Vol. 274, Issue 2, H494-H499, February 1998
Modulatory role of endothelial calcium level in vascular tension
of canine depolarized coronary arteries
Kazuo
Sato,
Jun
Yamazaki, and
Taku
Nagao
Laboratory of Pharmacology and Toxicology, Graduate School of
Pharmaceutical Sciences, University of Tokyo, Tokyo 113, Japan
 |
ABSTRACT |
The vascular
tension in the coronary artery is modulated by factors released by
endothelial cells. We investigated the relationship between the Ca2+ level in
endothelium and endothelium-mediated changes in smooth muscle tone in
high K+-depolarized canine
coronary arteries by measuring intracellular Ca2+ concentration
fluorimetrically with the Ca2+
indicator fura 2. Addition of Ca2+
(1 mM) caused an increase in endothelial
Ca2+ and relaxed the 30 mM
K+-depolarized arteries following
inhibition of Ca2+ influx in the
smooth muscle with diltiazem. This relaxation was inhibited by
NG-monomethyl-L-arginine. As
extracellular K+ concentration was
decreased, increases of endothelial
Ca2+ were augmented, whereas the
relaxation was decreased. Basal muscle tone was found to be decreased
in low K+ by measuring relaxation
by sodium nitroprusside. These results suggest the importance of
Ca2+ level in the endothelium in
playing a modulatory role in coronary tension through the production of
nitric oxide. The correlation of extracellular
K+ to
Ca2+ level in the endothelium
indicates a typical characteristic of the passive
Ca2+ entry pathway in the
endothelium, whereas the resultant relaxation appears to be restricted
by the basal muscle tone.
nitric oxide; diltiazem; potassium; fura 2
| |
INTRODUCTION |
VASCULAR SMOOTH MUSCLE TONE is known to be regulated by
endothelial mediators such as endothelium-derived relaxing factor. Endothelium-derived relaxing factor, also known as nitric oxide (NO) or
its analogous compound (8, 14, 16), is produced from
L-arginine by
Ca2+-calmodulin-dependent NO
synthase (17), activates guanylate cyclase [which causes an increase
in guanosine 3',5'-cyclic monophosphate (cGMP)
concentration in vascular smooth muscle], and leads to smooth muscle
relaxation (5). Additionally, Ca2+
influx in cultured endothelial cells has been shown to be a crucial step for the production of NO (12). Several endothelial
Ca2+ influx pathways have been
reported through which Ca2+ enters
down an electrochemical gradient, such as a nonselective cation
channel, a store-operated
Ca2+-permeable channel, and a
channel that is stretch or shear stress activated (1, 7, 15). In
cultured endothelial cells, the electrochemical gradient generated by
membrane potential modulates basal or agonist-induced
Ca2+ influx (2, 11, 13, 22).
The influx of Ca2+ in smooth
muscle cells through voltage-dependent
Ca2+ channels initiates
contraction of the depolarized arteries. However, the influx of
Ca2+ in endothelial cells may not
play a major role in the maintenance of vascular tone due to the
presence of a smaller electrochemical gradient for
Ca2+ in the depolarized
endothelial cells than those at resting membrane potential. This
appears to be theoretically predicted, although this has not been shown
in endothelial cells of intact vessels. The simultaneous measurement of
intracellular Ca2+ in endothelial
cells and tension of the smooth muscle serves as a useful approach in
understanding the role of endothelial Ca2+ levels and vascular tone (4,
21). To rule out the possibility of
Ca2+ entry in the smooth muscle,
use of Ca2+ channel blockers serves as an advantage, since
voltage-dependent L-type channels are not present in endothelial cells
(3, 9).
The aim of the present study was to measure endothelial
Ca2+ level simultaneously with
smooth muscle tension in different extracellular K+ concentrations in the intact
dog coronary artery. To determine the endothelial effect, the vessels
were treated with the Ca2+ channel
blocker diltiazem. In this study, we demonstrated that the
Ca2+ level in endothelial cells of
intact vessels regulates smooth muscle tension by releasing NO in an
external K+-dependent manner.
| |
MATERIALS AND METHODS |
Chemicals. The drugs used in this
study were the following:
CaCl2 · 2H2O
(Kanto Chemical, Japan), acetylcholine chloride (ACh, Daiichi
Pharmaceutical, Japan), diltiazem HCl (Tanabe Seiyaku, Japan), sodium
nitroprusside (SNP, Nacarai Tesque, Japan), cremophor EL (Nacarai
Tesque), tetrakis(2-pyridylmethyl)ethylene-diamine (TPEN, Molecular
Probes), and fura 2-acetoxymethylester (AM) (Dojin Chemical
Laboratories, Japan).
