1 Department of Pharmacology and Therapeutics and 2 Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan; and 3 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada
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ABSTRACT |
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The effects of
ruthenium red (RuR) on contractility were examined in skinned fibers of
guinea pig smooth muscles, where sarcoplasmic reticulum function was
destroyed by treatment with A-23187. Contractions of skinned fibers of
the urinary bladder were enhanced by RuR in a concentration-dependent
manner (EC50 = 60 µM at pCa
6.0). The magnitude of contraction at pCa 6.0 was increased to 320% of
control by 100 µM RuR. Qualitatively, the same results were obtained
in skinned fibers prepared from the ileal longitudinal smooth muscle
layer and mesenteric artery. The maximal contraction induced by pCa 4.5 was not affected significantly by RuR. The enhanced contraction by RuR
was not reversed by the addition of guanosine
5'-O-(2-thiodiphosphate) or a peptide
inhibitor of protein kinase C [PKC-(1931)]. The
application of microcystin, a potent protein phosphatase inhibitor,
induced a tonic contraction of skinned smooth muscle at low
Ca2+ concentration
([Ca2+]; pCa > 8.0).
RuR had a dual effect on the microcystin-induced contraction-to-
enhancement ratio at low concentrations and suppression at high
concentrations. The relaxation following the decrease in
[Ca2+] from pCa 5.0 to
>8.0 was significantly slowed down by an addition of RuR.
Phosphorylation of the myosin light chain at pCa 6.3 was significantly
increased by RuR in skinned fibers of the guinea pig ileum. These
results indicate that RuR markedly increases the
Ca2+ sensitivity of the
contractile system, at least in part via inhibition of myosin light
chain phosphatase.
phosphatase, skinned fiber, myosin light chain
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INTRODUCTION |
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THE RYANODINE RECEPTOR Ca2+-release channel (RyR) of the sarcoplasmic reticulum (SR) plays an obligatory role in excitation-contraction coupling in skeletal and cardiac muscles (6). Three isoforms (RyR-1, RyR-2, and RyR-3) have been identified (for a review, see Ref. 30). RyR-1 and RyR-2 are predominantly expressed in skeletal and cardiac muscles, respectively, whereas RyR-3 is widely expressed in other cell types, including neurons and smooth muscles (23). It has been reported that all these isoforms are expressed in vascular smooth muscles, but the physiological functions of RyRs in smooth muscles have not been fully elucidated (31).
Dantrolene, a specific inhibitor of RyR-1, suppresses the twitch contraction in skeletal muscle and is an effective treatment for malignant hyperthermia but has little or no effect on smooth or cardiac muscles (24, 31). Ruthenium red (RuR), [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6, blocks the SR Ca2+-release channel (34) and has been widely used as an inhibitor of Ca2+-induced Ca2+ release (25). The ability of RuR to impair Ca2+ release in neuronal cells has also been demonstrated (32). Because the inhibition of RyR-3 by RuR at relatively low concentrations (4, 9, 14) is similar to that of RyR-1 and RyR-2, the importance of RuR as a pharmacological tool to investigate the cellular functions of Ca2+ release through RyRs in the SR and endoplasmic reticulum of smooth muscles and neurons, respectively, has been increasing (22, 30).
Several additional effects of RuR have been reported (3, 21). An important finding with respect to the use of RuR as a pharmacological tool in smooth muscle is its inhibition of the binding of Ca2+ to calmodulin (26), which may cause inhibition of contraction. We report here that RuR markedly enhances the contraction induced by pCa 6.5-5.0 in skinned smooth muscle fibers of the guinea pig. Our results are not consistent with the results of Sasaki et al. (26) that would predict that RuR causes a decrease in the Ca2+ sensitivity of contraction. Ca2+ sensitization of smooth muscle contraction is involved in enhanced responses to some agonists without an increase in intracellular Ca2+ concentration ([Ca2+]i) (15, 27, 28). The present study was undertaken, therefore, to further investigate the effects of RuR on smooth muscle contractility and led to the characterization of RuR-induced Ca2+ sensitization in skinned smooth muscles.
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METHODS |
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Tension measurement.
