(Received for publication, January 2, 1995; and in revised form, May 22, 1995)
From the
In permeabilized smooth muscle, exogenously applied calponin
binds to myofibrils and reduces Ca-activated tension
(Itoh, T., Suzuki, S., Suzuki, A., Nakamura, F., Naka, M., and Tanaka,
T.(1994) Pflügers Arch. Eur. J. Physiol. 427,
301-308). A calponin peptide (calponin
Phe
-Arg
), which inhibits the binding of
calponin to actin, blocks the action of calponin and enhances the
contraction induced by submaximal Ca
in permeabilized
vascular smooth muscle. Unlike calmodulin, this peptide enhances the
Ca
-induced contraction without a corresponding
increase in the level of myosin light chain phosphorylation. These
results suggest that calponin decreases the sensitivity of smooth
muscle to Ca
at a given level of myosin light chain
phosphorylation.
Phosphorylation of myosin light chain by
Ca-calmodulin-dependent myosin light chain kinase is
believed to be responsible for the coupling between an increased
intracellular concentration of Ca
and contraction in
smooth muscle(1, 2, 3, 4) . However,
a dissociation of the relationship between the intracellular
concentration of Ca
and tension and that between
Ca
and myosin light chain phosphorylation in smooth
muscle contraction has been
reported(3, 5, 6) , and thus other mechanisms
that act in concert with, or without, the participation of myosin light
chain phosphorylation have also been suggested. Recently, much
attention has been paid to thin filament-linked mechanisms that are
mediated by the actin-binding proteins, calponin and caldesmon.
Calponin is a major component of the smooth muscle thin
filament(7) , and it binds to F-actin, tropomyosin, and
calmodulin. This protein inhibits the actomyosin ATPase activity of
smooth muscle through its binding to actin in
vitro(8, 9, 10) . Moreover, calponin is
known to be an excellent substrate for protein kinase C and
Ca-calmodulin-dependent protein kinase II in
vitro(9, 10) , the phosphorylation of calponin by
each kinase resulting in the loss of its ability to inhibit ATPase
activity (9, 10) . Three tryptic phosphopeptides of
smooth muscle calponin made using protein kinase C were recently
isolated and designated T
(Gly
-Lys
), T
(Phe
-Arg
), and T
(Gly
-Arg
)(11) .
Phosphorylation of Ser
(12) or Thr
(11) in the T
peptide accounts for over 50%
of the total calponin phosphorylation, and the phosphorylation is
competitively inhibited by actin, suggesting that peptide T
may be adjacent to the actin-binding region of calponin.
Here,
we present evidence derived from experiments using T peptide that calponin acts to lower the myofilament
Ca
sensitivity in smooth muscle contraction at a
given level of myosin light chain phosphorylation.
Figure 1:
Effect of exogenously applied calponin
and a calponin peptide (calponin Phe-Arg
;
T
) on Ca
-induced contraction in
permeabilized vascular smooth muscle. A, calponin (CaP) was applied during the maintained contraction induced by
1 µM Ca
. Subsequent application of
T
increased the tension to the level recorded before
application of calponin. B, concentration-dependent effects of
T
on contraction induced by 0.3 µM Ca
. After a recording of the maximum
Ca
-induced contraction (on application of 10
µM Ca
), 0.3 µM Ca
was applied. Subsequently, various
concentrations of T
(0.03-0.3 mM) were
applied on the Ca
-induced contraction, in ascending
steps. C, effect of simultaneous application of CaP and
T
on contraction induced by 0.3 µM Ca
. Both agents were applied during the steady
state contraction induced by 0.3 µM Ca
followed by a washout of T
only; 10 µM Ca
was finally applied to register the maximum
Ca
-induced response.
Protein kinase C phosphorylates calponin, causing
dissociation of calponin from actin and thus attenuates the
calponin-induced inhibition of actin-activated myosin Mg-ATPase in
reconstituted smooth muscle contractile
proteins(9, 10) . Calponin (3 µM)
phosphorylated by protein kinase C (0.6 mol of P/mol of
calponin) had less inhibitory action (0.95 ± 0.05 times control, n = 3) than nonphosphorylated calponin (0.48 ±
0.16 times control, n = 3, p < 0.05) on the
contraction induced by 1 µM Ca
. The
result was consistent with the previous findings(13) . Under
these conditions, the binding of the phosphorylated calponin to the
myofibril in permeabilized smooth muscle was decreased (0.67 ±
0.21 times that obtained by unphosphorylated calponin, n = 3). These results suggest that the action of exogenously
applied calponin is not nonspecific and that, on application, the
unphosphorylated form of calponin first binds to the myofibril and then
inhibits Ca
-induced contraction.