NG-monomethyl-L-arginine
(L-NMMA) was synthesized at the
Organic Chemistry Research Laboratories (Tanabe Seiyaku, Japan). The
fluorescent Ca2+ indicator fura
2-AM was prepared as a stock solution (1 mM) in dimethyl sulfoxide. The
other compounds were directly dissolved in distilled water.
Preparations. Mongrel dogs weighing
12-26 kg were anesthetized with pentobarbital sodium (30 mg/kg iv)
and then exsanguinated. The heart from each dog was
immediately excised, and the circumflex branch of the left coronary
artery (outer diameter, 2.0-2.5 mm) was dissected. The artery was
cleared of adhering connective tissue and cut into strips (1.5 × 10 mm) in physiological salt solution (PSS) containing (in
mM) 136.9 NaCl, 5.4 KCl, 1.5 CaCl2, 1.0 MgCl2, 20 HEPES, and 5.5 glucose
(pH = 7.4) and aerated with 100%
O2. In denuded preparations, the
endothelium was mechanically removed by gentle rubbing of the intimal
surface with a cotton swab.
Intracellular
Ca2+ and muscle
tension determination.
Cytosolic Ca2+ was measured with a
Ca2+-sensitive dye fura 2 (6). The
muscle strips were loaded with 7 µM fura 2-AM for 5-7 h in PSS
at room temperature. During the loading procedure, a noncytotoxic
detergent, cremophor EL (0.02%), was added to increase the solubility
of fura 2-AM. To eliminate the possible quenching effect of endogenous
heavy metal ions, 10 µM TPEN was also added. After the loading period
was completed, the strips were rinsed with PSS for 30 min.
The fura 2-loaded muscle strip was incubated with PSS and maintained at
37°C and aerated with 100%
O2. Fluorescence was measured by
pinning one end of the strip to a silicon rubber sheet laid on the
bottom of the organ bath (5 ml volume) and mounted on a dual-excitation
fluorescence spectrophotometer (CAF-100, Japan Spectroscopic, Japan)
(20, 21). The other end of the muscle strip was connected to a
strain-gauge transducer (SB-1T, Nihon Kohden, Japan) for the
measurement of isometric tension with a resting tension of 1.0 g. The
muscle strip was exposed alternatively (48 Hz) to the two excitation
wavelengths (340 and 380 nm), and the emission wavelength (500 nm) was
collected through a photomultiplier tube. The fluoresence signals
evoked by 340 and 380 nm were referred to as
F340 and
F380, respectively.
F340/F380
was used as an index of intracellular
Ca2+ in the present experiments.
Under this experimental condition, fura 2 signals tended to become
small with time (see Fig. 1). Although a
possible photobleaching of the fluoresence signals may have affected
the total amount of each signal,
F340/F380
was able to be recorded clearly at least up to 45 min, since such artifacts included in each signal should have been canceled in the
calculated ratio as an index of
Ca2+ level. We did not subtract
any background fluorescence from each signal, since the level of
background fluorescence was much lower compared with the fluorescence
of Ca-fura 2. To measure fluorescence that was mostly of an endothelial
origin, the endothelial surface was exposed to both excitation
wavelengths. In some experiments, the fluorescence was measured at the
adventitial surface.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Representative recording of change in fluorescence
(F340 and
F380) ratio
(F340/F380)
and tension induced by application of 30 mM KCl, 10 µM acetylcholine
(ACh), and 1 µM sodium nitroprusside (SNP) in canine coronary
arteries loaded with fura 2. Arteries were preincubated in normal
physiological salt solution (PSS) containing 1.5 mM
Ca2+. Intact, preparation with
endothelium; denuded, preparation without endothelium; reversed intact,
fluorescence was meaured at adventitial surface of intact preparation.