Male Hartley guinea pigs, weighing 300-350 g, were stunned by a
blow on the head and immediately exsanguinated. The urinary bladder,
the terminal portion of the ileum, and the fourth branches of the
mesenteric artery were isolated. Muscle strips (250 µm in width, 100 µm in depth, and 1.5-2 mm in length) were dissected from the
urinary bladder and ileum. Ring preparations (300 µm in width) were
dissected from the mesenteric artery. For tension measurement, the
muscles were maintained horizontally between two hooks and immersed in
a pool of solution (300 µl) that was rounded by surface tension on a
rotation plate 40 mm in diameter. Tension measurements were performed
as described previously (33). To prevent the change in surface tension
and ionic strength of the solution caused by evaporation, the muscle
was transferred from pool to pool at an interval of ~10 min. The
transfer occasionally resulted in a spikelike artifact in a trace. The
electrical signals of the tension recording were filtered with a
low-pass filter at 10 Hz (3 dB). Strips were allowed to
equilibrate at a predetermined optimal resting tension of 100-200
mg for ~60 min before the start of experiments. The preparation was
then repeatedly exposed to 142.9 mM
K+ solution at intervals of ~30
min until the contraction was reproducible. Experiments were carried
out at room temperature (21-24°C).
Skinned-fiber preparation.
After the steady contractions induced by the 142.9 mM
K+ solution were measured, the
strips were incubated in relaxing solution containing 2 mM EGTA (R2G)
for 15 min. The skinning of the smooth muscle preparations was achieved
by incubation with 60 µM -escin in a solution of pCa 6.3 at room
temperature. After the skinning, the solution was changed to R2G. The
pCa in R2G solution was <8.0, assuming that the
Ca2+ contamination of the solution
was <50 µM. The contractile response to caffeine was tested in
relaxation solution containing 0.1 mM EGTA (R0.1G). When the
Ca2+ sensitivity of the
contractile response was studied in detail, it was found that the
function of intracellular
Ca2+ storage sites was removed by
treatment with 10 µM A-23187 for 20 min in R2G solution after skinning.
Measurement of myosin light-chain phosphorylation. Tissue was frozen in acetone-5% (wt/vol) TCA on dry ice for 30 min, transferred to 100% acetone, incubated for 30 min at room temperature, washed repeatedly with acetone, dried, and incubated in an extraction buffer (20 µl/mg tissue dry wt) for 2 h on a shaker before centrifugation at 6,400 rpm for 10 min at room temperature. The supernatant was subjected to urea-glycerol gel electrophoresis. Myosin from chicken gizzards was used as the control. Electrophoretic transfer of proteins to nitrocellulose sheets was carried out in 25 mM Tris-192 mM glycine-20% methanol, pH 8.3, at 200 mA for 4 h at room temperature. The nitrocellulose was incubated overnight with blocking reagent, then with anti-myosin light chain (20 kDa; MLC20) antibody in 0.5% blocking solution. After being washed, the membrane was incubated with secondary antibody (anti-rabbit IgG-peroxidase conjugate) in 0.5% blocking solution. After further washing, immunoreactive protein bands were visualized with FUJIFILM immunoimage analyzer LAS-1000. Each band was scanned, and the peaks of integrated density were quantitated with FUJIFILM bioimaging analyzer Mac BAS, version 2.5.
Solutions.
The composition of the standard HEPES buffered solution was (in mM)
137.0 NaCl, 5.9 KCl, 1.2 CaCl2,
14.0 glucose, and 10.0 HEPES. The pH values of all HEPES-buffered
solutions were adjusted to 7.4 with NaOH. A
high-K+ solution was prepared by
replacing NaCl with equimolar KCl. A nominally
Ca2+-free solution was prepared by
replacing Ca2+ with 0.5 mM EGTA.
The solutions used for skinned-fiber experiments are listed in Table
1 and were prepared as described previously (33). The apparent stability constant of Ca-EGTA at 23°C and pH 7.0 was 106.35
M1, and the method for
calculating pCa (computer program SP 6802) was that of Horiuchi (12).
The ionic strength of the solution was maintained at 0.2 M by adjusting
the concentration of potassium methanesulfonate. The pH was adjusted to
7.0 with KOH at 23°C. The composition of the buffer for MLC
extraction was 6 M urea, 20 mM Tris, 22 mM glycine, 10 mM
dithiothreitol, 0.04% bromophenol blue, 10 mM EGTA, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 0.6 M KI.
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Statistics. Data are expressed as means ± SE. Statistical significance was examined by using the paired Student's t-test.
Drugs.
