenting Ca2+ efflux
through the sarcolemma. This is consistent with the fact that the
caffeine-induced contraction of venous rings with nondepleted SR was
reduced in the presence of endothelium (Table 2). This latter finding
suggests that either more of the
Ca2+ influx was extruded by the
sarcolemma instead of being pumped into the SR or more of the
Ca2+ influx pumped into the SR was
extruded by the sarcolemma through a vectorial
Ca2+ release process. Both
possibilities involve an augmented sarcolemmal Ca2+ efflux.
The previous considerations do not explain, however, why the
dissociation of fura PE3-detected
[Ca2+]i
from force production was heightened by endothelium removal (Fig. 3).
If it is presumed that the endothelium did not increase the apparent
Ca2+ sensitivity of the
contractile process, this latter finding suggests that the endothelium
reduced the proportion of
[Ca2+]i
in "noncontractile" myoplasm (i.e., devoid of myofilaments), such
as the subsarcolemmal space (1, 19). This interpretation is consistent
with the absence of active tone (as assessed by extracellular
Ca2+ removal) associated with the
increase in resting
[Ca2+]i
induced by endothelium removal (Table 1), as well as with an increased
sarcolemmal Ca2+ efflux, as
proposed above.
Together, the results suggest that after caffeine-induced SR
Ca2+ depletion the endothelium
reduces
[Ca2+]i
during 25K-PSS-induced Ca2+ influx
independently of Ca2+
pump-mediated SR Ca2+ uptake. We
propose that an increased sarcolemmal
Ca2+ efflux mediates this
endothelial effect.
Methodological issues.
In the experiments reported here, it is unlikely that changes in the
fura PE3 signal reflect compartmentalization of fura PE3 into the SR.
The physicochemical properties of this indicator dye support this
presumption. First, fura PE3 is a zwitterionic fura 2 analog that is
more resistant to compartmentalization into organelles than fura 2 (29). Second, fura PE3's
Kd is estimated to be 200 nM (29), whereas direct estimates of SR free
Ca2+ concentration in SM cells (8)
suggest that the latter remains in the micromolar range, even after SR
depletion. Therefore, any fura PE3 compartmentalized into the SR would
be permanently saturated with Ca2+
(29). The rapid decrease of the fura PE3 ratio that we observed on
extracellular Ca2+ removal and SR
depletion (Fig. 1) thus likely resulted only from the mere reduction of
myoplasmic Ca2+ concentration and
not directly from the concomitant decrease in SR free
Ca2+ (10).
During our fura PE3-loading procedure, endothelial cells, as well as SM
cells, were almost certainly loaded. However, it is unlikely that our
fluorometer collected any significant endothelium-derived signals. The
position of the tissue in the cuvette was such that the excitation
light beam focused on the adventitial side; the fluorescent light was
also collected from this adventitial side, but at a right angle to the
excitation beam. Analogous geometric considerations were shown to
virtually eliminate the endothelial contribution to the measured
fluorescent signal (23). In addition, all
[Ca2+]i
values were larger on endothelium removal, except in the case of
resting
[Ca2+]i
in the absence of extracellular
Ca2+ and with a depleted SR, where
they were equal (Table 1), arguing against the contribution of
endothelial
[Ca2+]i
to the recorded
[Ca2+]i.
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ACKNOWLEDGEMENTS |
We are grateful to Drs. Joseph E. Brayden and David W. Maughan for
comments on the manuscript.
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FOOTNOTES |
This work was supported by a research fellowship from the Medical
Research Council of Canada (R. Laporte) and by an operating grant from
the Heart and Stroke Foundation of Canada (I. Laher).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Laher, Dept.
of Pharmacology and Therapeutics, 2176 Health Sciences Mall, Faculty of
Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
(E-mail: laher{at}interchg.ubc.ca).
Received 25 March 1998; accepted in final form 23 March 1999.
| |
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Am J Physiol Heart Circ Physiol 277(2):H749-H755
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society
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ABSTRACT
INTRODUCTION
![[Ca<SUP>2+</SUP>]<SUB>myo</SUB> = <IT>K</IT><SUB>d</SUB> ∗ <FR><NU>F<SUB>380 max</SUB></NU><DE>F<SUB>380 min</SUB></DE></FR> ∗ <FR><NU>R<SUB>340/380</SUB> − R<SUB>min</SUB></NU><DE>R<SUB>max</SUB> − R<SUB>340/380</SUB></DE></FR>](/content/vol277/issue2/fulltext/H749/img001.gif)