Resting tension was set at 1.0 g before KCl was added. Baseline values
of
F340/F380
were 0.59, 0.52, and 0.55 in intact, denuded and reversed intact
preparations, respectively.
|
|
Experimental protocols. In the present
study, there were three types of vessel preparations:
endothelium-intact (intact preparation), endothelium-denuded (denuded
preparation), and endothelium-intact arteries with the adventitial side
facing the light (reversed intact preparation).
The effects of ACh (10 µM) were first tested to ensure that we
measured both intracellular Ca2+
in endothelial cells and muscle tension of the muscle strips. After we
incubated the muscle strips in PSS containing 1.5 mM Ca2+, a 30 mM
K+-containing solution (NaCl was
substituted with an equimolar amount of KCl) was added.
ACh was added to the bath after the contraction had reached a
steady-state level.
For experiments having Ca2+ and
SNP effects, muscle strips were first equilibrated in
Ca2+-free, 30 mM
K+-containing PSS (no
Ca2+-chelating agent was
added). Afterward, a 1 mM
Ca2+-containing solution was
added. In experiments where the
Ca2+ channel blocker diltiazem was
used, muscle strips were first equilibrated in
Ca2+-free, 30 mM
K+-containing PSS. Thereafter, 3 µM diltiazem was added to the baths to block
Ca2+ uptake in the smooth muscle.
Ten minutes after the addition of diltiazem,
Ca2+ (1 mM) or SNP (1 µM) was
applied to the baths. The NO synthase inhibitor
L-NMMA (0.1 mM) was applied 5 min before the addition of diltiazem. The same experiments were also
performed in 5.4 and 80 mM
K+-containing PSS, prepared by
replacing NaCl with an equimolar amount of KCl.
Statistics. Results are expressed as
means ± SE. Statistical comparisons between groups were carried out
using Student's t-test. Analysis of
variance and Bonferroni's multiple
t-test were used to compare more than
two groups. Values of P < 0.05 were
considered significant.
| |
RESULTS |
Effects of ACh on canine coronary
arteries. To validate our methodology of simultaneously
recording endothelial intracellular Ca2+ and smooth muscle tension,
the effect of ACh was tested in the three vessel preparations under
depolarizing conditions. Initially, the muscle strips were contracted
with 30 mM KCl (Figs. 1 and 2,
A and
B). Under these conditions, the high
K+ caused an increase in
F340/F380
and muscle tension in all three preparations. There were no discernible
differences of changes in
F340/F380
or muscle contraction among the three preparations. The increase in
ratio was indicative of an increase in intracellular Ca2+ in the smooth muscle.
Addition of ACh (10 µM) induced relaxation of the intact preparation
as well as an increase in
F340/F380, whereas the relaxation and
F340/F380
of the denuded preparation were inhibited (Figs. 1 and 2,
C and
D). This suggested that ACh caused
the increase in endothelial intracellular
Ca2+ and a resultant relaxation of
the smooth muscle. The SNP-induced relaxation was observed in the
endothelium-denuded preparation, indicating that the cGMP-mediated
response was not damaged in the smooth muscle. In the reversed intact
preparation, an increase in
F340/F380
was not detected, although the ACh-induced relaxation was obvious.
Therefore, the emitted fluorescence appears to be effectively collected
from the surface facing the excitation wavelengths.
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of 30 mM KCl (A and
B) and 10 µM ACh
(C and
D) on tension and
F340/F380
in canine coronary arteries loaded with fura 2. Arteries were
preincubated in normal PSS containing 1.5 mM
Ca2+. Intact, preparation with
endothelium; denuded, preparation without endothelium; reversed intact,
fluorescence was measured at adventitial surface of intact preparation.
A: resting tension was set at 1.0 g
before KCl was added. B: baseline
values of
F340/F380
were 0.57 ± 0.02, 0.58 ± 0.02, and 0.52 ± 0.01 in intact,
denuded, and reversed intact preparations, respectively. Changes in
tension and
F340/F380
before and after addition of ACh are shown in
C and
D, respectively. Each datum represents
mean of 45 experiments with SE.
** P < 0.01 compared with
intact arteries.
|
|
Effects of
Ca2+ or SNP on
canine coronary arteries.