The following compounds were obtained (sources are in parentheses): RuR
(Sigma, lot 126H1196; Wako Pure Chemicals Industries, lot
LEN0492), ATP disodium salt (Oriental), creatine phosphate disodium
salt (Wako Pure Chem. Indust.), EGTA and PIPES (Dojindo), protein
kinase C inhibitor peptide [PKC-(1931); Seikagaku],
microcystin-LR (Research Biochemicals International), and ML-9 (Sigma).
The MLC20 antibody was prepared in
Dr. M. P. Walsh's laboratory. Myosin from chicken
gizzards was obtained from Sigma. The purity of RuR from Sigma was
>40%, and the purity of that from Wako was >95%. There was no
marked difference between the potentiating effects on contraction in
skinned fibers of RuR from these two sources. All the data shown in
RESULTS were obtained with RuR from Wako.
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RESULTS |
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Opposite effects of RuR on intact and skinned smooth muscle fibers.
Figure 1 shows the effects of 100 µM RuR
on contractions induced by a
high-K+ solution in an intact
strip (A) and by a pCa 6.0 solution
in a skinned strip (B) of the guinea
pig urinary bladder. In intact muscle, a phasic contraction and a
subsequent tonic contraction were induced by 142.9 mM
K+. The second trial with 142.9 mM
K+ solution was performed in the
presence of 100 µM RuR added 5 min before the
high-K+ solution. Both phasic and
tonic contractions were markedly reduced by the application of 100 µM
RuR. The third trial with the
high-K+ solution after washout of
RuR showed that the inhibition by RuR was mostly removed. In a
preparation permeabilized with -escin and treated with A-23187, the
tonic contraction elicited by the pCa 6.0 solution was markedly
increased by the addition of 100 µM RuR (Fig.
1B). This effect was also reversed
on washout.
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Increase in
Ca2+ sensitivity
caused by RuR.
Figure 2A
shows the effects of 60 µM RuR on contractions induced by a
cumulative increase in
[Ca2+] in skinned
fibers of the guinea pig urinary bladder. In the presence of RuR, the
contractions induced by pCa 6.0 and 5.5 solutions were larger than
those in the control. Figure 2B shows
the relationship between pCa and relative tension in the absence of RuR
and in the presence of 60 µM RuR. The
[Ca2+] giving
half-maximal contraction was decreased from 2.14 ± 0.26 (n = 5) to 1.50 ± 0.19 µM
(n = 5) by the application of 60 µM RuR (P < 0.05). The relative
magnitudes of the maximal responses to pCa 4.5 in the absence of RuR
were 26.9 ± 14.1% of that induced by 142.9 mM
K+ solution before skinning and
22.9 ± 9.6% of that in the presence of 60 µM RuR
(P > 0.05). Therefore, the
Ca2+ sensitivity of skinned fibers
was increased by 60 µM RuR, whereas the maximal response was not
affected significantly. To confirm this, 100 µM RuR was added when
muscles were contracted in pCa 6.0 or 4.5 solution (Fig.
3A). The
contraction in pCa 6.0 solution was increased to 286.6 ± 22.6%
(n = 4) of control
(P < 0.01) and that in pCa 4.5 solution was not affected significantly (99.9 ± 0.1% of control,
n = 4, P > 0.05) by the addition of 100 µM RuR (Fig. 3B).
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Concentration-dependent effects of RuR.
The contraction of skinned fibers of the guinea pig urinary bladder in
pCa 6.0 solution was increased in a concentration-dependent manner by
the cumulative addition of 30-300 µM RuR (Fig.
4A). The
effects of 30-100 µM RuR were removed by washout, but those of
300 or 600 µM RuR were not removed completely, even after a 30-min
washout in R2G. Experiments were always performed in a paired fashion
in the presence (Fig. 4Aa) and
absence (Fig. 4Ab) of RuR, because
the effect of RuR developed slowly. In the time-matched control, the
relative amplitude of contraction in pCa 6.0 just before the solution
change back to R2G was 139.5 ± 22.6% of that measured 10 min after
the transfer to pCa 6.0 solution (n = 4; P > 0.05). Figure
4B shows the relationship between RuR
concentration and tension in the pCa 6.0 solution. The relative
magnitude of contraction in pCa 6.0 solution in the presence of RuR was
corrected for the time-matched control in each pair of preparations.