Figure 2:
A, effect of a synthetic calponin peptide,
T, on the fluorescence intensity of pyrene-labeled F-actin.
Pyrene-labeled F-actin (3 µM) in 20 mM Tris-HCl
(pH 7.4) containing 100 mM KCl and 0.1 mM CaCl
was preincubated for 1 h at 25 °C. Fluorescence intensity
(excited at 365 nm, emitted at 407 nm) was measured 15 min after the
addition of T
. The values are the mean of four
determinations with S.D. B, effect of T
on binding
of calponin or caldesmon to F-actin. T
(0.3 mM) or
T
(0.3 mM) was added to F-actin (3
µM) in 20 mM Tris-HCl (pH 7.5) containing 30
mM KCl, 2 mM MgCl
, 1 mM ATP, 1
mM dithiothreitol, and 0.2 mM CaCl
, and
the reaction mixture was incubated for 30 min at 25 °C following
the addition of either calponin (1.5 µM) or caldesmon (1.5
µM). Subsequent analysis for an actin cosedimentation
assay was performed as described under ``Experimental
Procedures.'' Each bar represents the mean of at least
four experiments, and each verticalbarabove indicates S.D. The squarebracket indicates
values that are significantly different (p < 0.01;
Student's t test). C, effects of T
peptide on actin-activated Mg
-ATPase activity.
The conditions for ATPase assay were as described under
``Experimental Procedures.'' Actin (2-8
µM) was added in the presence (
) or absence (
)
of 0.3 mM T
peptide. The rates of myosin
Mg
-ATPase activity are given as the mean of
triplicate measurements in the same assay conditions. D,
effects of T
and T
peptides on actin-activated
Mg
-ATPase activity. Conditions for the ATPase assay
were as described under ``Experimental
Procedures.''
Calponin and caldesmon simultaneously bind to F-actin, but calponin
uses both specific and common binding sites(22, 23) .
The effect of T peptide on the binding of calponin and
caldesmon to F-actin was investigated using the cosedimentation method.
T
peptide (0.3 mM) inhibited calponin binding to
F-actin compared with T
peptide (GASQAGMTAPGTKR). Moreover,
T
peptide had no effect on caldesmon binding to F-actin (Fig. 2B), indicating that T
peptide
interacts with F-actin at its specific binding sites. On the other
hand, T
peptide had little effect on actin-activated
Mg
-ATPase activity at the concentration of 0.3 mM (Fig. 2, C and D).
ITP is a substrate for myosin
ATPase but not for myosin light chain kinase(24) . In a
solution containing 5 mM Mg-ITP (with no ATP), T (0.3 mM) did not produce contraction in
Ca
-free solution containing 4 mM EGTA
(tension = 0.02 ± 0.02 times the maximum Ca
contraction in ATP-containing solution, n = 4). A
lack of action of T
on smooth muscle contraction was also
found when this peptide (0.3 mM) was applied in Mg-ITP
solution containing 10 µM Ca
(tension
= 0.03 ± 0.02 times the maximum Ca
response in ATP-containing solution, n = 4).
These results suggest that T
may enhance the contraction
induced by a given level of myosin light chain phosphorylation.