To investigate the relationship between the increase in intracellular
Ca2+ in endothelial cells and
smooth muscle tension in depolarized arteries, the effect of 1 mM
Ca2+ was determined. First, the
effect of the addition of Ca2+ was
studied in the absence of diltiazem (Fig.
3). Application of
Ca2+ caused an increase in
F340 / F380
and muscle tension in the intact preparation in the 30 mM
K+-containing PSS. Removal of
endothelium caused the contraction to become slightly stronger 15 min
after the addition of Ca2+,
although this was not statistically significant (0.166 ± 0.033 g in
the intact; 0.225 ± 0.011 g in the denuded preparation;
P > 0.05;
n = 6). On the other hand, the
increase in
F340/F380
was significantly inhibited in the denuded preparation 15 min after the
addition of Ca2+ (0.021 ± 0.005; P < 0.01;
n = 6) when compared with the intact preparation (0.066 ± 0.003; n = 6).
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of 1 mM Ca2+ on tension
(A) and
F340 /F380
(B) in absence of diltiazem in
canine depolarized (30 mM KCl) arteries loaded with fura 2. Arteries
were preincubated in nominally
Ca2+-free PSS.  , Intact;  ,
denuded. A: resting tension was set at
1.0 g before Ca2+ was added.
B: baseline values of
F340 /F380
were 0.43 ± 0.01 and 0.43 ± 0.01 in intact and denuded
preparations, respectively (B). Each
datum represents mean of 6 experiments with SE.
|
|
Second, the effect of the addition of
Ca2+ was studied in the presence
of diltiazem (Figs. 4 and
5). After an extensive washout of external
Ca2+ in the presence of 30 mM KCl,
the tissue tension was not changed by the addition of 3 µM
diltiazem. Application of 1 mM
Ca2+ to the tissue bath caused an
increase in intracellular Ca2+,
which was accompanied with relaxation of smooth muscle in the intact
preparation in the 30 mM
K+-containing PSS. The
Ca2+-induced relaxation was
significantly inhibited in the denuded preparation and in the intact
preparation pretreated with the NO synthase inhibitor
L-NMMA (0.1 mM). As shown in
Fig. 5A, 15 min after the addition of
Ca2+, the decrease in muscle
tension was 0.075 ± 0.005 g in the intact preparation, 0.014 ± 0.006 g in the denuded preparation (P < 0.01; n = 5) and 0.027 ± 0.005 g in the L-NMMA-treated
preparation (P < 0.01 vs. intact
preparation; n = 5). As
shown in Fig. 5B, 15 min after the
addition of Ca2+, the increase in
F340/F380
was significantly inhibited in the denuded preparation (0.000 ± 0.004; P < 0.01;
n = 5) compared with the intact
preparation (0.056 ± 0.010; n = 5). In contrast, F340/F380
was not altered in the
L-NMMA-treated preparation
(0.066 ± 0.014; P > 0.05 vs. intact preparation; n = 5).
These results suggest that an increase in
Ca2+ in endothelial cells precedes
NO production and the resultant relaxation of the coronary arteries.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Representative recording of change in
F340 /F380
and tension induced by 1 mM Ca2+
in presence of diltiazem in canine depolarized (30 mM KCl) arteries
loaded with fura 2. Arteries were preincubated in nominally
Ca2+-free PSS.
Ca2+ was applied 10 min after
addition of 3 µM diltiazem.
NG-monomethyl-L-arginine
(L-NMMA, 0.1 mM) was applied
5 min before the addition of diltiazem. Resting tension was set at 1.0 g before Ca2+ was added. Baseline
values of
F340 /F380
were 0.44, 0.43, and 0.43 in intact, denuded, and intact with
L-NMMA, respectively.
|
|
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of 1 mM Ca2+ on tension
(A) and
F340/F380
(B) in presence of diltiazem in
canine depolarized (30 mM KCl) arteries loaded with fura 2. Arteries
were preincubated in nominally
Ca2+-free PSS.
Ca2+ was applied 10 min after
addition of 3 µM diltiazem.
L-NMMA (0.1 mM) was applied 5 min before addition of diltiazem. , Intact; , denuded;  ,
intact treated with L-NMMA.
A: resting tension was set at 1.0 g
before Ca2+ was added.