The contraction in pCa 6.0 solution was enhanced by RuR in a
concentration-dependent manner in the range of 10-600 µM.
Maximal force was 324.9 ± 55.8% (n = 6, P < 0.01) of control, with
half-maximal effect at ~60 µM RuR.
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Effect of RuR on skinned fibers of ileum and mesenteric artery.
Figure 5A
shows the relationship between pCa and the relative amplitude of
contraction in skinned fibers of the urinary bladder, ileal
longitudinal smooth muscle layer, and mesenteric artery of the guinea
pig. The Ca2+ sensitivity of the
mesenteric artery was higher than those of the urinary bladder and
ileum. In pCa 6.0 solution, contractions of skinned fibers of the
mesenteric artery, ileum, and urinary bladder were 88.4 ± 2.0 (n = 5), 53.1 ± 5.4 (n = 5, P < 0.01 vs. the mesenteric artery
and urinary bladder), and 18.2 ± 3.2%
(n = 6, P < 0.01 vs. the mesenteric artery)
of the maximal contraction. Figure 5,
B and
C, shows the relationship between RuR
concentration and the relative magnitude of contraction for the ileum
(B) and mesenteric artery
(C) when RuR was added cumulatively
in pCa 6.0 and 6.3 solutions, respectively. The dotted lines indicate
the relationship for the urinary bladder at pCa 6.0 (see Fig.
4B). Although 300 µM RuR markedly
enhanced the contraction in the ileum (222.2 ± 46.3% of
control; n = 6, P < 0.05), this enhancement was
smaller than that for the urinary bladder (324.9 ± 55.8% of control; n = 5, P < 0.05). Moreover, in the
mesenteric artery, the contraction in pCa 6.0 solution was 88% of the
maximum contraction at pCa 4.5 and the enhancement by 300 µM RuR was
not significant (113.3 ± 5.8% of that before application of RuR;
n = 3). The contraction at pCa 6.3 in
this preparation was, however, markedly enhanced by 300 µM RuR (Fig.
5C) (461.8 ± 117.5% of control;
n = 6, P < 0.05). These results again
suggest that RuR increases the
Ca2+ sensitivity of the
contractile system without affecting the maximal response.
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Effect of GDPS on RuR-induced enhancement of
contraction.
It is widely accepted that smooth muscle contraction by agonist
stimulation is mainly due to an increase in
[Ca2+]i
and Ca2+ sensitization of
contractile elements (29), although additional Ca2+-independent mechanisms are
involved (1, 15). It has been reported that guanine nucleotide-binding
proteins (G proteins) are involved in the agonist-induced increase in
the Ca2+ sensitivity of
MLC20 phosphorylation and
contraction of smooth muscle (7, 18). This GTP-mediated
Ca2+ sensitization is inhibited by
nonhydrolyzable GDP analogue guanosine 5'-O-(2-thiodiphosphate) (GDP
S). The contraction of
skinned guinea pig ileum strips enhanced by 100 µM ACh was reduced by
the addition of 100 or 300 µM GDP
S (Fig.
6Aa). On
the other hand, the enhancement of contraction by 100 (n = 5; not shown) or 300 µM
(n = 4) RuR was not affected by the
addition of GDP
S (Fig. 6Ab).
Figure 6B shows the summarized
results. The magnitude of the enhancement of contraction by 100 µM
ACh was smaller than that by 300 µM RuR (to 209.1 ± 26.7 vs.
387.8 ± 70.3% of control, respectively;
n = 4). The ACh-induced enhancement of
contraction was significantly reduced by 300 µM GDP
S, whereas the
enhancement by 300 µM RuR was unaffected.
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Effect of PKC inhibitor on RuR-induced enhancement of contraction.
The activation of PKC induces contraction in intact and permeabilized
smooth muscle preparations via an increase in
Ca2+ sensitivity and/or a
Ca2+-independent pathway (5, 29,
36). The application of 3 µM phorbol 12,13-dibutyrate (PDBu), a
potent activator of PKC, strongly enhanced the contraction at pCa 6.3 in skinned smooth muscle strips of the ileal longitudinal layer (Fig.
7Aa).
The PDBu-induced enhancement was abolished by PKC-(1931), a peptide inhibitor of PKC. To investigate the involvement of PKC in
Ca2+ sensitization caused by RuR,
the effect of PKC-(19
31) was studied (Fig.