To
test this hypothesis, the relationship between Ca and
tension and that between Ca
and myosin light chain
phosphorylation was determined in the presence and absence of
T
, and the effects were compared with those obtained with
calmodulin (Fig. 3). Calmodulin (6 µM) enhanced
both the contraction and myosin light chain phosphorylation induced by
submaximal concentrations of Ca
without any change in
the maximum responses induced by 10 µM Ca
. The enhancing action of calmodulin was
apparent at concentrations over 0.3 µM Ca
(Fig. 3). The Hill coefficient and ED
value
for Ca
in producing contraction were 1.7 ± 0.1
and 0.6 ± 0.1 µM, respectively, in control (n = 4) and 2.4 ± 0.5 (p < 0.05) and 0.31
± 0.02 µM (p < 0.05), respectively, in
the presence of calmodulin (n = 4). In contrast, 0.3
mM T
enhanced the Ca
-induced
contractions without a corresponding increase in the level of myosin
light chain phosphorylation; this action was apparent on the
contractions induced by Ca
at concentrations over 0.1
µM. T
had no effect on either the maximum
contraction or myosin light chain phosphorylation induced by 10
µM Ca
. The Hill coefficient and
ED
value for Ca
in producing contraction
in the presence of T
(n = 4) were 1.5
± 0.4 (p < 0.05 against control) and 0.4 ±
0.1 µM (p < 0.05), respectively. These results
indicate that T
peptide enhances the contraction without a
change in the level of myosin light chain phosphorylation at a given
concentration of Ca
(0.1-1.0 µM).
Figure 3:
Effects of T peptide and
calmodulin on the relationship between Ca
and tension (A) and on that between Ca
and 20-kDa myosin
light chain (MLC) phosphorylation (B) in
-escin-permeabilized vascular smooth muscle. A,
increasing concentrations of Ca
(0.03-10
µM) were cumulatively applied from low to high in
Ca
-free solution containing 4 mM EGTA. B, measurement of phosphorylation of MLC was performed as
described under ``Experimental Procedures.'' The
phosphorylation of MLC is expressed in mol of PO
/mol of
MLC. In A and B:
, control;
, 6 µM calmodulin;
, 0.3 mM T
. Results shown
are each the mean of 4-5 observations with S.D. The slope of the
concentration-response relationship (of Ca
against
tension and against MLC phosphorylation) is shown by the Hill
coefficient (N) and midpoint position (pK =
(-log K), where K is the dissociation
constant). These parameters were obtained by fitting the data points
for each curve to the following equation by a nonlinear least squares
method. F/F
= (C/K)
/(1 + (C/K)
), where C represents the concentration of Ca
. In A, F is the amplitude of contraction at any given
concentration of Ca
, and F
is
the maximum response evoked by 10 µM Ca
expressed as a relative tension of 1.0. *, values that are
significantly different with a corresponding control (p <
0.05).
Figure 4:
Effect of calmodulin and T
peptide on the relationship between tension and MLC phosphorylation in
-escin-permeabilized smooth muscle. The curves were
obtained by fitting the data points to the following equation by a
nonlinear least squares method: F/F
= (C/K)
/(1+ (C/K)
). N, Hill
coefficient; K, dissociation constant. C and F/F
represent phosphorylation of MLC (mol
of PO
/mol of MLC) and relative tension, respectively.
Results shown are each the mean of four observations with S.D. The
fitted values of N and K were, respectively, 2.4 and
0.26 mol of PO
/mol of MLC in control (r =
0.996) and 1.6 and 0.13 mol of PO
/mol of MLC in the
presence of T
(r = 0.992).
, control (n = 4);
, 6 µM calmodulin (n = 4);
, 0.3 mM T
(n = 4);
, 1 mM T
(n = 4);
, 1 mM T
with 6 µM calmodulin (n = 4).
It has been
suggested that the phosphorylation of myosin light chain does not
entirely depend on the cellular concentration of Ca.
In permeabilized smooth muscles, agonist with GTP and GTP
S alone
both enhance, at a given concentration of Ca
, the
phosphorylation of myosin light chain and the tension produced,
possibly through an inhibition of myosin phosphatase (6, 25) . Phosphorylation of myosin light chain kinase
by Ca
-calmodulin-dependent protein kinase II causes
an increase in the concentration of Ca
-calmodulin
required for the activation of myosin light chain
kinase(26, 27) . These two processes increase and
decrease, respectively, the Ca
-sensitivity of myosin
light chain phosphorylation without a change in the relationship
between tension and myosin light chain phosphorylation. However, our
study has raised the interesting point that, in contrast, T
peptide did change the tension-myosin light chain phosphorylation
relationship.
In conclusion, under physiological conditions,
calponin acts to lower the myofilament Ca sensitivity
in smooth muscle contraction at a given level of myosin light chain
phosphorylation.