B: baseline values of
F340/F380
were 0.43 ± 0.01, 0.44 ± 0.01, and 0.43 ± 0.01 in intact,
denuded, and intact with L-NMMA,
respectively (B). Each datum
represents mean of 5 experiments with SE.
|
|
Application of the NO donor, SNP, induced a significant relaxation of
the smooth muscle and a slight decrease in
F340/F380 in the 30 mM K+-containing PSS
without external Ca2+. Removal of
endothelium or pretreatment with
L-NMMA had no effect on the
SNP-induced relaxation. Fifteen minutes after the addition of SNP, the
decrease in tension for the intact, denuded, and
L-NMMA preparations was 0.076 ± 0.007, 0.072 ± 0.017, and 0.071 ± 0.010 g, respectively
(n = 4), whereas the changes in
F340/F380
were 0.011 ± 0.005, 0.000 ± 0.003, and 0.000 ± 0.003, respectively (n = 4).
Effects of extracellular K+
concentration on Ca2+- or SNP-induced
relaxation and F340/F380 changes in canine
coronary arteries. To investigate the effect of changing
extracellular K+ concentration on
Ca2+ influx in endothelial cells,
the Ca2+-induced relaxation and
changes in
F340/F380
were measured in the presence of diltiazem (Table
1). The
Ca2+-induced change in
F340/F380
increased as extracellular K+
concentration was decreased. The increase in
F340/F380
in 5.4 mM K+ was significantly
greater than that observed in 80 mM
K+. On the other hand, the
Ca2+-induced relaxation decreased
as extracellular K+ concentration
was lowered. The relaxation observed in 5.4 mM K+ was significantly
smaller than that observed in 80 mM
K+.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effects of extracellular K+ concentration on 1 mM Ca2+- and 1 µM SNP-induced
relaxation and increases in F340/F380 in intact
canine coronary arteries
|
|
To test the resting tone in those three extracellular
K+ concentrations, the SNP-induced
relaxations in canine coronary arteries were also measured under the
same condition described above (Table 1). The SNP-induced changes in
F340/F380
were similar under all three conditions, whereas SNP-induced relaxation
tended to decrease as extracellular
K+ concentration decreased. The
amount of relaxation measured at each
K+ concentrations was similar to
that induced by 1 mM Ca2+.
| |
DISCUSSION |
The aim of the present study was to investigate the relationship
between Ca2+ level in endothelial
cells and the endothelium-mediated regulation of smooth muscle tension
in the K+-depolarized canine coronary arteries. Application
of Ca2+ to the endothelium intact
muscle strips caused a significant increase in the fura
2-to-Ca2+ fluorescence ratio
(F340/F380)
and muscle contraction. More importantly was our initial observation
that removal of endothelium significantly inhibited an increase in
fluorescence ratio, giving us an idea that an endothelium-dependent
Ca2+ signal is present in the
K+-depolarized preparation. Using
a Ca2+ channel blocker to rule out
the possibility of Ca2+ entry in
the smooth muscle, we demonstrated in the present study that the
Ca2+ level in endothelial cells of
intact vessels regulates smooth muscle tension through NO release in an
external K+-dependent manner.
Our results measuring ACh-induced changes in endothelial intracellular
Ca2+ and smooth muscle tension
clearly demonstrated that this method is successful in determining both
parameters simultaneously. When the muscle strip was depolarized with
30 mM KCl, an increase in F340/F380
was observed in both intact and denuded preparations, suggesting that
the increase in intracellular Ca2+
represents the K+-induced
Ca2+ influx in smooth muscle
cells. Subsequent application of ACh caused an additional increase in
F340/F380
and a corresponding relaxation of smooth muscle relaxation, which was
endothelium mediated. Thus, under our experimental condition, we were
able to measure Ca2+ influx in the
endothelium and also the subsequent modulation of the muscle tone. This
conclusion was further supported by the effectiveness of SNP-induced
smooth muscle relaxation in the denuded preparation with no apparent
damage in the muscle preparation.
To confirm that the fluorescence measurements were obtained from
endothelial cells, we also measured the fluorescence ratio from the
adventitial side by exposing either side of the muscle strip to the
excitation wavelengths. ACh caused an increase in F340/F380
in the endothelial surface but was without effect in the adventitial
surface. This indicates that more fluorescence is emitted from
endothelial cells when they are exposed to the excitation light and
further supports the previous findings in rat and rabbit aortae (4,
21).