7Ab). The enhanced contraction was
not affected significantly by PKC-(19
31). Figure
7B shows the summarized results of the effects of PKC-(19
31) on PDBu- and RuR-induced contractions. The
application of 3-30 µM PKC-(19
31) decreased the PDBu-induced enhancement of contraction in a concentration-dependent manner (n = 5, P < 0.05 vs. control at 10 µM and
P < 0.01 vs. control at 30 µM) but
did not affect the RuR-induced enhancement of contraction (n = 4, P > 0.05 vs. control).
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Effects of microcystin-LR on RuR-induced contraction.
Contraction of smooth muscle is triggered by
Ca2+ binding to calmodulin, which
activates MLC kinase (MLCK). Activated MLCK phosphorylates Ser-19 of
MLC20, resulting in activation of
the myosin MgATPase by actin (8). The principal mechanism of relaxation
involves dephosphorylation of
MLC20 by MLC phosphatase.
Therefore, the inhibition of MLC20
phosphatase is a possible mechanism of
Ca2+ sensitization (28). The
application of microcystin-LR, a potent protein phosphatase inhibitor
(11), induced a concentration-dependent tonic contraction of
permeabilized guinea pig ileum strips in R2G solution (Fig.
8Aa).
The relationship between the concentration of microcystin and
contraction in the presence of 300 µM RuR was examined (Fig.
8Ab). RuR exhibited a dual action on
microcystin-induced contraction: 300 µM RuR significantly enhanced
the effect of microcystin at low concentrations (30 and 50 nM) and
significantly reduced that at high concentrations (1 and 3 µM). The
contraction induced by 30 nM microcystin was significantly increased in
the presence of 300 µM RuR (from 2.7 ± 1.0 to 10.2 ± 3.5%,
n = 6, P < 0.05) (Fig.
8B). The concentrations of
microcystin required for half-maximal effect were 84 and 44 nM
(n = 6, P < 0.05) in the absence of RuR and
in the presence of 300 µM RuR, respectively. The minimum
concentration of microcystin required to induce a detectable
contraction was also decreased. The maximum contraction obtained at 1 µM microcystin was significantly decreased from 42.4 ± 4.6 to
27.4 ± 4.2% (n = 6, P < 0.05) (Fig.
8B).
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Effect of RuR on the relaxation rate.
The rate of relaxation following the decrease in
[Ca2+] from pCa 5.0 to
nominally free (R2G solution; pCa > 8.0) was measured (Fig.
9A). To prevent MLC phosphorylation,
100 µM ML-9, an inhibitor of MLCK, was added to the solution. Under
these conditions, the relaxation mainly depends on the
dephosphorylation rate of phosphorylated MLC by phosphatase (28, 33).
The relaxation phase was well described by a function of
single exponential with a time constant () of 15.8 ± 2.7 s.
Although the peak amplitude of the contraction in the pCa 5.0 solution
in the second trial decreased to 60.1 ± 4.4% of that of the first
one (n = 6), the rate of relaxation did not change (
= 15.7 ± 2.5 s,
n = 6, P > 0.05 vs. the results of the 1st
trial). The effect of 100 µM RuR on the relaxation phase was examined
in the second trial (Fig. 9Ab). In
the presence of RuR, the contraction at pCa 5.0 slightly but
significantly increased (67.4 ± 4.5% of that in the 1st trial;
P > 0.05 vs. the 60.1 ± 4.4%
measured in the 2nd trial in the absence of RuR; Fig.
9Aa;
n = 6). The relaxation phase in the
presence of 100 µM RuR was slower than that of the control (Fig.
9B) and was also described by a
single-exponential function (
= 27.3 ± 3.7 s; P < 0.01 vs. the results of the 2nd
trial in the absence of RuR; Fig.
9C).
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Effect of RuR on MLC20 phosphorylation.
Figure
10A
shows the detection of unphosphorylated and phosphorylated
MLC20 bands with the
MLC20 antibody after separation by
urea-glycerol gel electrophoresis. When skinned ileum strips were
exposed to the pCa 6.3 solution, the extent of phosphorylation of
MLC20 was 20.7 ± 1.9%
(n = 6; Fig.
10B). The addition of 300 µM RuR
significantly increased the level of phosphorylation to 28.1 ± 1.5% (n = 6, P < 0.05 vs. the results in the
absence of RuR; Fig. 10B).