In the intact preparation, an increase in intracellular
Ca2+ of endothelial cells is
expected to decrease the
K+-induced contraction due to the
role of Ca2+ in initiating the
release of relaxing factors such as NO. We found, however, that the
removal of the endothelium caused a small change in smooth muscle
contraction. It may be that the contractile response is too strong to
observe any clear and definite smooth muscle relaxation mediated by the
endothelium. To circumvent this problem, the
Ca2+ channel blocker diltiazem was
used to block Ca2+ entry in the
smooth muscle, and in this manner we could observe the modulation of
tension by the endothelium. We can also rule out any effect of
diltiazem on endothelial cells, since they do not possess a
voltage-dependent Ca2+ channel (3,
9). Moreover, it seems to be advantageous that the resting tone is
still preserved under our experimental condition in the presence of
diltiazem and nominally Ca2+-free
PSS (10, 23, 24).
In the presence of diltiazem, application of
Ca2+ caused a significant
sustained increase in intracellular
Ca2+, which was accompanied with
relaxation of the smooth muscle in the intact preparation. The increase
in intracellular Ca2+ and
relaxation was significantly inhibited in the denuded preparation. When
the intact preparation was pretreated with the NO synthase inhibitor,
L-NMMA, the
Ca2+-induced relaxation was
significantly inhibited but the
Ca2+ signal remained unaffected.
These results suggest that the cytosolic Ca2+ level of endothelial cells
increases initially and this leads to the production of NO, which
subsequently leads to smooth muscle relaxation. Removal of endothelium
or pretreatment of L-NMMA did not appear to have an effect on smooth muscle relaxation, since SNP-induced relaxation or Ca2+
signals were not affected.
It is interesting to note that
Ca2+ level in endothelium can
cause a marked relaxation in the presence of 30 mM
K+ due to the presence of a
smaller driving force for Ca2+ in
the depolarized cells. Therefore, we examined the correlation between
Ca2+-induced change (i.e.,
F340/F380)
or Ca2+-induced relaxation and
extracellular K+ concentrations
(5.4, 30, and 80 mM). The
Ca2+-induced change in the
F340/F380
increased as the extracellular K+
concentration decreased, suggesting that the
Ca2+ level in the endothelium of
intact vessels is likely to be determined by the electrochemical
driving force for Ca2+, which is
determined by membrane potential. Our result also suggests that the
membrane depolarization in 80 mM
K+ solution still provides the
driving force for Ca2+ influx to
increase the endothelial Ca2+
concentration. Cannell and Sage (2) reported that in cultured bovine
pulmonary artery endothelial cells, intracellular
Ca2+ under unstimulated conditions
was augmented by hyperpolarization. Moreover, it was suggested that
Ca2+ influx in endothelial cells
occurs via a passive permeability pathway, i.e., via a channel rather
than through an antiporter system (e.g.,
Na+/Ca2+
exchange), since Ca2+ influx
induced by bradykinin was attenuated by depolarization (11, 22). The
resting potential in endothelial cells is known to vary greatly even in
the same cell type (between 
10 and 70 mV) due to two
types of ion conductances, i.e.,
K+ and
Cl
(15). Although the
resting potential cannot be estimated in each
K+ concentration because of the
heterogeneous expression of these channels, the present results suggest
that changes in the membrane potential are an important regulator of
intracellular signal transduction in the endothelium of intact vessels
by modulating the driving force for transmembrane
Ca2+ fluxes.
In contrast to the increase in intracellular
Ca2+ measured in endothelium, the
Ca2+-induced relaxation decreased
as extracellular K+ concentration
was decreased. We have previously found in canine coronary arterial
ring preparations that the degree of relaxation is dependent on the
resting tone, which is affected by extracellular K+ concentration (23). Similarly,
in the present study, SNP-induced relaxation decreased as extracellular
K+ concentration was lowered. The
amount of SNP-induced relaxation was almost identical to that induced
by Ca2+ in all three extracellular
K+ concentrations tested. These
results indicate that a smaller resting tone may restrict an available
Ca2+-induced relaxation, even
though Ca2+ influx in endothelial
cells is increased in the lowest extracellular K+ concentration.