MLC20 phosphorylation in intact
strips, which were extensively contracted in 142.9 mM
K+ solution, was 37.7 ± 3.0%
(n = 8, P < 0.01 vs. the phosphorylation in
pCa 6.3 solution). These results indicate that RuR significantly increased MLC20 phosphorylation
along with the enhancement of contraction. The relationship between
relative tension and MLC20 phosphorylation was obtained from skinned fibers in the absence of RuR
and in the presence of 300 µM RuR (Fig.
10C). The increase in
[Ca2+] over the range
pCa 6.3-4.5 increased both relative tension and MLC20 phosphorylation. The
increase in the relative tension produced by 300 µM RuR was also
coupled with the enhancement of
MLC20 phosphorylation. It is,
however, also noteworthy that the phosphorylation
level at pCa 6.3 in the presence of 300 µM RuR (28%) tends
to be smaller than those at pCa values of 5.5 (31.6 ± 3.0%, n = 4;
P > 0.05 vs. 28%) and 4.5 (35.6 ± 3.5%, n = 4;
P > 0.05 vs. 28%) in the absence of
RuR, whereas the corresponding relative tensions are comparable.
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DISCUSSION |
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The present results clearly show that RuR increases the Ca2+ sensitivity of the contractile system in skinned smooth muscle preparations. This observation was unexpected in light of the report that RuR suppresses Ca2+ binding to calmodulin (26). Although the reduction of the high-K+-induced tonic contraction by RuR (Fig. 1) confirmed the previous observation (26), our interpretation of the cause of the reduction is different. Sasaki et al. (26) suggested that RuR enters the cytoplasm and inhibits Ca2+ binding to calmodulin. Electrophysiological experiments using whole cell voltage-clamp techniques, however, indicate clearly that RuR inhibits the voltage-dependent Ca2+ channel current in urinary bladder myocytes with an IC50 of ~3 µM (10). This strongly suggests that the RuR-induced reduction of high-K+-induced contraction is mainly attributable to the blocking of Ca2+ entry through voltage-dependent Ca2+ channels. The complete recovery of high-K+-induced contraction after washout of RuR (Fig. 1) supports this interpretation. The direct effects of RuR on the contractile system can be clarified only by use of skinned smooth muscle fibers. The concentration of RuR required for the increase in Ca2+ sensitivity in skinned fibers (>30 µM) was higher than that required for interaction with Ca2+-binding proteins {calmodulin, Ca2+-ATPase, the ryanodine receptor, and the D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [~10 µM]} or for the inhibition of Ca2+ current (IC50 ~3 µM). This would suggest that the site of action of RuR is not a Ca2+-binding protein.
RuR blocks Ca2+-release channels in the skeletal muscle SR membrane and thereby reduces Ca2+ release through the channel (19, 34). It has been reported that RuR inhibits smooth muscle Ca2+-release channels reconstituted into planar lipid bilayers (9) and caffeine-induced contraction of skinned smooth muscle strips (14). Ca2+ release via Ins(1,4,5)P3 receptors is also inhibited by RuR, presumably because RuR competes with Ca2+ at the binding sites. It has also been reported that SR Ca2+ pump activity is reduced by RuR. The RuR-induced enhancement of contraction is, however, not due to changes in SR Ca2+ uptake and/or release, because it was observed in skinned fibers in which SR Ca2+ storage and release functions were destroyed by A-23187.
The enhancement of contraction by RuR was observed only at relatively low [Ca2+] values (pCa 6.0 and 5.5). The maximal contraction at pCa 4.5 was not affected by RuR, strongly suggesting that RuR increases the Ca2+ sensitivity of the contractile system without a change in the maximal interaction between actin and myosin. It is also noteworthy that a significant enhancement of contraction by RuR in the mesenteric artery was observed at pCa 6.3 but not at pCa 6.0, because the contraction at pCa 6.0 was 88.4% of the maximum and further significant enhancement was not available. The possibility that 30-300 µM RuR changes pCa in the solutions via direct interaction with 5 mM EGTA seems to be low. It is clear, therefore, that RuR increases the Ca2+ sensitivity of the contractile system but does not change the maximal contractile ability in both phasic and tonic smooth muscles: the urinary bladder, ileal longitudinal layer, and mesenteric artery. A smaller but significant increase in the Ca2+ sensitivity of the contractile system produced by RuR in cardiac muscle has been reported, but the mechanism remains unknown (37).