We cannot rule out the possibility that the
Ca2+-induced change in
F340/F380
may have been underestimated by a possible decrease of
Ca2+ in smooth muscle cells due to
NO. However, this seems unlikely because the SNP-induced changes in
F340/F380
were similar in all three extracellular
K+ concentrations. It has been
reported that SNP or nitroglycerin inhibits high
K+-induced contraction not only by
inhibiting Ca2+ influx but also by
decreasing the sensitivity of contractile elements to
Ca2+ (18, 19, 25). Therefore, the
present study suggests that any change in intracellular
Ca2+ of smooth muscle by NO
released from endothelium is negligible.
In conclusion, endothelial Ca2+
level of canine coronary arteries can be successfully monitored from
simultaneous measurements of intracellular
Ca2+ employing the
Ca2+-fluorescent dye fura 2 and
muscle tension. We provide evidence that the
Ca2+ level in endothelium of
intact vessels is dependent on the external K+ concentration, indicating
passive Ca2+ entry pathway in the
endothelium, and the resultant NO-mediated relaxation of smooth muscle
cells appears to be restricted by the resting muscle tone.
| |
ACKNOWLEDGEMENTS |
The authors thank Drs. Hideaki Karaki and Koichi Sato (University
of Tokyo) for advice on the fluorescence measurement and Dr. Victor
Ruiz-Velasco (University of Nevada Reno) for critical reading of the
manuscript.
| |
FOOTNOTES |
This study was supported by a Grant-in-Aid from the Ministry of
Education, Science, Sports and Culture, Japan.
Address for reprint requests: T. Nagao, Laboratory of Pharmacology and
Toxicology, Graduate School of Pharmaceutical Sciences, University of
Tokyo, Tokyo 113, Japan.
Received 27 June 1997; accepted in final form 16 October 1997.
| |
REFERENCES |
1.
Adams, D. J.,
J. Baraakeh,
R. Laskey,
and
C. van Breemen.
Ion channels and regulation of intracellular calcium in vascular endothelial cells.
FASEB J.
3:
2389-2400,
1989[Abstract].
2.
Cannell, M. B.,
and
S. O. Sage.
Bradykinin-evoked changes in cytosolic calcium and membrane currents in cultured bovine pulmonary artery endothelial cells.
J. Physiol. (Lond.)
419:
555-568,
1989[Abstract/Free Full Text].
3.
Colden-Stanfield, M.,
W. P. Schilling,
A. K. Ritchie,
S. G. Eskin,
L. T. Navarro,
and
D. L. Kunze.
Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells.
Circ. Res.
61:
632-640,
1987[Abstract/Free Full Text].
4.
Demirel, E.,
J. Rusko,
R. E. Laskey,
D. J. Adams,
and
C. van Breemen.
TEA inhibits ACh-induced EDRF release: endothelial Ca2+-dependent K+ channels contribute to vascular tone.
Am. J. Physiol.
267 (Heart Circ. Physiol. 36):
H1135-H1141,
1994[Abstract/Free Full Text].
5.
Griffith, T. M.,
D. Edwards,
M. J. Lewis,
and
A. H. Henderson.
Evidence that cyclic guanosine monophosphate (cGMP) mediates endothelium-dependent relaxation.
Eur. J. Pharmacol.
122:
195-202,
1985.
6.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract/Free Full Text].
7.
Himmel, H. M.,
A. R. Whorton,
and
H. C. Strauss.
Intracellular calcium, currents, and stimulus-response coupling in endothelial cells.
Hypertension
21:
112-127,
1993[Abstract/Free Full Text].
8.
Ignarro, L. J.,
G. M. Buga,
K. S. Wood,
R. E. Byrns,
and
G. Chaudhuri.
Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.
Proc. Natl. Acad. Sci. USA
84:
9265-9269,
1987[Abstract/Free Full Text].
9.
Jayakody, R. L.,
C. T. Kappagoda,
M. P. J. Senaratne,
and
N. Sreeharan.
Absence of effect of calcium antagonists on endothelium-dependent relaxation in rabbit aorta.
Br. J. Pharmacol.
91:
155-164,
1987[Medline].
10.
Kikkawa, K.,
S. Murata,
and
T. Nagao.
Endothelium-dependent calcium-induced relaxation in the presence of Ca2+-antagonists in canine depolarized coronary arteries.
Br. J. Pharmacol.
98:
700-706,
1989[Medline].
11.
Laskey, R. E.,
D. J. Adams,
A. Johns,
G. M. Rubanyi,
and
C. van Breemen.
Membrane potential and Na+-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria.
J. Biol. Chem.
265:
2613-2619,
1990[Abstract/Free Full Text].
12.
Luckhoff, A.,
U. Pohl,
A. Mulsch,
and
R. Busse.
Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells.
Br. J. Pharmacol.
95:
189-196,
1988[Medline].
13.
Luckhoff, A.,
and
R. Busse.
Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential.
Pflügers Arch.
416:
305-311,
1990[Medline].
14.
Myers, P. R.,
R. L. Minor, Jr.,
R. Guerra, Jr.,
J. N. Bates,
and
G. Harrison.
Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide.
Nature
345:
161-163,
1990[Medline].
15.
Nilius, B.,
F. Viana,
and
G. Droogmans.
Ion channels in vascular endothelium.
Annu. Rev. Physiol.
59:
145-170,
1997[Medline].
16.
Palmer, R. M. J.,
A. G. Ferrige,
and
S. Moncada.
Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.
Nature
327:
524-526,
1987[Medline].
17.
Palmer, R. M. J.,
D. D. Rees,
D. S. Athton,
and
S. Moncada.
L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation.
Biochem. Biophys. Res. Commun.
153:
1251-1256,
1988[Medline].
18.
Pfitzer, G.,
F. Hofmann,
J. Disalvo,
and
J. C. Ruegg.
cGMP and cAMP inhibit tension development in skinned coronary arteries.
Pflügers Arch.
401:
277-280,
1984[Medline].
19.
Pfitizer, G.,
L. Merkel,
J. C. Ruegg,
and
F. Hofmann.
Cyclic GMP-dependent protein kinase relaxes skinned fibers from guinea-pig taenia coli but not from chicken gizzard.
Pflügers Arch.
407:
87-91,
1986[Medline].
20.
Sato, K.,
H. Ozaki,
and
H. Karaki.
Change in cytosolic Calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator fura 2.
J. Pharmacol. Exp. Ther.
246:
294-300,
1988[Abstract/Free Full Text].
21.
Sato, K.,
H. Ozaki,
and
H. Karaki.
Differential effects of carbachol on cytosolic calcium levels in vascular endothelium and smooth muscle.
J. Pharmacol. Exp. Ther.
255:
114-119,
1990[Abstract/Free Full Text].
22.
Schilling, W. P.
Effect of membrane potential on cytosolic calcium of bovine aortic endothelial cells.
Am. J. Physiol.
257 (Heart Circ. Physiol. 26):
H778-H784,
1989[Abstract/Free Full Text].
23.
Yamazaki, J.,
K. Ohashi,
F. Ohara,
Y. Harada,
and
T. Nagao.
Calcium, barium and strontium cause endothelium-dependent relaxation of coronary arteries: species difference and the underlying mechanism.
In: The Biology of Nitric Oxide. Physiology and Clinical Aspects, edited by S. Moncada,
M. Feelisch,
R. Busse,
and E. A. Higgs. Essex, UK: Portland, 1994, part 3, p. 119-122.
24.
Yamazaki, J.,
F. Ohara,
Y. Harada,
and
T. Nagao.
Barium and strontium substitute for calcium in nitric oxide production in endothelium of canine coronary arteries.
Jpn. J. Pharmacol.
68:
25-32,
1995[Medline].
25.
Yanagisawa, T.,
M. Kawada,
and
N. Taira.
Nitroglycerin relaxes canine coronary arterial smooth muscle without reducing intracellular Ca2+ concentrations measured with fura 2.
Br. J. Pharmacol.
98:
469-482,
1989[Medline].
AJP Heart Circ Physiol 274(2):H494-H499
0363-6135/98 $5.00
Copyright © 1998 the American Physiological Society
Top
Abstract
Introduction