An increase in the Ca2+
sensitivity (Ca2+ sensitization)
of the contractile system elicited by the addition of GTP and ACh was inhibited by GDPS, as previously reported (17). The involvement of
the small GTPase, Rho, in the Ca2+
sensitization of smooth muscle contraction has been shown (2, 7, 16,
18). On the other hand, Ca2+
sensitization by RuR was not affected by GDP
S, suggesting that the
activation of a small GTPase is not involved in the mechanism of
RuR-induced Ca2+ sensitization.
The activation of PKC by phorbol ester induces or enhances the tonic
contraction of intact and permeabilized smooth muscle preparations via
the inhibition of MLC20
phosphatase (20) and also via the phosphorylation of actin-binding
protein calponin through a pathway independent of
MLC20 phosphorylation (13, 35,
36). Although the mechanisms of agonist-induced
Ca2+ sensitization have not been
fully clarified, the inhibition of MLC20 phosphatase by PKC is
implicated as one of the major components (29). In the present study,
however, the addition of 3-30 µM PKC-(1931), a selective
peptide inhibitor of PKC, did not reduce the
Ca2+ sensitization induced by 100 µM RuR, whereas it did inhibit PDBu-induced Ca2+ sensitization. The activation
of PKC, therefore, is not the major cause of RuR-induced
Ca2+ sensitization.
MLC20 phosphorylation was significantly increased in skinned fibers treated with RuR at submaximal [Ca2+]. This could result from the activation of MLCK or the inhibition of MLC20 phosphatase. The latter is more likely because RuR has actually been reported to reduce MLCK activity via inhibition of Ca2+ binding to calmodulin (26). Moreover, the concentration-response relationship of Ca2+-independent, microcystin-induced contraction was affected significantly, suggesting that RuR may affect MLC20 phosphatase: the addition of RuR reduced the EC50 of microcystin for contraction from 84 to 44 nM and, interestingly, reduced the maximum response to microcystin. RuR may have a lower potency of phosphatase inhibition than microcystin and could compete with microcystin for inhibition of the phosphatase. Of potential relevance here is the observation that high concentrations of RuR (>1 mM) caused contraction in the absence of Ca2+ and microcystin (data not shown). The relaxation following the change in [Ca2+] from pCa 5.0 to nominally free (R2G; pCa > 8.0) was significantly slowed down by the treatment with RuR, under conditions in which MLC20 phosphorylation was blocked by ML-9. These results strongly suggest that RuR reduces MLC20 phosphatase activity to increase Ca2+ sensitivity in these smooth muscles.
Although the increase in Ca2+ sensitivity by RuR is mainly due to the increase in MLC20 phosphorylation (Fig. 10), the extent of the increase in MLC20 phosphorylation may not completely explain the enhancement of the relative contraction. An additional mechanism for RuR-induced enhancement of contraction may be via an increase in actomyosin ATPase activity through an actin-linked pathway that was not examined in the present study. For example, if RuR directly enhances the activity of calponin or inhibits calponin phosphatase, the result could be a decrease in the calponin-mediated inhibition of actin-activated myosin MgATPase activity (36).
In conclusion, RuR has a novel Ca2+ sensitization effect on the contractile apparatus of smooth muscle. This is the first report of such a sensitization effect. The effect is not mediated by the activation of a G protein or PKC. Results rather indicate that RuR inhibits MLC20 phosphatase. Although the concentration of RuR required for Ca2+ sensitization is higher than that for inhibition of the RyR, these findings are important because of the frequent use of RuR as a pharmacological tool.
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ACKNOWLEDGEMENTS |
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This work was supported by Monbushio International Scientific Research Program through Joint Research Grants 08044313 and 10044313. M. Watanabe and Y. Imaizumi are also supported by Grant-in-Aid for Scientific Research on Priority Areas 09273101 and by the Japanese Ministry of Education, Science, Sports and Culture.
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FOOTNOTES |
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M. P. Walsh is a Medical Scientist of the Alberta Heritage Foundation for Medical Research.
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: Y. Imaizumi, Dept. of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Nagoya City Univ., 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan (E-mail: yimaizum{at}phar.nagoya-cu.ac.jp).
Received 16 July 1998; accepted in final form 16 November 1998.
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